BioinorganicBIOINORGANIC CHEMISTRY K. Hussain Reddy Associate Professor of Chemistry Sri Krishnadevaraya University Anantapur NEW AGE NEW AGE INTERNATIONAL (P) LIMITED, PUBLISHERS New Delhi e Bangalore e Chennai e Cochin e Guwahati e Hyderabad Jalandhar « Kolkata @ Lucknow @ Mumbai e RanchiCopyright © 2003 New Age International (P) Ltd., Publishers First Edition : 2003 Reprint : 2005 NEW AGE INTERNATIONAL (P) LIMITED, PUBLISHERS 4835/24, Ansari Road, Daryaganj, New Delhi - 110 002 Visit us at : www.newagepublishers.com Offices at: Bangalore, Chennai, Cochin, Guwahati, Hyderabad, Jalandhar, Kolkata, Lucknow, Mumbai and Ranchi This book or any part thereof may not be reproduced in any form without the written permission of the publisher. ‘This book cannot be sold outside the country to which it is consigned by the publisher without the prior permission of the publisher. Rs. 225.00 ISBN : 81-224-1437-0 2345678910 Published by New Age International (P) Ltd., 4835/24, Ansari Road, Daryaganj, New Delhi-110 002 and printed in India at A.P. Offset, DelhiContents Preface 1.__Introduction 2. L1___Trace elements 12 Complex formation 1.3 Hard and soft acids and bases (HSAB' 14 Inert and Labile complexes 1.5 Ligand substitution in octahedral complexes 1.6 Substitution in square planar complexes L7___The trans effect 8 = : Protei L9 Structure of proteins 1.9.1 Primary Structure 1.9.2 Secondary Structure 1.9.3 Tertiary Structure 1.9.4 Quaternary Structure 110 The Peptide Bond 1.10.1 Binding Sites in Peptide Lil Enzymes 1.12 Nucleosides, Nucleotides and Nucleic Acids 113 Carbohydrates (Sugars) L14__Blood L1S5__Plasma L16 The Cell-Structural and Functional Unit L.16.1 Structure of Eucaryotic Cell 1.16.2 Structure of prokaryotic cell 1.16.3 Membrane Structure Bibliography Physicochemical and Spectral Methods 2.1 Electronic spectra 2.1.1 Spin States 2.1.2 Russel-Saunders Coupling Scheme 2.1.3. Selection Rules 2.1.4 Jahn-Teller Effect 2.1.5 — Spectrochemical Series 2.1.6 Charge Transfer Bands < RREBBBEERREGREEG BE Benaviii__Contents 2.2 ___ Nuclear magnetic resonance (NMR) spectroscopy 42 2.3 Electron spin resonance (ESR) spectroscopy 45 2.4 Infrared and Raman spectroscopy 49 2.5 Optical rotatory dispersion and circular dichromism 50 2.6 Méssbauer spectroscopy 52 2.7 Extended X-ray absorption fine structure (EXAFS) 54 2.8 X-ray structure determination!? 59 2.9 Cyclic voltammetry 60 2.10 Potentiometry 62 Bibliography and References 8 Perspectives of Essential Trace Elements 64 3.1 The Concept of Essentiality 66 3.2. The Evolution of Essential Trace Elements 67 3.3 Future Essential Trace Elements SC 3.4 Role of Bulk or Structural Elements 70 3.5 Role of Minerals 70 3.6 Working of Essential Trace Elements 7 3.7 Essential Ultratrace Metals 72 28 Essential Ultratrace Nonmetals 3.9 Antagonism and Synergism among Essential Trace Elements 75 References 76 Bibliography 76 4. The Alkali Metal and Alkaline Earth Cations 77. 4.1 Coordination chemistry of alkali cations 7 4.2 Relationship between ionic specificity and field strength 79 4.3 The Ligands of Alkali Cations 82 4.3.1 Naturally Occurring Ionophores 82 4.3.2 Synthetic Complexing Agents 86 4.4 lon Transport 92 4.4.1 Facilitated Transport 94 4.4.2 Active Transport of Cations 96 4.4.3 Models for Active Transport 98 444 Sodium Dependent Transport 99 References 5.__Non-Redox Metalloenzymes 102 5.1 Carboxypeptidase A 102 S.1.1 Structure of Carboxypeptidase A (CPA) 103 412 Mechanism of Action of CPA Sd‘ 5.1.3 Specificity of Enzymatic Reactions 104 5.1.4 Model Compounds of CPA 106 5.2 Carbonic Anhydrase 107 5.2.1 Structure of Carbonic Anhydrase (CA) 107 5.2.2 Metallocarbonic Anhydrases 109Contents ix 5.2.3__Mechanism of Action of CA __109 524 Inhibi 0 525 Kinetic Studi 5.2.6 Model Compounds of Carbonic Anhydrase 112 5.3___ Alcohol dehydrogenases 113 5.3.1 Structure of Liver Alcohol Dehydrogenase (LADH) 113 5.3.2 Mechanism of Action of LADH SA 5.3.3 Model Compounds 11s Reference: 117 Further Reading 118 6. Respiratory Proteins and Model Compounds 119 6.1 Properties of respiratory Pinsent A comparison 119 62 Myoglobin and Hemoglobin’ 119 6.2.1 Functions of Myoglobin 121 Y Oxygenation Reactions 123 Structure Function Relationship 125 Oxygen Molecule 128 Structural Models for Dioxygen Binding 128 Synthetic Models for Oxygen Binding 129 6.2. 7 Models for Hemoproteins (Mb & Hb) 131 63 Hemerythrin 135 6.3.1 Structural Studies 138 64 Hemocyanin 139 Oxygen and Hemocyanin 140 Oxygenation Curves 140 Bohr Effect 142 Dissociation and Association of Hemocyanin 142 Spectral Properties 144 Metal Binding Groups/structure 144 Model Compounds 144 References 146 Further Reading 147 7. Redox Metalloenzymes 148 7.1 Biological Redox Reactions 148 7.2 Types of heme 150 7.3 Cytochromes 150 7.3.1 Cytochrome c 151 7.3.2. Cytochrome ¢ Oxidase 156 74 Cytochrome P-450 162 7.4.1 Synthetic Models for the Active Site 164 7.5 Superoxide and Bovine Superoxide Dismutase 165 7.5.1 Superoxide Toxicity 166 7.5.2 Structure of Cu, Zn-SOD 167X_Contents 7.5.3. Enzymatic Activity and Mechanism 7.5.4 Models for Superoxide Dismutase 7.6 Peroxidase and Catalase 7.7 Blue-copper proteins 7.7.1 Type | Copper Type Il Copper Type IH Copper 7.74 — Blue-oxidases with Types I, II and [IT Cu 7.75 Blue Electron-transfer Proteins with a Single Type I Copper 78 — Non-blue Proteins References Further Reading The Chemistry of Vitamin B,, and Model Compounds 8.1 Structure of vitamin By» 8.2 Derivatives of By» 83 Biochemical functions of B,» 8.3.1 Enzymes Using Coenzyme B,2 8.3.2. Enzymes Utilizing Methylcobalamin 84 Oxidation States 8.5 Properties of B,>, and By», 8.6 Model Compounds 8.7 Electrochemistry 8.7.1 Thermodynamics of the Co(II - Co(Il) - Co{I) System 8.7.2 Role of the Base-off/base-on Reactions and of Other Ligand — Exchange Reactions 8.7.3 Kinetics of Electron Transfer—Trans Effects 8.8 Chemistry of the carbon-cobalt bond 8.8.1 Synthesis of Alkyl Cobalt Compounds and Stereochemistry of Alkylation of Co(I) 8.8.2 Axial Isomers of Alkyl B,, Compounds 8.8.3 Alkyl Transfer Reactions 8.9 — Coordination Chemistry 8.10 Chemical Equilibria 8.11 Photochemistry of vitamin B,2 derivatives 8.12 The cis and trans Effects 8.13 Kinetics of Ligand Exchange 8.14 Spectroscopy of cobalt corrinoids 8.14.1 Electronic Spectroscopy 8.14.2 IR Spectroscopy 8.14.3 Electron Spin Resonance Spectroscopy 8.14.4 Nuclear Magnetic Resonance Spectroscopy References Bibliography 168 169 170 173 173 173 173 173 176 180 180 182 183 184 185 186 186 191 193 194 195 196 200 201 203 205 220 222 222 228 230 233Contents xi 9. Nitrogen Fixation and Iron-Sulphur Proteins 234 9.1 Nitrogen Fixing Microorganisms (In Vivo Nitrogen Fixation) 235 9.2 Nitrogenase 236 9.2.41 Reactivity of Nitrogenase 238 9.2.2 Redox Properties of Nitrogenase 239 9.3 Postulated Mechanisms for Biological Nitrogen Fixation 240 9.3.1 Reduction Mechanism Involving Nitride Intermediate 240 9.3.2 Reduction Mechanism Involving Diazene/or Hydrazine Intermediate 241 9.4 — The Nitrogen Molecule, Dinitrogen Complexes and their Reactivity (in vitro nitrogen Fixation) 242 9.5 Iron-Sulphur Proteins 247 9.5.1 Rubredoxin (Rd); (1Fe-OS*) Proteins 247 9.5.2 2Fe-2S* Ferredoxins 248 9.5.3. 4Fe-4S* and 8Fe-8S* Proteins 250 9.5.4 Synthetic Analogues of the Fe,S, Cluster 251 References 253 Bibliography 253 10. Metal Jon Transport and Storage 255 10.1 Iron storage and transport 255 10.1.1 Transferrin 235 10.1.2. Ferritin 258 10.2 Synthetic iron-oxo aggregates 260 10.3 Iron transport in microbes 260 10.3.1 Siderophores 261 10.3.2 Hydroxamate Siderophores 262 10.3.3 Phenolate Siderophores 262 10.3.4 Models for Siderophores 266 10.4 Other storage and transport systems 267 10.4.1 Ceruplasmin and Serum Albumin 267 10.4.2 Metallothioneins and Phytochelatins 268 10.5 Vanadium Storage and Transport 269 References 271 Further Reading on 11. Photosynthesis 272 11.1 Chloroplast 272 11.2 Light Reactions 274 11.3. Structure of Chlorophyll 274 11.4 Photosynthesis in Bacteria 277 11.4.1 The Pheophytin-quinone Reaction Center (Type II Reaction Center) 21xii__Contents 11.4.2 The Iron-sulphur Reaction Center (Type I reaction center) 278 11.5 Photosynthesis in Green Plants (Z-Scheme) 279 11.6 ATP Synthesis 281 11.7 Role of Manganese complex in evolution of oxygen 282 11.8 Dark Reaction (calvin cycle) 282 11.9 Features of Chlorophyll 285 11.10 Model System for Chlorophyll 287 Bibliography 288 12. Coordination Compounds in Medicine 289 12.1 A. Chelation therapy 289 12.1.1 BAL (Dimercaprol) 290 12.1.2. Pencillamine 290 12.1.3 Polyaminopolycarboxylic Acids 290 12.1.4 Aurine Tricarboxylic Acid 291 12.1.5 Desferrioxamine 292 12.1.6 Cryptates 292 12.2 Gold compounds and Rhematoid Arthritis 292 12.3 Anticancer Drugs 293 12.3.1 Platinum Complexes 293 12.3.2 Gold Complexes 295 12.3.3 Metallocenes and their Halides 295 12.3.4 Other Complexes 295 12.3.5 Structure Function Relationship 296 12.4 Antimicrobial agents 297 12.5 Metal complexes as Radiodiagnostic Agents 299 12.6 Magnetic Resonance Imaging 301 References 301 Further Reading 302 13. Transition Metal Complexes and Their Interaction with Nucleic Acids 303 13.1 Structures of Nucleic Acids 304 13.2 Interactions of Metal Complexes with Nucleic Acids 307 13.2.1 Coordination 307 13.2.2 Intercalation 307 13.2.3 Hydrogen Bonding 308 13.3. Fundamental Reactions with Nucleic Acids 309 13.3.1 Redox Chemistry 309 13.3.2. Hydrolytic Chemistry 311 13.4 Nuclease Activity of Classical Tris(phenanthroline) Metal Complexes 313 13.4.1 Binding Interaction with DNA 313 13.5 Techniques to Monitor Binding 316 13.5.1 Visible Spectroscopy 316Contents _ xiii 14. 13.5.2__NMR Spectroscopy 317 13.5.3 X-ray Diffraction 13.5.4 Gel Electrophoresis 13.5.5 Other Techniques 13.6 Applications of Nuclease Activity of Metal Complexes 13.6.1 Spectroscopic Probes 13.6.2 Metallofootprinting Reagents 13.6.3 Conformational Probes 13.6.4 Cleavage Probes 13.7. Metal-nucleic Acid Intractions in Nature 13.7.1 Structural Role 13.7.2. A Regulatory Role 13.7.3. Pharmaceutical Role 13.8 Miscellaneous/Recent Studies 13.8.1 Metal Oximates 13.8.2 Copper Hydroxamates 13.8.3 Copper(II) Complexes of Carbamic Acids and Thiocarbamic Acids 13.8.4 Mixed Ligand Copper(II) Complexes 13.9 Conclusions References Effects of Inorganic Pollutants on Biological Systems 14.1 Impact of toxic metal ions on enzymes 14.2. Cadmium 14.3. Mercury 14.4 Lead 14.5 Arsenic 14.6 Carbon Monoxide 14.7 Nitrogen Oxides 14.8 Sulphurdioxide 14.9 Cyanide Bibliography 317 317 321 321 321 322 324 324 325 325 326 328 328 330 330 331 332 333 333 337 337 338 339 341 342 342 343 343 344 345 Index 8G‘The author gratefully acknowledges the use of figs from the following sources: 5.6 63 7.24 ouret R.W. Hay, Bioinorganic Chemis page 32. ’, Ellis Horwood, Chic! , 1984, FA, Cotton, G. Wilkinson, Advanced Inorganic Chemistry, Wiley- Interscience, New York. K. Wilson and J. Walker, Practical Biochemistry, Cambridge Unvversity Press, 1995. -do- ~-do- -do- -do- E. Frieden, Biochemistry of the Essential Ultratrace elements, Plenum Press, New York 1984. Pure and Applied Chemistry, 20 (1969) 93. JE. Huheey, E.A. Keiter and R.A. Keiter, Inorganic Chemistry, Harper College Collins Publishers. FA. Cotton, G. Wilkinson, Advanced Inorganic Chemistry, Wiley- Interscience, Yew York. J. Huheey, E.A. Keiter and R.A. Keiter, Jnorganic Chemistry, Harper College Collins Publishers. FA. Cotton, G. Wilkinson, Advanced Inorganic Chemistry, Wiley- Interscience, New York. R.W. Hay, Bioinorganic Chemistry, Ellis Horwood, Chichester, 1984 -do- T. Spiro, Metal ion Activation of Oxygen, Wiley-Interscience, New . 1980. -do- Journal of Chemical Education 62 (1985) 991. Journal of Chemical Education 62 (1985) 998. Journal of Chemical Education 62 (1985) 1000. -do-7.25 8.6 8.7 8.12 8.13 8.14 8.15 8.16 8.17 RIK 10.1 10.9 11.6 17 8 13.4 13.5 13.6 13.8 13.9 13.10 13.14 13.15 13.16 Journal of Chemical Education 62 (1985) 999. D. Lexa and J.M, Saveant, Ace, Chem, Res., 16 (1983) 235. -do- -do- S.J. Lippard (ed.), Progress in Inorganic Chemistry, vol. 18, Interscience, New York 1973. -do- -do- -do- -do- -do- -do- -do- DE. Fenton, Biocoordination Chemistry, Oxford University Press, Oxford, 1995. ~do- DO. Hall and K.K. Rao, Photosynthesis, Cambridge University Press, 1994 -do- ~do- Bertini, Gray, Lippard and Valentine, Bioinorganic Chemistry, Viva Low-priced edition, New Delhi 1998. ~do- -do- -do- -do- -do- -do- -do- J. Stubbe and JW. Kozarich, Chem. Rev., 87 (1987) 1107. Reproduced with permission form American Chemical Society.CHAPTER 1 Introduction Bioinorganic Chemistry, as the name implies, is the Inorganic Chemistry o1 life. It is concerned with the functions of all metallic and most non-metallic ele- ments in biology. Metal ions play a vital role in a vast number of widely differing biological processes. Some of these reactions are very specific in their metal ion requirements. Only certain metal ions in specified oxidation state can fulfill the necessary catalytic or structural requirements, for example, the role of metal ions in respiratory pigments (haemoglobin, myoglobin, hemerythrin and hemocyanin) and vitamin B,, derivatives (coenzyme B,, and methyl cobalamin). Although bioinorganic chemistry is diverse field, it is largely concerned with a limited number of inter-related and interesting issues. These are: 1. What are the roles of metals and non metals in biological system? How do they work? 2, What are the chemical and three dimensional structures of biological molecules? How do they form these structures and how do their properties/ functions vary with them? 3. What are the molecular mechanisms of enzymatic catalysis. How do proteins work? 4. How do nucleic acids interacts with metal ions/metal complexes, and what are the applications in the field of medicine. These issues are previewed here and further illuminated in later chapters. How- ever, the aim becomes obvious as you read further. In all the cases the knowl- edge is extensive as it is, is dwarfed by my ignorance. The scope of bioinorganic chemistry is broad. It stretches from chemical physics to clinical medicine. The subject has developed dramatically during the last three decades. The growth of this interdisciplinary subject is due to (1) improved analytical techniques, (2) the recognition of the role of essential trace elements in plant, animal human nutrition, (3) rapid preparative methods, (4) sophistication of spectroscopy and diffraction techniques, (5) the improved and facile synthesis of simple inorganic complexes used to model or mimic of various aspects of biological molecules, (6) the use of metal complexes as therapecutic agents and (7) the increased concern about the environmental hazards caused by some metal ions. Living organism consists of a relatively small number of elements, C, H, O, N, P and S, all of which readily form covalent bonds. These elements comprise some 92% of the dry weight of living things. The balance consists of elements that are mainly present as ions and occur in trace quantities. The perspectives of essential trace elements are discussed in Chapter 3 (of this book). The pre- dominance of carbon in living matter is no doubt a result of its tremendous2 _ Bioinorganic Chemistry chemical versatility compared with all the other elements. Carbon has the unique ability to form a virtually infinite number of compounds as a result of its capacity to make as many as four highly stable covalent bands. Thus, of the over 13 million chemical compounds known, nearly 90% are organic (carbon containing) substances. Life is known to have existed for at least 3500 million years. Most probably life is preceded by a period of chemical evolution lasting about 500 million years. It is generally believed that all the molecules essential for life were successfully synthesized during the period of chemical evolution. A set of inorganic and organic reactions took place leading to the formation of the amino acids and nucleotides, the precursors of proteins and nucleic acids. Similarly during this period of chemical evolution a mixture of inorganic and organic polyphosphates was synthesized. These compounds are required to provide the primary chemical reserves of free energy for further evolution. As a result of polymerization, organisation and development, the first cell evolved. The cell requires about 100 different proteins for the metabolism and anaerobic energy production. Presumably a series of metal ions are incorporated into the cell. Some ions are required for the integrity of the cell, such as osmotic pressure, others assumed functions of catalytic centres. The main metal ion cata- lyst must have been the magnesium ion, as all the reactions involved in funda- mental cell reactions such as protein synthesis and anaerobic energy production require Mg(Il) ions. Magnesium ions are also known to catalyse several prebiotic condensation reactions. Further support for the assumption that Mg(II) ions were important in bio-evolution arises from its presence in sea water and sea sediment, an environment similar, it is believed, to that form which our cell system once evolved (Table 1.1), (It is a well known assumption that the life is originated {rom sea water). Another important biological process which requires magne- sium is photosynthesis. In photosynthesis, chlorophyll (magnesium porphyrin complex) captures light quanta and then utilizes this energy to fix carbon dioxide and to evolve oxygen (see Chapter 11). The abundance of Mg(II) ions led to the formation of chlorophyll. Table 1.1. lon concentration* in sea water and extra cellular blood plasma fon Soa water Biood plasma Na* 470 136 Mg? 50 1 Ca* 10 3 K 10 4 cr 35 100 HPO?- 0.001 1 soz 28 1 Fe 0.0001 0.02 Zn 0.0001 0.02 cue 9.001 0.015 Co* 105 0.002 Ni 106 o "mMIntroduction 3 The atomosphere during the time of chemical evolution has been a reducing one, characterized by a moderate partial pressure of methane (0.01 to 0.001 atm) and a very low oxygen pressure. Thermodynamic data revealed that the redox potentials in the seas were as low as -0.325 V (pH 8.1), that is close to the hydrogen electrode (0.480 V at pH 8.1). Inorganic sulphur would be in the form FeS.(s), the major excess of iron would be in the form Fe,O,(s), and there would be a certain amount in solution as Fe(II) (< 0.2 mM Fe(I)). It is believed that the concentration of Mn) was much higher in primordial seas, perhaps as high as 50 mM. Thus these two metals would become of importance in early redox processes. Some of the first metalloproteins appear to have been the iron-sulphur proteins. The relatively high concentrations of Fe(II) and Mn(IT) in the oceans would lead to their incorporation into other ligands such as the porphyrins. This led to the synthesis of hemoproteins. Some blue-green algae contain upto 12 atoms of Mn per reaction centre. These various components, along with chlorophyll, were necessary for photosynthesis which led to the release of oxygen into the atmosphere (Chapter 11). 1.1 TRACE ELEMENTS Majority of trace elements function as key components of essential enzyme systems or of other proteins (eg. hemoproteins) which perform a vital bio- chemical reaction. Most of the metal ions [Mn(I), Fe(II), Fe(II), Co(II), Ni), Cu(I), Zn(I), Mo(VD)] are present in biological systems in the form of coordi- nation compounds. The perspectives of essential trace elements are given in Chapter 3. 1.2 COMPLEX FORMATION The interaction of metal ion with the ligand can be expressed by the equilib- rium _ [ML] M™ IL] where K is the formation constant of the complex. Coordination chemists have been aware of trends in the formation constants of metal complexes. For exam- ple, the Irving Williams series of stability is very useful to understand and estimate the formation constants. For a given ligand, the formation constant with a divalent metal ion are in the order Ba®* < Sr? Zn? Figure 1.1 illustrates the above order of stability of metal complexes in solution. ‘The complexing ability of a cation depends on the ratio of charge to radius and in the case of transition metal ions, on the ligand field stabilization energy. These factors are illustrated in Fig. 1.2. A list of effective radii on metal ions in octahedral field is given in Table 1.2. M™ +L <= ML™K ab4 Bioinorganic Chemistry Log stability constant Oa SF Ga Wg Wi -Fe Go NI Co Zn Fig. 1.1 The Irving Williams series. The stability increases in the series barium to copper decreases with zinc. Curves (1) 2-Aminoethanethiol, (2) Oxalate, (3). Glycine and (4) Ethylenedimine. [From A. Sigel and D.B. McCormick, Acc. Chem. Res., 3 (1970) 201] +18 =| & 3 2 3 , 438 g} a vtT 6 2 | 368 sf 22 =| 35 \ i 3 72 |.0Mn Fe Co Ni Cu Zn wn/ Fe Co Ni Cu \ zn A 8 Fig. 1.2 Factors leading to the Irving William series. A. lonic radius, B. Ligand field stabilization energy.Introduction 5 Table 1.2. Effective radii (r) (pm) of metal ions in octahedral coordination’ Group | Group II Group Ill Group IV ur 74 Be 35 BY 23 sit 26 Nat 102 Mg? 72 Ar 53 Ti" 60 a 138 ca* 100 ‘Sc 73 ze T2 Rb* 149 sr 116 Nie 89 HY 1 cst 188 Ba** 136 Las 106 Ge* 54 cur 96 2n* 74 Ga* 62 Sré* 69 Ag 115 cat 95 In 79 Po 7 Au 137 Hg? 102 Bad 88 Transition metals Te 86 vee 79 NR 70 = o* 73{LS) Mn?" 67(LS) Fe? 61(LS) Co* 65 (LS) 82(HS) 82(HS) T7(HS) 73 (HS) Te 67 hee 64 cr 62 = Mn? 58(LS) Fe* S5(LS) Co 52(LS) Ne 56(LS) 65(HS) 64(HS) 61(HS) 60(HS) “From Shannon and Prewit, Acta Crystallogr, B25 (1969) 925. 1.3. HARD AND SOFT ACIDS AND BASES (HSAB) Ligands are Lewis buses and metal ions are Lewis acids. Pearson has devel- oped the concepts of hardness and softness to describe systematically the inter- action between them. A hard metal ion is one which retains its valence electrons very strongly. Hard cations are not easily polarized and are of small size and high charge. Correspondingly a soft cation is relatively large, does not retain its valence electrons firmly and is readily polarized (Table 1.3). Table 1.3, HSAB classification of metal ions Herd Ht, Lit, Nat, Kt, Mg? Ga’, Mn®, Cr, Fe, Com Border line Zr, Cu, Nit, Fe%, Co, Sné, Phi Soft Cut, Ag’, Ti, Pd2*, Pe, Ca, Hg? Ligands having highly electronegative donor atoms (O, N, F) are difficult to polarize and are classified as hard bases. ee Such ligands include amines, ammonia, water and ions such ° o. as phosphate or sulphate (Table 1.4). Readily polarizable ( i) ligands containing phosphorus, arsenic or sulphur donor at- So oms, act as soft bases. As a general rule, the formation of rN _/ stable complexes results from interaction between hard acids \-0: and hard bases or soft acids and soft bases. Hard-soft interac- ay tions are weak. The choice of chelating agent for complexing various metal ions can be rationalized on this basis. The cation K* is hard with a noble gas structure and will therefore interact with hard donors such as oxygen. The bonding will be primarily electrostatic. The ligand (1.1) [18] crown-6 has a hole diameter of 2.6 — 3.2 A which provides a good fit for K* (2.88 A) leading to relatively large formation constant.6 _ Bioinorganic Chemisty Table 1.4, HSAB classification of ligands Hard HO, OH", ROH, OR, R,0, NH;, NCS", Cr, PO}, SO}, F, NO, CO? Border line Pyridine, ANH, Nz, Nj, NOx, Br Soft RSH, RS", R,S, RyP, RAs, CO, CN, SCN, S,03-, Hy, Fr The chemistry of alkali metal complexes with different crown ethers is given in Chapter 4. The ligand diethyldithiocarbamate contains readily polarizable sulphur donors and is suitable for chelation of soft acids such as Cuil), CdD) and Hed). 1.4 INERT AND LABILE COMPLEXES Metal complexes may be classified as kinetically inert or kinetically labile. Inert complexes undergo slow substitution, while labile complexes undergo rapid ligand substitution. It may be noted that there is no relationship between kinetic inertness and thermodynamic stability (as determined by a large forma- tion constant). Thus [Ni(CN),)> has a very high formation constant, but the CN" ligands undergo rapid exchange with “CN in aqueous solution. Practi- cally, labile complexes are those whose reactions are over in less than one minute at 25°C for concentrations around 0.1 mol dm™, while inert complexes are those whose reactions may be readily followed under these conditions, having half-life greater than one minute. Much work has been carried out on the reactions of inert complexes particularly those of cobalt(III) and chromium(II}), but increasing attention is being paid to the reactions of labile complexes. This is mainly due to the advent of techniques for following very fast reactions in solutions, relaxation techniques and magnetic resonance tech- niques. The use of relaxation techniques permits the study of reactions having first order rate constants up to 10! s+. 1.5 LIGAND SUBSTITUTION IN OCTAHEDRAL COMPLEXES For this nucleophilic substitution, two basic mechanisms are available. They are (1) Associative mechanism (A) and (2) the Dissociative mechanism (D). The A mechanism involves a seven coordinate transition state while the D mechanism involves a five-coordinate transition state. The mechanisms are shown below for reaction (1.2), in which ligand X is replaced by ligand Y~. L is neutral, unidentate ligand. Rate laws for the two pathways are shown in equations (1.2) and (1.4). {CoL,X}* + Y" —> [CoL,Y}* +X a2) A mechanism ICoL, XP*+ Y° —S22> [CoL,XY] * [CoL; XY]* —=%4 [CoL,Y}* Rate = k, [CoLs X”*] [Y] (1.3)Introduction 7 D mechanism [CoL, X}** —Sl0" > [CoL.]** +X" [CoL, XP* + Y° By [CoL, YP* Rate =k, [CoLs X”*] (1.4) The rate laws (1.3) and (1.4) suggest that the two mechanisms may be read ily distinguished from each other on kinetic grounds. However the mechanism is more complicated particularly in aqueous medium due to the participation of water in some reactions. Reaction (1.2) may occur by formation of an intermedi- ate aquo complex, in which X~ is replaced in an A mechanism, followed by fast replacement of H,O by Y~. [CoL. X}* + HO —Slx4 [CoL, H,O]* + X° (1.5) [CoL, H,O}* + Y —2%9 [CoL, Y}* + H,O (1.6) However as the concentration of the solvent water is effectively unchanged it will not appear in the rate law, which will then be identical to (1.4). This corresponds to a D mechanism even though the mechanism is an associate one. Ton pairing is another complication, particularly if the reactions are carried out in non-aqueous solvents. The cationic complex {CoL,X]** and the anion Y~ may well exist as the ion pair [CoL;X]**... Y~ which may be the species undergoing reaction. In this case the rate law must include a dependence upon the concentration of the ion pair, which will in tur depend on the concentra- tion of complex and entering group, quite independently of the mechanism of the actual act of substitution. In the light of above problems, mechanisms have been assigned indirectly. The indirect method involves the observation of the effect on the rate of reac+ tion of various modifications to the complex, and then comparing these with the differing requirements of the A and D transition states. For example, a D reaction should be speeded up by the presence of bulky ligands. Similarly the A reaction should be slowed due to the overcrowding consequent to the pres- ence of bulky ligands. More direct approaches have involved the setting up of competition experiments in which the five-coordinate intermediate of the D mechanism is able to distinguish between different nucleophiles. Such studies have confirmed the existence of the D mechanism, but there is no uncompli- cated evidence for the A mechanism. It may be noted that many reactions likely to lie between these two extremes. Another example for associate mechanism is the base hydrolysis reaction. [CoL.,X]°* + OH” —> [CoL,OH]** +X” (7) Rate = k , [Col.sX]™* [OH]. Although much controversy has centred around this reaction, it is generally accepted now that it takes place via a dissociative process, despite the second- order rate law. The reactive species is the conjugate base of the complex, the concentration of which depends upon the concentration of the proton-accepting base, in this case [OH8 Bioinorganic Chemistry [Co(NH,),Cl}* + OH” <== [Co(NH,),(NH,)CI]*+ H,0 (1.8) {Co(NH,),(NH,)CI* —S5 [Co(NH,),(NH,)P°* + Clr (1.9) (Co(NH,),(NH,)?* + H,O —E8\5 [Co(NH,),OH}** (1.10) Electron transfer mechanism has been suggested for this reaction, which is of interest as it may have some general relevance to the biochemical systems. Hydrolysis of cobalt(II) complexes by OH™ has certain special features. Thus this reaction differs from those of the analogous chromium(II}) and rhodium(IID) complexes, in that base hydrolysis is very much faster than acid hydrolysis, and that stereochemical change occurs frequently. A number of reactions of cobalt(III) complexes are known to involve redox catalysis. All this suggest that base hydrolysis may also proceed via a cobalt(II) intermediate and generation of a radical. In the following scheme Y represents base. [L,Co™X]* + Y == {IL,Colxyyy"* aap Jon pair [L,Col™X]@* + Y =a (IL.Co"xyy yo (1.12) Jon pair {{L,Col"X] ¥"}O* = {fLColXyy yO 4 xo (1.13) | | Col + SL+¥ [L,CoMy]* ‘The overall reaction pathway will then depend upon the lability of the Co(II) intermediate and the oxidizing powers of the radical Y', For Y~ = OH’, the rate~ determining step is electron transfer from OH™ to Co(II), presumably to an excited state of Co(II) to avoid the generation of a low-spin cobalt(II) state. This scheme offers an explanation of the special features of base hydrolysis of cobalt(II) complexes discussed above. Cobalt(Il) differs from chromium(III] and thodium(IID) in the case of reduction of the metal to the bivalent state. This explains why base hydrolysis is much faster than acid hydrolysis of cobalt(II). In general, it should be noted that the larger the ligand field strength, the smaller the differences in rates of acid and base hydrolysis for cobalt(III). Stroger ligand fields stabilize cobalt(III) over cobalt(II) and hence increase the redox potential for cobalt(II) - cobalt(III). Example for such electron transfer reaction is given below. Co* + “OOH > Co + ‘OOH. The replacement of coordinated water by an entering ligand group is a reaction of fundamental importance. The aquo-complexes of alkali alkaline earth and transition metals are six coordinate. The alkali metals form com- plexes faster than any other metal ions in the Periodic Table, but the complexes are weak and so the studies have been carried out at high concentration. How- ever, the rate of substitution varies linearly with the ionic radius of the cationintroduction 9 (Table 1.5) suggesting that the rate determining step is the loss of water mol- ecule. The larger the metal ion (as the charge is constant), the poorer its hold over the water molecule and the faster the rate. The D mechanism is confirmed by only a very slight dependence of rate constant on the nature of the entering ligand. The rate constants for the reactions of the alkaline earth ions show a much wider spread. The reactions of the aquo-complexes of Cr** and Ba”* are very fast. Together with Ca** they appear to show a correlation between rate constant and ionic radius as observed in Group I. The rate difference between Ca and Mg** are significant in helping to understand the different behaviour of these ions as enzyme activators. Table 1.5. First order rate constants for complex*® formation (298 K) 107 ky (Ss) k, S* ky 8) ur 47 Be* 107 ver 30 Nav a8 Mg’ 10° ce 10° K 15 Ca 10° Mn 3x 107 Fo* 23 sr x 108 Fe® 3x 108 cst 35 Bat 9x 10 Co* 2x 10% Ce 5x 10° NR 2x 108 Hg* = 3x 10% Cu* 2x 10% Po 6x 10% 2n* 3x 10" Crystal field stabilization energy effects are dominant in the case of transi- tion metal ions. The rates of exchange of coordinated water have been measured by NMR for certain divalent transition metal ions. The results are as follows: V2" < Ni* [PtL X, YJ7+X™ 1.16) In general the lability of any group in square planar complexes is deter- mined by the ligand trans to it. This is trans effect, and it is possible to write a sequence of ligands in order of increasing trans effect, such as CN" > CO > NO; >I > Br > Cl > NH; > OH” > H,O ‘The effect may be explained in terms of the associative mechanism. The T-acceptors at the strong end of this series aid bond making in a trans position to themselves by withdrawing electron density from the metal and trans ligand, thus aiding the nucleophilic substitution at that point. The other ligands operate through a o effect and assist bond breaking for the leaving sans ligand. The trans effect is of practical significance. It is useful, for example, in designing synthetic pathways to derive desired product ie. either pure cis or pure trans isomers. The reaction schemes are given below. YT a cl cl Ci Pt Pi ’ / Re / + NH; —7 EL, / . Cis- cr cl cl NH, NHs NH, (1.17) HN ;p 7 Ha} NH, — NH, / PU /ecr—s Mf ie, Re Trans~ Hy HN Cl HAN NH; cl (1.18) Considerations of this type have been used in assessing the reactions of cis- and trans-Pt(NH;),Cl, with nucleic acid bases as discussed in Chapter 13, in the context of their anti-tumour properties. 1.8 AMINO ACIDS AND PROTEINS It is hardly surprising that much of the early biochemical research was con-Introduction 14 cerned with the study of proteins. Proteins form the class of biological macro- molecules that have most well-defined physicochemical properties and conse- quently they were generally easier to isolate and characterize than nucleic acids. The proteins are macromolecules of great biological importance. Pro- teins particularly in the form of enzymes have obvious biochemical functions. Proteins are made up of amino acids of L-configuration, linked together via peptide bonds -CO-NH-. By suitable treatment they can be degraded to smaller peptides and finally to the constituent amino acids. Therefore amino acids are monomeric units of proteins. Amino acids are also energy metabolites and many of them are essential nutrients. The analysis of a vast number of proteins from almost every conceivable source have shown that all proteins are composed of the 20, ‘srrandard’ amino acids listed in Table 1.6. Each naturally occurring polypeptide or protein in- volves specific sequence of amino acid residues, which may be determined by chemical and biochemical methods. The sequence of amino acid residue will determine the physical and chemical properties of the protein in terms of the chemical and physical interactions occurring between the side chains R in NH,CH(R)COOH. The important side chains in this connection are those in- volving aromatic groups, SH, ~NH,, -OH and -COOH groups. The nature of the residues may generate hydrophobic or hydrophilic environments in certain regions of the protein chain. The pK values of the 20 standard a-amino acids of proteins are tabulated in Table 1.6. Here pK, and pK,, respectively refer to the a-carboxylic acid and @-amino groups and pK, refers to the side groups with acid-base properties. Data in Table 1.6 indicate that the pK values of the @-carboxylic acid group lie around 2 so that above pH 3.5 these groups are almost entirely in their carboxylate forms. The @-amino groups all have pK values near 9.4 and are therefore almost entirely in their ammonia ion forms below pH 8.0. This leads to an important structural point. In the physiological pH range, both groups are completely ionized. An amino acid can therefore act as either an acid or a base. Substances with this property are said to be amphoteric and are referred to as ampholytes. Molecules that bear charged groups of opposite polarity are known as Zwitter ions or dipolar ions. Thus amino acids exist as Zwitter ions (1.2) in the physiological pH range. R H,N* —C —COO- i 1) The @carboxyl and amino functions are involved in the formation of the peptide link, and so each peptide has terminal -NH, and -COOH groups, together with peptide links and side chains. These are all possible metal binding sites. The molecular weights of proteins are in the range 10*- 10°. The determination of the chemical structure and the spatial configuration of proteins12 _ Bioinorganic Chemistry Table 1.6. Naturally occurring amino acids NHy-CH{R)—COOH S.No, Name of R Average pK, pK, pKa amino acid occurrence in a=COOH a side proteins (%) NH2 chain Amino acids with nonpolar side chains 1 Glycine H 72 2.35 978 ane 2 Alanine CH, 78 235 987 — 3 Valine (CH).CH- 66 229 974 — 4 Leucine (CH),CH-CH,- 94 233 974 — 5 Isoleucine CHyCH,CH(CH,)~ 53 232 976 — 6 Methionine CH{SCH,CH,- 22 243 928 — 7 Proline” 62 1.95 10.64 — 8 Phenylalanine 39 220 931 — 8 Tryptophan 14 248 9410 ‘Amino acids with uncharged potar side chains 10 Serine HO-CH,_ 638 219 9210 — Ww ‘Threonine CH,HC(OH) 59 2.09 9.10 -— 12 Aspargine NH_-C(O)CH,_ 43 21 B72 13 Glutamine NH,-G(0)CH.CHs. 43 217 913 — 14 Tyrosine 32 220 921 10.46 15 Cysteine —CH,-SH 19 1.92 10.70 837 Amine acids with charged polar side chains 16 Lysine NHp(CH,)gCH)__ 59 216 905 10.54 17 Arginine 51 1.82 899 12.48 HON, > —NH(CHg)CHy HN 18 ine cL 23 1.80 9.33 6.00 ee H 19 Aspartic acid ~=— —CH,_COO~ 53 1.99 990 3.90 20 Glutamic acid —OO0CCHCH,_ 63 2.10 947 4.07 * Complete structure of protein (inchuding R) is given is very important aspect as this is linked with the biological function of the proteins. In the era of X-ray diffraction techniques, an increasing number of protein structures has been determined to a high degree of resolution. The structure of protein is discussed in terms of primary, secondary, tertiary and quaternary structure.Introduction 13 1.9 STRUCTURE OF PROTEINS Proteins are polymers containing large number of amino acids joined to each other by peptide bonds. For establishing the structure of a protein we will have to consider the following points. i. The nature of amino acids. ii. The number of each particular amino acid present in one molecule of the protein. iii. The sequence in which the various different amino acids are arranged in the molecule. iv, The shape of the peptide chain i.e., whether it is linear, cyclic, branched or arranged in the form of helix. vy. The forees with which the peptide chains are held together. vi. The way in which the individual peptide chains are arranged in definite manner. ‘The number of peptide chains and arrangement in natural protein. Fig. 1.3 a-Holical structure of protein. vii. The first three points constitute the primary structure, the point (iv) consti- tutes the secondary structure, v and vi constitute the tertiary structure and vii constitutes the quaternary structure. 1.9.1 Primary Structure It refers to the number, nature and sequence of amino acid residues along the peptide chains. The determination of complete primary structure involves (a) purification, (b) determination of amino acids composition, (c) end group determination, (d) disulphide bond cleavage (if present) and (e) determination of the position of the disulphide bonds. The primary structure of protein has strikingly important biological implica- tions, even in very closely related proteins. For example, if at one point (B-6) in the chain of the haemoglobin, a glutamic acid is replaced by a valine unit, a different haemoglobin commonly known as “haemoglobin S” is obtained. The latter causes the inherited genetic disease known as sickel cell anemia, charac- terised by sickel shaped red blood cells. The normal RBC are spherical. Polypeptide from Haemoglobin A His-Val-Leu-Leu-Thr-Pro-Glu-Glu-Lys14 Bioinorganic Chemistry Polypeptide from Haemoglobin S His-Val-Leu-Leu-Thr-Pro-Val-Glu-Lys The genetic disorder is caused by a mutant gene. 1.9.2 Secondary Structure It deals with conformation of the peptide chain present in the protein molecule. Two different conformation are found to exist. They are a-helix and f-confor- mation. a-Helix The a-helical structure arises due to resonance (1.3) in the peptide linkage and hydrogen bonding (1.4) between NH and >C=O groups along the protein chains. 20: :0: |. I. —C-N- > —C=N- I | H H (1.3) Resonance | o=c— | N—H- --O—C | | N-H | (1.4) Hydrogen bonding In the @-helical structure of the protein each turn of the helix has nearly 3-7 amino acids and it is at a distance of 5.4 A from the other. Two adjacent turns are linked by means of hydrogen bonds between NH group of one amino acid and the carbonyl group of another amino acid. The hydrogen bonding prevents the free rotation and so the helix is rigid. The £-conformation This conformation was proposed by Pauling ef al. (1951). The polypeptide chain extends and chains are held together by intermolecular hydrogen bonds. This results in two types of pleated sheets known as parallel and antiparallel (Fig. 1.4). In parallel pleated sheet, all the polypeptide chains run in the same direction e.g. Keratin, while in antiparallel the chains run alternatively in oppo- site directions eg. Fibroin. The existence of the pleated sheet structure in solid state has been confirmed by X-ray analysis. The calculated bond distance between two CHR on the same side of a chain is 7.2 A, while the experimental value is 7.0 A. This differenceIntroduction 15 is due to the crowding caused by the side chains (R), which in turn prevents the chain from being fully extended. (A) Parallel \ /C4—Ne } ‘ (8) i ; Anti.paraliel i Z Fig. 1.4 Structure of protein in B-conformaton. (A) Parallel pleated and (B) Anti-parallet pleated. 1.9.3 Tertiary Structure Tertiary structure of a protein refers to its three dimensional structure, i.¢., the coiling (folding) of the long peptide chain with or without a helical region into the final structure. The tertiary structure describes the overall spatial arrange- ment of the polypeptide chain (or chains) and thus gives an exact account of molecular shape in most of the small and medium sized proteins. Two major molecular shapes have been found viz. fibrous and globular. Fibrous proteins have a large helical content and are essentially rigid molecules of rod-like shape. On the other hand, globular proteins have a polypeptide chain which consists partly of helical sections and folded about the random coil section to give a spherical shape. The tertiary structure of a protein is best determined by X-ray studies which is achieved in many years by experts. Other methods used for elucidating the tertiary structure are viscosity measurements, diffusion, light scattering, ultra centrifuge method and electron microscopy.16 Bioinorganic Chemistry 1.9.4 Quaternary Structure Proteins exist as an association of several chains. In such cases, the protein is known as oligomeric and the individual chains as protomers or sub-units. The association of sub-units can be disrupted by reagents which do not break covalent bonds and thus two chains joined by disulphide bonds should not be consid- ered as two sub-units. Each sub-unit has its own primary, secondary and terti- ary structures. Two or more sub-units may have identical structures. The Haemoglobin molecule is made up of four sub-units (two identical a-chains and two identical f-chains): each chain binds a heme group and there are minor differences in folding between the a and B-chains. The chains fit together in an approximately tetrahedral arrangement in the quaternary struc- ture of haemoglobin. Structures of protein are schematically represented in Fig. 15. ° R i a AN ye Ng As 4 I l ° R Primary J wR Ml a a) on. Secondary Tertiary Quatemary Fig. 1.5 illustration of primary, secondary, tertiary and quaternary structure of protein. 1.10 THE PEPTIDE BOND The peptide bond is planar and trans. Its structure is revealed by X-ray diffrac- tion studies on the crystals of small peptides. This structure (Fig. 1.6) has been found for all peptide bonds in proteins apart from a few rare exceptions. This planarity is due to the delocalization of the lone pair of electrons on nitrogen. The C-N bond is consequently shortened and has double-bond character. Twist- ing of the bond prevents delocalization and some 75-88 kJ mol"! of resonance energy is lost. Amides are reasonably acidic and the pK, value of the ionization of the peptide hydrogen on the simple dipeptide glycylglycine is estimated to be about 14.5. Metal ions may interact with peptide oxygen or peptide nitrogen. If a metal ion binds to the peptide nitrogen, the resonance energy of the peptide bond isa eS te ——— 30 ———_ > Introduction 17 al al on sae 123° at Fig. 1.6 The peptide bond. Distances in pm. lost. The resonance energy (ca. 40 KJ mol) can be retained if the peptide hydrogen ionizes and such deprotonated peptide complexes are observed with many metal ions (Cu**, Ni”, Pt’*, Pd). This type of complexation can be illustrated by the reaction of copper(II) with glycylglycine. If the pH of a 1:1 mixture of the glycylglycine and the metal ion is increased, a new band appears in the visible spectrum at 625 nm (Fig. 1.7) due to the formation of the com- plex which has € = 84 L mol cm at 625 nm. 2.0 a i Absorbance 6 e a 0.0 == 600-700 2 (nm) Fig. 1.7. Visible spectra of Cu(ll!-glycylglycine in aqueous solutions. (a) pH = 3.75; (b) pH (d) pH = 4.75; (e) pH 07; (c) pH = 4.52; 00; (f) pH = 7.76.18 Bioinorganic Chemistry 1.10.1 Binding Sites in Peptide In addition to the peptide bond itself the available metal binding sites in protein are the N-terminal NH, group, the C-terminal CO; group and the functional side chains of amino acid residues such as histidine (His), glutamic acid (Glu), tyrosine (Tyr), cysteine (Cys), aspactic acid (Asp). The metalloenzyme, carboxypeptidase A which contains zinc(II) was one of the first metalloenzymes in which the ligand binding sites were established (Fig. 1.8). The ligating residues of various Zn(II) metalloenzymes as evident by X-ray data are given in Table 1.7. 4 ! ~% —N~suBSTRATE oo | “ft H cH, “¥ awu-72 Fig. 1.8 The coordination of zino(ll) ion at the active site of CPA. Table 1.7. Lagating amino acid residues in various zinc enzymes Enzyme Metal ion Ligating residues Atcohol dehydrogenase Zn Cys-46, Cys-174, His-67 zn Cys-97, Cys-100, Cys-103, Cys-111 Carboxypeptidase A Zn His-69, His-196, Glu-72. Carbonic anhydrase B Zn His-94, His-96, His-119 Carbonic anhydrase C Zn His-93, His-95, His-117 Superoxide dismutase Zn His-61, His-69, His-78, Asp-81 Cu His-44, His-46, His-61, His-118 Thermolysin Zn His-142, His-146, Glu-166 “Zing in inactive structural unit. 1.11 ENZYMES Enzymes are large proteinous molecules. They specially bind (organic) substrates through geometrically and physically complementary interactions. This permits enzymes to be absolutely stereospecific, both in binding substrates and in catalyzing reactions. The molecular weights of enzymes vary from ca 1.2 x 10* to 5 x 10‘, The substrate molecules are normally small (M < 10°) and so can interact with only a small portion of the enzyme (Fig. 1.9). X-ray data on a variety of enzymes indicate that they often have a cleft to which the substrateIntroduction 19 ES Enzyme-substrate Complex Product +E => E—Product Fig. 1.9 Illustration of enzyme catalyzed reaction. molecules can bind. This cleft is the active site which contains the catalytic side chains of the amino acids and other functionalities such as metal ions which are required for catalysis. The enzyme must not bind the products strongly, or the enzyme will be inactivated as the enzyme-product complex. Since the enzyme is a chiral molecule, reaction of the enzyme with only one enantiomer of the substrate is expected, the unnatural enantiomer does not fit into the active site in the required manner. The specificity of enzymatic action may be illustrated by an analogy i.c., basic lock and key theory. This represents the complex three dimensional relationship between an enzyme and the substrate on which it acts. This relationship requires the use of certain cofactors or activators before the reaction is complete. The result of this is to activate the substrate so that it is able to react in the required way. X-ray studies on enzymes have shown, in all cases, the presence of cleft at the active site into which the substrate must fit. The lock and key theory is, however, inadequate in that it does not indicate other effects such as conformational changes that occur when the substrate bound to enzyme (for eg. carboxypeptidase). X-ray studies have also suggested that the initial interaction between substrate and enzyme induces further interactions that cause other parts of the enzyme to close in upon the substrate. Several factors influence the actual source of catalytic power. They are proximity and orientation of the effects and electron-push-pull-effects. How- ever it is difficult to explain these effects on a quantitative basis. 1.12 NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS Nucleosides are composed of a purine (1.5) or pyrimidine (1.6) base attached to the sugar ribose via the N-9 and C-1 atoms respectively. In nucleotides the sugar is linked to a phosphate group, which may be a mono, di- or tri-phos- phate species. The important nucleotide, adenosine tripohspahte (ATP, Fig. 1.10), illustrates the structural features. This molecule contains the purine base, adenine.20 _ Bioinorganic Chemistry i Il Wl ° ° ° OH OH Fig. 1.10 Structure of adenosine triphosphate (ATP). nN ww ee to H Purine (1.5) Pyrimidine (1.6) The nucleic acids are polymers built up from nucleotides via phosphodiester bond formation between the 3’ hydroxyl group of one nucleotide and 5’ hydroxyl group of the adjacent nucleotide. The sequence of the nucleotides is extremely important as it constitutes the genetic code in DNA. Different nucleotides vary in the nature of the purine and pyramidine base. The four important bases in RNA (ribonucleic acid) are the two purines (adenine and guianine) and two pyramidines (cytosine and uracil). In dexoxy ribonucleic acid (DNA), thymine (5-methyl uracil) is present instead of uracil. The structures of various nucleic acid bases are shown in Fig. 1.11. dy.09.0 tr Adenine Guanine Cytosine Uracil sip Fig. 1.11 Structure of nucleic acid bases. The interaction of nucleosides and nucleotides with metal ions has been much studied, particularly by NMR techniques. Base, phosphate and ribose groups are all potential ligands. Complexes of adenine nucleotide have been particularly well studied. Mg™* and Ca?* bind only the B and yphosphate of ATP, while Zn*, Mn™*, Cu** and Ni’* also bind to the N-7 nitrogen atom of the base in addition to the phosphate. The complex, Cu; (2’-deoxy guanosine), (OH),4H,O has been the first example of a nucleoside coordinated to a metal via sugar group.Introduction 21 RNA is a single strand polymer (Fig. 1.12) but DNA consists of two inter twined helices with each heterocyclic base hydrogen bonded to a base on the other strand in famous “double helix’ structure. Replication of DNA involves unwinding into separate strands, with formation of complementary strands for each of the original two, so that two new DNA double helices are formed. The DNA acts as a template for the synthesis of messenger RNA in the process of transcription. This messenger RNA thus carries the genetic code, which is conveyed through trinucleotide sequences or codons, which code for a specific amino acids, eg. the group CUU codes for phenylalanine. These codons associate with trinucleotide sequence of transfer RNA which contain the heterocyclic bases that are complementary to those in the codon. This takes place on the surface of ribosomes. The transfer RNA also binds the specific amino acid corresponding to the original codon. In the example given above, the transfer RNA for phenylalanine contains the nucleotide sequence GAA, which is complementary to CUU. Thus the code in the messenger RNA is translated into the correct sequence of amino acids, which is followed by the formation of peptide links. These essential processes of replication, transcription and translation are dependent on metal ions. ° cH, \ ©. base, oe ° O\. base, Fig. 1.12 Illustration of single strand in RNA.22 _ Bioinorganic Chemistry 1.13 CARBOHYDRATES (SUGARS) Carbohydrates form complexes with hard cations such as Ca*. The approxi- mate formation constant of the calcium complex of epi-inositol is ca 3M7. Structure of the complex (1.7) is given below. (7) Electrophoresis and nmr techniques are generally used to detect the complex formation. At high pH, sugars migrate towards the anode, as they are partially ionized. However, in the presence of Ca they migrate to the cathode due to complex formation, ‘The sugars such as allose, talose, ribose form complexes with metal ions. Further, stable complexes of metal ions with sugars are relatively rare in na- ture. However, polysaccharides offer the possibility of complexing to more than three oxygen atoms. Alginic acid is a polysaccharide which forms strong gels in the presence of Ca**. This molecule is made up of long chains of B-D- mannuronic acid a-L-guluronic acid, the better the gel-forming properties of the alginic acid. A possible structure of the polysaccharide calcium complex is given in Fig. 1.13. Fig. 1.13 Part of the structure of calcium complex with alginic acid (From Pure Appl. Chem., 35 (1973) 145). 1.14 BLOOD Mammalian blood consists chiefly of a suspension of living cells (the red and white corpuscles) and particles, known as platelets in an aqueous solution ofIntroduction 23 protein (ca 7%), containing metal salts (ca 1%), glucose (ca 0.1% in humans) and other nutrients (vitamins), hormones and waste products of metabolism. Within a few minutes of removal from an animal, blood becomes viscous, and then clots owing to enzymic activity (triggered by Ca”*), which results in the formation of a network of solid fibrous protein (fibrin). On standing, a clot of blood contracts as the fabrin network shrinks. The clear yellow liquid (plasma deprived of its fibrin) which exudes from the clot is known as serum. 1.15 PLASMA Plasma can be separated from the cells in it by centrifugation. The plasma of human blood is a pale, straw coloured, transparent fluid (pH 7.4) containing some 9% of total solids. Plasma has an osmotic pressure equal to that of a 0.9% sodium chloride solution. Such a solution which is isotonic with blood is known as physiological saline. ‘The proteins present in plasma account for some 7% of the plasma. Exclud- ing Water, proteins are the major constituents. The plasma proteins differ in their isoelectronic points and when subjected to electrophoresis, they can be separated into six main fractions (Table 1.8). The albumin, which can be crystallized, ac- counts for more than 50% of the total plasma protein. 's molecular weight is lower than that of other protein, it is largely responsible for the osmotic and buffering effects of the plasma proteins. Unlike the albumin and fabrinogen, the globulin fractions are not homogeneous and can be fractionated into many other proteins. Table 1.8. Plasma proteins Protein fraction 9/100 om? plasma Albumin 4.04 #Globulin 0.81 Globulin 074 ‘Globulin 0.48 Fabrinogen 0.34 %-Globulin 031 6.72 (Total) Plasma contains all the amino acids necessary for cell nutrition, gluatmine, glutamic acid, alanine and lysine being the most important. The total amino acid contents of human plasma is ca 35 mg per 100 cm, one third of this amount being glutamic acid and glutamine. Assuming an average amino acid molecular weight of ~150, the amino acid concentration is 2 x10 M. Glycine and alanine constitute about one fourth of the total amino acid content and there are only tracer amounts of methionine. Human plasma may be considered as typical of mammalian plasma, there being no marked differences between different species. A range of inorganic ions are found in blood plasma and their concentrations are already summa- rized in Table 1.124 Bioinorganic Chemistry 1.16 THE CELL-STRUCTURAL AND FUNCTIONAL UNIT The fundamental role of cell in the organism has come from investigations for the past 300 years by several Scientists. The history of ‘cell science’ begins with the description of a thin section of cork by Robert Hooke in 1665. Using microscope for the first time, Hooke described that cork has a honey-comb like structure and called each compartment as a cell which were separated by a wall. As cork is dead material, cells were empty. Hooke’s observation prompted many scientists of that period to observe a variety of materials under micro- scope. Around the same time, Anton Von Leewenhoek described the existence of unicellular organisms. Next important discovery was the report on the presence of nucleus in the cells by Robert Brown in the year 1831. During the same period, Schelden (1804-1881) observed that all plants have cells separated by a cell wall. In 1838, he proposed that cells are the fundamental structural units in all plants. Surprisingly Theodre Schwann (1810-1882) made almost similar observation using animal tissues. He observed the presence of cells in all the animal tissues. Both Schlieden and Schwann discussed their results and proposed Cell theory in 1838—which states that all living organisms are made of cells. In 1855, Rudolph Virchow observed the process of cell division and proposed that cells are formed from pre-exisitng cells and this was added to cell theory. Meanwhile, methods were developed to analyse the chemical composition of small amounts of materials. Using these methods chemical composition of a wide variety of cells was studied. Surprisingly, it was observed that cells from different types of organisms have the same chemicals in them. They have nucleic acids (DNA and RNA), a large variety of proteins, carbohydrates, ions etc. These studies indicated that all the cells are made up of same chemicals and this was added to cell theory. Studies on the physiology of cells revealed that cell carry physiolgical. functions like—oxidation of food materials to derive energy, excretion of waste material, proliferation of cells, defence mechanism against disease. In other words, functions essential for the survival of organisms are carried out even at the level of cell. Functions of organisms depend on the functions carried out by cell. This concept was added to cell theory. As this theory states that all the organisms are made up of cells, we can consider the cell as the basic structural unit of all organisms. Since the func- tions of an organism depends on the functions carried out by its cells, we can state that the cell is the basic functional unit in all organism. By putting these two statements together, it may be inferred that cell is the basic structural and functional unit of all the living organisms. The presence of plasma membrane and protoplasm are common to all the cells from bacteria to man or plant. However, the bacterial cell differs from the cells of plants, animals and fungi. Cells of these organisms have several intracellular structures known as organelles such as mitochondria, nucleus, lysosomes, gogli complex, plastids. Each organel has its own membrane. TheIntroduction 25 structure, function of different organelles are different. In contrast to these cells, bacterial cell has no such organelles. The major and striking difference between bacteria cells and cells of other organisms is the absence of nucleus in bacteria. Because of this difference, bacteria are called procaryotes. The ab- sence of nucleus and other organelles represents primitive condition, but the bacteria cells carry out the same functions as eucaryotic cells. 1.16.1 Structure of Eucaryotic Cell The size and shape of eucaryotic cells differ considerably The unicellular or- ganisms have cell in the size of 1 um to 2 ym. The cells of most multicellular organisms are in the range of 95 um to 100 pm. Largest single cell of a multicellular organisms is ostrich egg (15 to 20 cm). The shapes of multicellular organims varies considerably. Various types cucaryotic cells are given in Fig. 1.14 and the structure of a typical cucaryotic cell is given in Fig. 1.15. Plasma membrane and various organells of cell are described below. Fig. 1.14 Various types of Eukaryotic (animal cells). (1) Sperm, (2) Diatom, (3) Liver cell, (4) Coblet cell, (5) Muscle cell, (6) Nerve cell. 1, Plasma membrane Alll the cells have an outer covering made up of lipids and proteins called plasma membrane separates the cell from surroundings and from other cells. This helps the cells to maintain several molecules inside without getting lost to the extracellular environment.26 Bioinorganic Chemistry _— Golgi Complex _- Centrosome with Centrioles _ Free Ribosomes 4 i L Plasma Membrane = Nuclear Membrane _— Nucleus _ Smooth Endoplasm Reticulum Nucleolus Rough Endoplasmic Reticulum Microtubules Mitochondria Fig. 1.18 Structure of Eukaryotic (animal) coll. 2. Protoplasm Inside the cell, a jelly or fluid like substance fills the entire volume of the cell. This is called protoplasm. The major chemical components of protoplasm are water (70 to 80%), protein, nucleic acids, amino acids, carbohydrates, lipids and ions (Na*, K* Mg”* etc.,). Small amounts of vitamins are also present in the protoplasm. ‘The presence of plasma are common to all cells from bacteria to man or plants. The major striking difference between bacterial cells and other organ- isms is the absence of nucleus in bacteria, while a distinct nucleus is present in other cells. Because of this difference, bacteria are called prokaryotes while others (algae, fungi, higher plants and animals) are called eukaryotes. 3. Nucleus Of the organells in cell, nucleus is the most prominent. It can be seen with an ordinary microscope. It is covered by two membranes which are together called nuclear envelope. Inside the nucleus, there is diffuse thread like material called chromatin. It is made up of DNA and proteins. DNA is the genetic material. It has the information required for the synthesis of proteins and other vital func- tions in the cell. This information is present in discrete units called genes. Within the nucleus, a dark staining body called nucleolus is present. Nucleus controls and organises all the functions in a cell. Besides nucleus, there are other organelles in the cytoplasm, which perform different functions. They are mitochondria, lysosomes, ribosomes, endoplasmic reticulum, gogli complex and cytoskeleton.Introduction 27 4. Mitochondria These are spherical or long thread like organelles. They are called power houses of the cell, as they generate energy required for various functions of the cell. They generate the energy by oxidising carbohydrates and fatty acids. They have a central matrix covered by two membranes. They contain small amount of DNA and ribosomes. Mitochondria are smaller than the nucleus by a factor of upto hundred. 5. Lysosomes ‘These are spherical in shape and are covered by a. single membrane. They contain enzymes which can degrade carbohydratres, proteins, lipids, and nu- cleic acids. They help in breaking down complex molecules to simple mol- ecules which can be used by the cell for generating energy or for growth. Lysosomes also participate in the digestion of food materials. Under some disease conditions structure of lysosomes is destroyed and this may lead to the death of the cell. Lysosomes are therefore called suicidal bags of the cell. 6. Ribosomes These are pear or heart shaped structures and are not covered by the mem- branes. They are made up of RNA and proteins, They may be free in the cytoplasm or in groups (called polysomes) or attached to the membranes of endoplasmic reticulum, These are involved in protein synthesis. 7. Endoplasmic reticulum These are tube like structures present in the cell. They extend all over the cell. Empty space inside the tube is called LUMEN and is covered by a single layer of membrane. There are two types of endoplasmic reticula in the cell—Rough endoplasmic reticulum (attached by ribosomes) and smooth endoplasmic reticulum to which ribosomes are not attached. The former participates in protein synthesis while the latter is involved in lipid synthesis. 8. Gogli complex These are also tube like structures with a central lumen covered by a single- layer of membrane. It is involved in the secretion of proteins from the cell. 9. Cytoskeleton This is a net work of fibres present all over the cell. They give mechanical support of the cel] and also participate in cell moments. There are three types of fibres in cytoskelelton. They are microtubules, microfilaments and interme- diate filaments. Some of the eukaryotic cells may also have several short hair-like process called Cilia or one or two long hair like processes called Flagellum. These two structures help in locomotion.aa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.Introduction 29 Protein —_—_ CN ee l angE ~~ Fig. 1.17 The trlamellar structure of the membrane. Lipid ws lS represented in Fig. 1.18 which serves also to demonstrate the tail-to-tail struc- ture of bilayer. Fig. 1.18 The Fluid Mosaic Model for the membrane (From Science, 175 (1972) 720). Recent reports reveal that the membrane consists of a lipid bilayer in which globular proteins float as “ice bergs”. These proteins are postulated either to span the lipid layer or to be embedded in it and so be accessible from one side only. The integral proteins will always have hydrophilic and hydrophobic sec- tions, so that the hydrophobic section is then in contact with lipid, while hydrophilic layer would be exposed to the aqueous medium outside the mem- brane. Proteins that span on membrane would have two hydrophilic segments sepa- rated by a section of hydrophobic character which is long enough to span theaa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.aa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.32 Bioinorganic Chemisiry coefficient, € for each peak can be calculated according to the Beer-Lambert Law. A= Logy CJD = el (2.1) where A is the absorbance, c is the molar concentration and | the path length in centimeters. The colours most often associated with transition metal complexes arise from transitions between different energy levels corresponding to a redistribu- tion of electrons in the partially filled d-orbitals. These are referred to as d-d transitions. In the absence of any ligands around the metal, the energies of all five d-orbitals of a transition metal are equal, that these they are degenerate. ‘The- presence of ligands will result in an increase in the energy levels of all these orbitals, Anything but a completely spherical distribution of clectron density evenly spaced around the metal will result in removal of the degen- eracy, because some ligands and orbitals will interact more strongly than oth- ers. The exact form of the interaction and hence energies of the d-orbitals depend on the type, number and special arrangement of the ligands. The energy level diagrams of some common geometries/shapes are given in Figure 2.1. A fev fe aa ~_ fa) ) © (3) Fig. 2.1. The energy level diagrams for (a) tetrahedral (b) octahedral (c) distorted octahedral and (d) square-planar geometry. Based on the splitting of orbitals we would expect a single electronic transi- tion for a d! transition metal complex of octahedral or tetrahedral geometry, corresponding to the excitation of the single electron from the ground to ex- cited state. For a d! metal in distorted octahedral or square planar geometries a larger number of transitions are possible. The visible spectrum of [Ti(H,O),J** shows a band at ca. 500 nm corre- sponding to a single transition. This wavelength corresponds to the green part of the visible spectrum and therefore the complex appedrs purple in colour. 2.1.1 Spin States The octahedral splitting of the metal d-orbital results in ty and an eg set of orbitals. However, placing electrons in these levels may result in further inter- actions resulting in a number of different spin states. In the absence of any ligands around the metal, the ground state of the free ion can be written in a short-hand fashion known as a spectroscopic term symbol according to the Russel-Saunders coupling scheme.Physicochemical and Spectral Methods 33 2.1.2 Russel-Saunders Coupling Scheme In the ground state, the single electron of a d' metal ion, according to Hund’s rules, will occupy the orbital with the highest magnetic quantum number m, = 2 as shown below. (tT TTT) m, 1 0-1 = ‘The total angular momentum (L), given by the sum of m, values, is therefore 2 and this state is labelled D, according to the scheme given below. L = 0 1 2 3 4 5 Label = s P D F G H The spin multiplicity is given by (2S + 1), where ‘S’ is defined as the sum of m, values (the spin of the electron). In this case there is only one electron (m, = 1/2) so the total spin is 1/2 and therefore the spin multiplicity is 2. Thus the spectroscopic term symbol according to the Russel-Saunders notation for a d! metal ion is 7D (which is said as doublet D). This scheme may be used to determine equivalent symbols for all the possible ground state ions as given in Table 2.1. Table 2.1. Spectroscopic term symbols for d' metal ions configuration Term symbol a =D & a & F a D a &s dé *D qd F ae oF a *D a? 1g The arrangement of electrons within a complex can be obtained in two different ways depending on the relative importance of the crystal field split- ting and the interelectronic interactions. In the weak field approach we con- sider the interelectronic repulsions as being the dominant effect and build their effects on the free metal ion splitting. In the strong field approach the crystal field splitting is the most important factor and electronic repulsions are consid- ered as secondary to this effect. The ground state electronic configurations and their counter parts in an octahedral field are given in Table 2.2.34 Bioinorganic Chemistry Table 2.2. The splitting of Russe!-Saunders states by the crystal field Ground State label ‘Octahedral label A Ty E+T, Agt Ty) +T2 Ay +E+T,4+To E +21, +Tp ITOnoVwMH A single d-d transition should be observed for d!, d*, d® and d? octahedral configurations while three transitions are anticipated for d?, d°, d” and d* com- plexes (Fig. 2.2). Th 1oré 4or9 ® Octahodral 2 or 7 gore 4or9 1oré d”Tetrahedral 3 or 8 2or7 Fig. 2.2. The splitting of energy levels for high-spin octahedral and tetrahedral d" configurations. Although we can approximate the relative energies of these electronic states, the exact ordering is determined by the relative magnitude of A, and electronic repulsion energies. The latter interactions are normally defined by a number of variables which are brought together in two Racah parameters B and C. For the free metal ions B is typically about 1000 cm”! and C is around 4 x B. There are two common types of energy plots for electronic states. They are (i) Orgel diagram and (ii) Tanabe- Sugano diagram. AE Orgel diagram shows how the elec- £5 tronic levels change as a function of A,, such as that shown in Fig. 2.3 for d' ion. The diagrams assume that the = interelectronic repulsion are insignifi- cant compared to crystal field splitting. The Tanabe-Sugano diagram is a plot of orbital energies as a function of the Racah parameter B versus AB. A sim- tetrahedral 4 octahedral plified diagram for the electronic states Fig. 2.3 Orgel diagram for d’ ion of the d? ion is given in Fig. 2.4. ‘These diagrams may be used either to determine A, values (once d-d transi- tions have been identified) or they may be used to predict the position ofaa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.aa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.Physicochemical and Spectral Methods 37° being laporte allowed for the tetrahedral complex where it is not for the octahe- dral complex. Visible spectra of these two complexes are shown in Fig. 2.7. (a) y' 6 es Absorbance ° 2 & = 400 800 Wavelengthinm Fig. 2.7 Visible spectra for (a) tetrahedral [CoCI,]*- and (b) octahedral (Co(H,O),J** cam- plexes, For high spin d° complexes all d-d 4 transitions are formally forbidden by all & ER & three selection rules. ,They are forbid- > den since AJ = +1, they are Laporte AL is bag toy forbidden, and any electronic transition must result in a change in the spin mul- tiplicity of the complex as shown in Fig. 2.8. Any d-d transition in a high spin d° complex will result in change in spin multiplicity. For these reasons complexes containing high-spin d transition metal ions such as Mn** containing complexes tend to be very weakly coloured (Fig. 2.9). We can use selection rules to predict the relative intensities of the transitions of compounds. For example, the intensity of the d-d transitions of (A) IMCI,}?>, (B) trans-{M(H,O),Cl,] and (C) cis-[M(H,0),C1,} complexes may be predicted. Fig. 28 Any d-dtransition in a high-spin 4° complex lead to change in spin muttiplicity. A 0.18) \ ono J 400 800 \Wavelength/nm Fig. 2.9 Electronic spectrum of [Mn(H,0),]**. The large number of weak bands correspond to forbidden transitions. Absorbance 2 aaa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.aa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.aa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.Physicochemical and Spectral Methods 41 Table 2.4. Absorption spectra of some metalloenzymes Metalloenzymes Bond position, nm Intensity , e(M™' cm) Cu(ll) carboxypeptidase 790 <100 Cu(Il) carbonic anhydrase 580 50 750 100 300 7 Cu(ll) carbonic anhydrase + CN" 700 130 900 20 Cu(ll) alkaline phosphatase ~750 ~ 100 Laccase [Cu(II)} 730 ~ 500 615 1400 632 ~ 300 ‘Azurin (psuedomonas blue protein) (Cu(l)] 808 ~ 600 621 2800 - 3500 524 ~ 300 467 ~ 400 Ascorbic acid oxidase {Cu(t)] 606 770 42 ~~ 500 Coruplasmin [Cu(|!)} 605, 1200 370 ~500 Co(ll} carboxypeptidase 500 = 555, 160 572 160 940 ~25 Co(ll) carbonic anhydrase 520 205 555 340 61S 230 640 240 200 ~25 Coll!) carbonic anhydrase + CN 310° M5 450 520 845 350 570 885 650 Co(!!) carbonic anhydrase + acetazolamide 465 515 520 550 350 570 570 560 600 590 500 Co(ll) alkaline phosphatase 640 260 60S 220 585 378 510 335 Co(II) alkaline phosphatase + HPO} 640 120 535 360 480 260 oj!) alkaline phosphatase + HASOz 500 ~240 550 ~260 ® Figures in this column determined from band positions in the C.D. spectra. metalloproteins are not common, it is possible to substitute cobalt(II) at active site of some zinc(II) metalloenzymes with the retention of catalytic activity. The spectrum of cobalt(II) enzyme is similar to that expected for a tetrahedral complex rather than that of an octahedral complex (Fig. 2.12).42 Bioinorganic Chemistry te | eg?) 600 5 B10! a £10 /\ ‘500 . ge <—— / | —~ 4400 8 : | z = 2 gst 00 & 3 | 2 3 ab '200 3 si (octatedeal) \ atnnaven a Zar JS 100 3 I r 0 [ ° 400 500 700 800 Wavelength (nm) Fig. 2.12 The visible spectra of (A) [Co(H,0),J** and (B) [CoCl*. X-ray structure of the active site of the enzyme establishes that the metal in the zine enzyme tetrahedrally ligated by the imidazole rings of three histidine residues. The fourth ligand is probably a water molecule or hydroxide ion, depending on the pH. Both the X-ray data and the visible spectrum are ent with a distorted tetrahedral geometry about the metal ion. The ini cyanide and acetazolamide are both believed to displace the coordinated water molecule. The shift to a narrower more intense spectrum on inhibitor binding has been interpreted as a shift to a more regular tetrahedral geometry. 2.2 NUCLEAR MAGNETIC RESONANCE (NMR) SPECTROSCOPY It is widely used and important physical technique available to the modern scientist working in different research areas. It gives invaluable chemical and structural information and therefore it is widely applied to solve the different kinds of problems, The technique is frequently associated with proton and carbon nuclei. A nucleus whose spin quantum number (1) is not equal to zero possess magnetic moment. In the absence of a magnetic field all the magnetic states of that nucleus are degenerated. If such nucleus is placed in an external magnetic field, it gives a total of (21 + 1) possible orientations, It is the splitting of the otherwise degenerate energy levels in a magnetic field that makes NMR spectroscopy more informative. ‘The most widely studied nuclei ('H, "°C, '°F *'P) all possess a spin quantum number of 1/2, and so when these nuclei are placed in a magnetic field two spin states arise (m = +1/2, m = -1/2). Transition between these two energy levels is possible and the difference between these two states is given by = 1HBy ae= 18 (2.3)Physicochemical and Spectral Methods 43. where B, is the strength of the applied magnetic field, and y the gyromagnetic ratio for the nucleus under study. The frequency of radiation that corresponds to this energy is called the resonance frequency and is given by 7hBy Tt (2.4) 2mv= YB, (2.5) The magnetic field strength usually applied will be in 2-14 tesla and this corresponds to radiation within the radio-frequency part of the electromagnetic spectrum. The motion of the electrons surrounding the nucleus will result in induced magnetic fields. Therefore, the applied magnetic field (B,) to a sample and effective magnetic field (B,,) experienced by the nucleus are not necessarily be the same. The nucleus is therefore said to be shielded from the external field by its surrounding electrons, AE = hv= By = B, U- 9) (2.6) where ois the shielding constant which will vary and depends on chemical envirnonment. The difference between the resonance frequency of the nucleus in unknown (¥V,,.) and in a reference compound (¥,,,) be determined. This difference is very small and so it is multiplied by a million. The frequency will also vary depending on the strength of the magnetic field so this quantity is divided by the frequency of the standard compound to provide the 6 or ppm (parts per million) scale which is a dimensionless quantity and independent of field strength. Vay; 8, ppm = fasta! x 10° (2.7) Veep The shielding of a nucleus is influenced by the type of atoms surrounding it and the bonding arrangements. The nuclei of a particular group in one nuclei will resonate at a similar frequency in another molecule. Thus it is possible to use the chemical shift of a particular nucleus under study. The chemical shift value (8) depends on the molecular environment of a proton and is measured relative to an internal standard. The frequently used internal standard is tetramethylsilane (TMS). The field axis is on a scale from 0 to 10 8 with TMS at minimum value. The type of proton may thus be identified by the absorption peak position, i.e., its chemical shift and the area under each peak being proportional to the number of such protons in a particular group. 'H-NMR spectrum of ethyl alcohol is given in Fig. 2.13. Three peaks are observed in the spectrum. The peaks (from low 6 value to high 6 value) corresponds to three methyl, two methylene and one alcohol group protons, the peaks appear in the area proportions 3:2: 1. Another important piece of information that is available from NMR spectra which helps further in spectral assignment is hyperfine splitting. This arises from the fact that non-equivalent magnetically active nuclei couple to each other. The magnetic field arising from one nucleus can influence the magnetic field experienced by the other non equivalent nuclei that are near by in the44 Bioinorganic Chemistry t \_-H or a H-C—H ae (a) PAA" Hq (b) a Fig. 2.13 'H-NMR spectrum of ethanol (a) under low resolution and (b) under high resolution. molecule. The hyperfine splitting gives information about the environment of immediate neighbour nucleus. The essential components of an NMR instrument are shown diagramatically in Fig. 2.14. The layout is almost identical with that of ESR, except that instead of a Klystron oscillator being present to generate microwave radiation, two sets of coils, a transmitter and a receiver are used to for generation and reception respectively of the appropriate radiowave frequency. RFO|| RFD CRO Sample Fig. 2.14 Schematic diagram of an NMR spectrometer CRO = Cathode ray oscilloscope RFO = Radio frequency oscilator RFD = Radio frequency detector Samples in solution are contained in sealed tubes that are rotated rapidly in the cavity to climinate irregularities and imperfections. In this way an averagePhysicochemical and Spectral Methods 45 and uniform signal is reflected to the receiver to be processed and recorded. Solid state and highfield NMR are more recent and rapidly advancing tech- niques enabling to solve difficult problems. The latest developments allow two dimensional NMR to be performed to determine even more complicated struc- tures. The study of molecular structure, conformational changes and certain types of kinetic investigations are the main uses of NMR in the biological field. Most work is done in solution and in order to eliminate solvent effects the equivalent deuterated solvent would be used. The use of the technique in drug metabolism studies is of increasing importance, particularly when coupled with IR and X- ray diffraction data. The development of high field magnets and solid state NMR has improved the usefulness of the technique for the biological scientist. The biological ac- tion of antibiotics such as gramicidin and valinomycin has been understood largely from NMR studies. The isotopes '°C and *'P exhibit nuclear resonance and NMR has been used extensively in studies of porphyrins and phosphate metabolism respectively. High field n.m.r. measurements to proteins and enzymes have been made to understand the structural features in solution and in solid state. The use of NMR shift reagents gave impetus to probe the structural information of biomolecules. FT-NMR spectrometers operating 300 MHz or greater coupled with a variety of new difference spectroscopy technique has made it possible to carry out procedures normally used in the examination of the n.m.r. spectra of small molecules, Proteins such as lysozyme, cytochrome c, cytchrome ¢;, triose phosphate and some blue copper proteins and calcium proteins have been studied in some detail by high resolution nmr. technique. The inspection of comparative structural features by 'H-NMR is very fast as. the spectrum provides a structural finger print of the molecule. For example, the active site of human leukaemia lysozyme was shown to be closely related to that of hen egg-white lysozyme in work which took a single day. NMR experiments established that the main chain fold of lysozyme in solution is similar to that in the solid state. One of the body scanners used for the location of tumours is based on NMR using 'H-resonance in water, which appears to be in greater concentration in rapidly dividing cells in tumour mass. By using appropriate Computer Tomography (CT-Scan) methods, it is possible to identify tumour and to iden- tify its shape. 2.3 ELECTRON SPIN RESONANCE (ESR) SPECTROSCOPY An electron possesses a magnetic moment by virtue of the fact that it is a moving charge. Therefore, moving (unpaired) electron will generate a magnetic moment. The magnetic moment is much stronger than that generated inside the nucleus so the energy gap between the ground and excited state is much greater than it is for the nucleus. This energy gap changes to Boltzman distributionaa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.aa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.aa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.Physicochemical and Spectral Methods 49 2.4 INFRARED AND RAMAN SPECTROSCOPY These two spectroscopic methods are complementary, giving similar informa- tion but the criteria for the phenomena to occur are different for each type. It is also true that, for asymmetric molecules, absorption will give rise to both types and the same information could be gained from either. However, for symmetric molecules having centre of symmetry, the fundamental frequencies that appear in the Raman do not appear in the infrared and vice versa. Thus the two methods are truly complimentary. The bonds between atoms in a molecule may be considered as flexible springs so that the atoms are in constant vibration motion i.e., the molecule is not fixed and rigid. Bonds, can either stretch or deform (bend) and theory predicts that if the molecule contains n atoms there will be 3n-6 fundamental vibrations (3n-5 in the case of linear molecule) in total of these 2n-5 cause bond deformations and n-1 cause bond stretching. The region of the electromagnetic spectrum ranges from the red end of the visible to the microwave lengths. The criterion for an infrared spectrum is that the vibration should cause change in the dipole moment. ic. change in charge displacement. Conversely, if there is a change in the polarisability of the mol- ecule, a portion of the scattered radiation will have a frequency different from that of incident radiation. These different frequencies constitute Raman spectra. The fundamental frequencies observed are characteristic of the functional groups concerned and are absolutely specific. These groups of certain bands regularly appear near the same frequency and may be assigned to specific molecular groupings just as particular chromophores absorb in the UV-visible regions. Such group frequencies are extremely valuable in structural diagnosis. ‘The frequency associated with a particular group varies slightly, owing to the influence of the molecular environment. This is extremely useful in struc- tural biochemistry studies as it is possible to distinguish between C-H vibra- tions in methylene (-CH,-) and methyl (-CH,-) groups. The most common source of i.t. radiation is a nichrome alloy coil wire heated to incandescence. This region of the electromagnetic spectrum contains the heat waves. Detectors are of the heat recognition type. The Golay cell contains a gas or liquid whose expansion is registered when the energy is absorbed. Thermal detectors such as thermocouples can also be employed. Analysis using a Michelson inferometer allows Fourier Transform Infrared Spectroscopy (FT-IR) to be performed. The instrument involves fixed and rotating mirrors (made up of alkali halides) that split incident beam into two. The beams are recombined after passage through the sample but as the two path lengths are different, interference patterns arise that maybe analysed by FT methods. Samples of all physical states viz., solid, liquid gas may be analysed using vibrational spectroscopy. Samples are cither prepared in mulls such as nujol and held as layers between salt plates such as NaCl or pressed into KBr discs. Non covalent materials must be used for sample containment and also in the optics as these materials are transparent to infrared.aa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.aa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.aa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.Physicochemical and Spectral Methods 53 T T T - ‘Ae (Human C and B) Ae (Bovine B) HCAB‘$ 0.2 = de 40.5 L L L oO 300 400 500 600 700 4 A, mu Fig. 2.22 Visible C.D of three isoenzymes and species variants of Co(!|) carbonic anhydrase (—) human isoenzyme C ( bovine isoenzyme B (-e-e) human isoenzyme B (From J.E. Coleman, Progress in Bioinorganic Chemistry, Vol 1. F.T. Kaiser and FJ, Kezdy (eds), Wiley- Interscience (1971). The yray energy from radioactive nucleus may be modulated by giving a Doppler velocity to the source. The Doppler effect, observed in all wave forms (sound and electromagnetic) is recognized as the apparent change in frequency that occur when the source is moving relative to the detector (observer). The change in frequency is proportional to the source velocity and any velocity may be chosen to give the required frequency. Gamma-rays of discrete energy can be resonantly absorbed by appropriate nuclei. The source used is usually *’Co; this emits a range of rays with different energies, an appropriate one of which may be selected. The selected ray is then modulated by the imposed Doppler phenomenon. A simplified diagram of the arrangement required to perform Mossbauer spectroscopy is given in Fig. 2.23. Usually, because of the energies and [yray | | Absorber source, ;-}—————_»> Detector eo "o| (sample) i Vibrator Fig. 2.23 Layout of simple Mossabuaer spectrometeraa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.aa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.aa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.Physicochemical and Spectral Methods 57 Fourier transforms for the multishell systems | Mo(NHSC,H,);, MoO(S,CNEt,),, MoO,[CH,SCH,CH,N(CH,CH,S),], MoO {(CH;);NCH, CH,N(CH,CH,S),] and MoO ,(NH,CH,CH,NHCH,CH,NH,) are shown in Fig. 2.27. The second complex clearly shows distinct Mo-O and Mo-S shells, but the remaining complexes show decreased resolution of individual shells and it becomes difficult to assign individual shells to particular scattering groups. In order to get accurate structural data from multishell systems various curve fitting procedures have been developed. The curve fitting procedures provide an approximate value for the number of scattering atoms and a precise value of their distances. Results for some Mo complexes are listed in Table 2.5, where it can be seen that EXAFS are comparable to those of X-ray data. “| \\ | MoO(S,CNEt,)2 | % & Relative Magnatude _ =e, ft a Bk ok | wt pe Fig. 2.27 Fourier transform for multishell systems [From "Molybdenum and molybdenum containing enzymes’, ed. M.P.Coughlan, Peraman, Press Oxford). As ESAFS data are precise (Table 2.5) it is possible to determine bond distances accurately in complexes of unknown crystal structure. EXAFS data for MoO[CH,SCH,CH,N(CH,CH,S),] are given in Table 2.6, The crystalaa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.aa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.aa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.Physicochemical and Spectral Methods 61 -1.50 Current Fig. 2.30 Cyclic voltammogram of O, at a mercury electrode in acetonitrile with Agi0.01 M— AQNo,, reference electrode supporting electrolyte (C2H,),N‘CIO; with Eye = ~1.22 V. [From J. Chem. Ed. 60 (1983) 290). half-peak potential (E,,.) and the half-wave potential (E,,.). The equation E,, = E, + (RT/nF)in(RD,)'* AL. is adapted from classical polarography. In the above equation, E° is the formal potential related to the ionic strength used, D, and Dg are the diffusion coeffi- cients of the oxidized and reduced forms, and n is the number of electrons in the half reaction. Since D, ~ Dg, Ey; Value will be usually within few mV of E° The half wave potential (E,,.) for a reversible couple is centered between E,, and E,. Ey, = epee (2.12) The values of i, and i,, should be identical for a simple reversible couple. However, the ratio of peak currents can be significantly influenced by chemical reaction coupled to the electrode process. Electrochemical irreversibility is caused by slow electron exchange of the redox species with working electrode. The reduction of O, is a reversible process. The electron transfer reaction at the electrode surface is so rapid that equilibrium conditions are maintained, even with a substantial net current and a rapidly changing potential. There are several criteria for reversibility. They are: (i) AE, =E,, - E,, = 56/n mV; (ii) Exo — Ex. = 56.5/n mV values of which must be independent of the scan rate and concentration (iii) the E,, is situated exactly (with 2/n mV) midwayaa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.aa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.aa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.Perspectives of Essential Trace Elements 65 elements. The dominance of the eleven common structural elements, which in human account for over 99% of the atoms is shown in Fig. 3.1. The three prominent biologically active metals are iron, zinc and copper. All the remaining essential trace elements are considered ultratrace since they are present less than 10 mg in the adult human. Position of essential elements in the ‘Periodic Table’ is shown in Fig. 3.2. Elemental Composition of the ‘Agutt Human Booy 24 -F gfe 8 oan Trace Elements S g 2 € £ 3 g xt ‘So Nn Ba Go As N 4B Mocs) tra micro esncrpp Trace co Elements Fig. 3.1 Elemental composition of the human adult expressed on a logarithmic scale (From Reference 1) [Louk elements H [__] Essential Trace Elements He [_] S2ocntie! Trace ements BICIN/O|F|Ne. INa|Ma) (Proposed) Alsi P[s|ci]ar K |ca]Se| ni [vJ[cr]uol Felco] niculzal cal Ge]as|Sel Bx] Kr Rb] Sr| ¥ [Zr |No|Mol To [Ru| Rh] Pa] Agi Calin [Snisb|Te| 1 [xe |Cs/Ba| Hf | Ta] W |Re|Os} Ir | Pt | Auj Hg] Ti |iPbj Bi |Po] At| An Fr [Ra] \[>tanthanides Actinides Fig. 3.2 Present status of the essentialty of the elements and the periodic table,aa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.aa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.aa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.Perspectives of Essentia! Trace Elements 69 Williams* has enumerated a number of general chemical parameters which influence the selection of metal ions by living cells. These include charge-type, ionic radius, donor atoms of ligand, preferential coordination geometry, spin- pairing stabilization, kinetic controls and the chemical reactivity of metal ions in solution. Proper understanding of these factors® would enable us to speculate about possible future essential trace elements. 3.3. FUTURE ESSENTIAL TRACE ELEMENTS All of the non-metals, excluding the inert gases with an atomic weight less than bromine are now considered to be essential. Bromine is probably the most likely remaining non metal to qualify as an essential element. The division be- H ieacieuas’ fe tween the metals and non metals is i ae[8 [c NIO|FINe shown in Fig. 3.4. Most of the remain- ing lighter elements are metals and most MaMa ALSIP TS 1G [ar of the recognized trace elements can be K |Ca|Ga\Ge|As| Se) Br| Kr regarded as wansition metals. The pre- Rb} Sr} in |Sn|SbjTe) | [xe transition metals include the alkali met- Cs/8a! Ti Pb! Bil Po] At/Rn als (Group IA), the alkaline earth metals (Group IIA) and the Group ITI elements. Many of these metals are essential -Na, Fig. 34 Part of the periodic table of the K, Mg, Li, Rb, Sr and Ba are the strong- finland a - een metals and nonmetals. est remaining candidates that may even- tually be found useful biologically. The most prominent groups of trace elements are observed in the transition and the post-transition metals. The transition metals have d orbitals that are increasingly filled with electrons, leading to a gradual transition in metallic properties. They form stable complexes with sulphur, nitrogen and oxygen ligands especially amino acid residues of proteins. Iron occupies a dominant position among the transition metals in the vertebrates as the metal choice for hemoglobin and other heme proteins. All the first row transition metals (except titanium) are considered to be essential. The neighbouring post-transition metal (copper and zinc) are also important trace elements. They serve as coenzyme or prosthetic groups for a large number of enzymes. Of the heavier transition elements, only molybdenum has been proven es- sential. The remaining transition metals are poorly absorbed by cells and rela- tively non-toxic. Tungsten has been reported to be biologically active as a component of the enzyme formate dehydrogenase in several bacteria.° Probable additional essential ultratrace elements are Ni, Cd, Sn and Pb. Several other metals (Au, Ag, Pt, Hg) have played significant role in the history of our civilization, but they have not been proved to be essential. It is hoped that these metals may qualify for consideration as essential elements in near future. Fr|Ra Metalsaa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.aa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.aa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.Perspectives of Essential Trace Elements 73 H HN L ii G—CH, opo;- j HANAN, OH s$ $ NN if ™ mM x" Fig. 3.5 The proposed structure of the molybedenum cofactor, molybdopterin amide-phosphate-ribose side chain to a 5,6-dimethylbenzimidazole group which coordinates with cobalt ion. These compounds serve as cofactor in various enzyme reactions in which a hydrogen atom is interchanged with a substituent on an adjacent carbon atom. The best example of cobalt containing mammalian enzyme is methylmalonyl coenzyme A. This is a mutase that catalyses the rearrangement to succinyl coenzyme AS Thus for the metalloenzymes have provided the best model for determining how the trace metal ions operate. However, data supporting the metalloenzyme model for other ultratrace metals (Cr, V, Cd, Sn, Pb and Li) are lacking. Chromium functions in vivo as an organic peptide chromium complex. Its biological role is to potentiate insulin activity. In addition to chromium, this organic complex may contain nicotinic acid and glutathione as its constituents. Vanadium compounds inhibit numerous enzymes, particularly ATPases and phosphotransferases. This has suggested a role for the vanadate ion in the contol of the sodium pump. Vanadium also serves as a biocatalyst for oxida- tion of substrates invoived in cholestrol metabolism. Except for metallothionein induction, most biological effects of cadmium have been inhibitory. Alleviators of lead deficiency resulted in the correction or prevention of changes in iron metabolism and hematology. Data on the possible action of tin and lithium are lacking. Thus we have no definitive explanation for the action of these ultratrace elements. The challenge to bioinorganic chemist is to solve these problems clear, 3.8 ESSENTIAL ULTRATRACE NONMETALS As shown in Table 3.5, these essential nonmetals comprise a much more het- erogeneous group than do the transition metals, whose properties are closely related. The two essential halogens, fluorine and iodine, are highly specific and very different. Fluoride has a remarkable anti-dental caries effect. This may be related to its ability to replace OH, thereby stabilizing the structural matrix of bones and teeth. Fluoride also can inhibit strongly certain key enzymes enolates, pyrophosphatase. One of the major surprises among non metals is provided by seleniam. Despite its high toxicity (0.2 mg/d), selenium has been shown to be a compo- nent of several enzymes involved in essential oxidation-reduction reactions. One enzyme, glutathione peroxidase (GSH) play a major role in the protection of red blood cells against the effects of hydrogen peroxide which is readilyaa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.aa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.aa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.CHAPTER 4 The Alkali Metal and Alkaline Earth Cations ‘The alkali (Na*, K*) and alkaline earth metals (Mg** Ca"*) constitute about 1% of human body weight, whereas the trace metals (Cr, V, Mn, Fe, Co, Cu, Zn, Mo) represent less than 0.01%. Paradoxically trace metals have been studied most frequently and quite early. The solution chemistry of the groups IA and IIA was mainly developed quite recently except for organo-lithium and organo- magnesium reagents. The slow start of investigations on the bulk metals may be due to (i) the lack of complexes of these cations with simple neutral ligands (such as those with ammonia, ethylene diamine etc. form complexes readily with transition metal ions) and (ii) the absence of spectroscopic (UV visible) and magnetic properties. The development of X-ray diffraction methods and the discovery that naturally occurring macrocyclic antibiotics which display both ligand as well as carrier properties towards alkali cations opened a new field of investigations. Alkali metal ions along with magnesium and calcium ions play an essential role in biological systems. They are involved in several physiological processes such as (i) transmission of nervous impulses, (ii) nervous control of secretery and muscular functions, (iii) protein synthesis, (iv) enzymatic regulation in metabolism and so on. The four cations, Na*, K*, Mg’ and Ca” are widely distributed in all living organisms. However, Na* and K* are most abundant in cells. Their concentration (mM Kg™) in various biological systems respec- tively, is given in parenthesis. Human heart muscles (40, 78); rat muscle (27,101), crab nerve (168, 158), dog liver (38, 81), E. coli cell (20, 250). In Table 4.1, ‘one may note that the extra and intra cellular concentrations of Na‘, Kt, Mg** are different. In general, the concentration of potassium ions inside a cell is much higher than the sodium ions, while the reverse is true outside a cell. This observation raises the questions: (1) How is the dissymmetry established and maintained? (2) What is the purpose of this distribution? Some answers to these questions are given here. This asymmetry of ion distribution is necessary for the maintenance of the resting potentials of most cells. It is particularly important in nerve since excit- ability depends on the ionic gradient. The maintenance of the ionic gradient involves the energy-requiring outward movement of sodium and the inward transport of potassium ions. To elucidate the mechanisms of all these processes requires the understanding of (i) the transport mode of ions across the mem- brane, (ii) the principles by which transport sites in membrane distinguishaa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.aa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.aa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.The Alkali Metal and Alkaline Earth Cations 81 stability of complex increases with the decreasing metal ion-radius. This con- cept has been discussed by Eigen er al.'* and is demonswated in Figure 4.2. Size dependence on the free energy of hydration is represented by Curve 1. Curves 2 and 3 represent two different size dependencies of the free energy of ligand binding. The shapes of Curves 2 and 3 are controlled by the ligands due to their steric fixation. The free energy of ligand binding increases monotonically regardless of whether the stability constant increases or decreases. The free energy difference between the ligand-binding curve and hydration curve deter- mines the stability constant of the metal complex. 120- 8 a & ——?—_? ~AG, kcal/mole Cs"Rb"K” Na* 0.5 15 1.0 (radius), A* Fig. 4.2 Schematic representation of the dependence of binding energy and solvation en- ergy on the reciprocal radius of alkali cation. Eigen’s model suggests that it is possible, in principle, to synthesize or isolate a multidentate ligand to give the maximum stability constant for a chosen cation size. Experimental data on alkali metal complexes of natural and synthetic macrocyclic compounds suggest that the selectivity patterns observed can be rationalized by the superposition of the following effects'*"”. (i) Solvation energy of metal ion, (ii) solvation energy of the ligand (energy required to change the conformation of ligand to a complex configuration) and (iii) the configuration of the ligand to provide maximum effectiveness for metal-ligand interactions. These effects can be described by the following equation. ART InK = A Gyjnding ~ 4 Gyoiy (M*) — A Gyoiy (L) — A Googe L) (4.5) where K = stability constant of the complex AGyinting = free energy of metal ligand bond AG, oi, (M*) = free energy of metal ion solvation AG,,jy (L) = free energy of ligand solvation AG oar (L) = free energy of conformation of ligand. In fact, the solvation energy and the conformational energy of the ligands are closely related to each other particularly for macromolecules. Experimentalaa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.aa You have either reached a page that is unavailable for viewing or reached your viewing limit for this book.84 Bioinorganic Chemistry (b) Table 4.3. Stability constants* of alkali ~ metal ion compiexes of macroteirolides Ligand ur Nat Kt RY Cs Solvent Temp °C Nonacin <0S 24 41 41 32 MeOH 5 = 22 (38 — — MeCH 20 Monactin <03 26 44 44 33 MeOH 6 = 34 54 = os MeOH 30 Dinactn =§<03 «3.0 46 4.6 3.6 MeOH 25 Trinaction <03 32 50 49 40 MeOH 25 * Stability constants expressed in log K values decreased. This trend is reversed after the potassium ion is reached. The data suggest that the solvation energy of the metal ion is important among other factors, in determining the stability of the compiex. The coordi- nated solvent molecules are completely substituted by the macrotetrolide ligand. This is supported by NMR data’. In addition, X-ray crystallo- graphic data for KSCN-nonactin showed that no solvent molecule is co- ordinated with the potassium ions in the complex. Complex formation studies” confirm the important role of the solvation energy of the metal ion in the relative stability of the complexes formed with a given ligand. Within the group, for a given metal ion, the complex stability shows the trend: trinactin > dinactin > monactin > nonactin. Trinactin, which is the most hydrophobic in this group, is likely to have the lowest solvation energy in a relatively polar solvent like methanol. Further, complex for- mation would shift the trinactin to a more hydrophilic form. It has been shown that these ligands exist in more than one structural form in both solution and solid. However, unlike other antibiotics, these conforma- tions are not stabilized by intrahydrogen bonds. Kinetic data”’ suggest that at least two conformations are present in solution prior to the addi- tion of the metal ion, A sizable conformational change of the nonactin molecule due to complex formation with an alkali ion was detected by proton magnetic resonance studies. The X-ray data” of pure nonactin and its potassium complex suggest changes in configuration due to complex formation. The structure of K* nonactin complex (Figure 4.4) is one in which the potassium ion is surrounded by four furane oxygen atoms and four keto oxygen atoms to form approximately cubic octa-coordinated complex. The 32 member ring can be described as resembling the seam of a tennis ball with potassium ion at the center of the ball and the hydrocarbon group on the surface. The dynamic study of complex formation of macrotetrolides suggest that these molecules are very flexible and undergo a conformational transition with a relaxation time of about 6 n sec before the complex formation takes place. Cyclodepsi peptidases: Valinomycin is a typical example of cyclodepsi peptidases. It has 36 atoms in its ring containing ester and peptide bonds. Its K* complex (Fig. 4.6) shows the very efficient encircling of the cat- ion”’. Note that binding is achieved in this case by only six donor sites.The Alkali Metal and Alkaline Earth Cations 85. Fig. 4.6 Structure of (a) valinomycin and (b) its K* complex. CD and NMR measurements” indicate that valinomycin undergoes con- formational changes due to complex formation and polarity of solvent. The enniatin class of depsipeptidases has only 18 atoms in the ring. The K* complex of enniatin has a very simple structure (Figure 4.7). Fig. 4.7 Structure of K* complex of eniatin. i) Open-chain Carboxylic Acid lonophores A large number of antibiotics belong to this class. They have a carboxylic acid function which may be ionized at physiological pH values. Nigercin, monensin and grisorixin belong to this category. The uncharged open-chain antibiotics such as gramicidin and so on have also been studied. The structures of these ionophores are given in Fig. 4.8. The structure of sodium complex of monensin is shown in Fig. 4.9. The complex has a cyclic structure which is maintained by the intramolecular hydrogen bonds”. This is a general characteristic of all these types of antibiotics. It is important that the resulting complex with a mono valent cation is neutral. Lasaloacid can form a complex with mono and divalent cations. However,this ligand has low selectivity”. Tonophores that have high specificity for divalent cations are rare. Calcimycin is an example for this category. It transports calcium across membranes and can form L:1 or 1:2 cation:ligand complexes. The latter complex is found to be neutral”.86 Bioinorganic Chemistry Monensin: R, = CH(Me)CO,H; Rp = Et Ry CO:H Antibiotic A 23187 Fig. 4.8 Structures of open chain antiboitics. | Fig. 4.9 Structure of Na"—Monensin complex | 4.3.2 Synthetic Complexing Agents Several hundreds of ligands have been synthesized and investigated them as ligands/carriers for alkali and alkali earth cations. Unlike the transition metal ions, the alkali metal ions do not form stable complexes with simple ligands such as ammonia, ethers, thioether, halides, cyanides and so on.The Alkali Metal and Alkaline Earth Cations 87 Synthetic (polydemate) ligands can easily replace water molecules, achieve strong complexation and provide an optimal donor atom distribution around the cation. This discussion will be concerned with several types of synthetic Jigands and will be shown how the structural constitution of ligand affects the stability of complex obtained. @ Conventional Ligands Ligands such as P,O#, POS, EDTA*, 1,3-diketonates etc, are well known complexing agents. An extensive review of these compounds is available”, (ii) Linear Polydentate Ligands These compounds are structurally simple and may be obtained by short step syntheses. The oligoethylene glycol dimethyl ethers (glymes) are the simplest neutral complexing agents for alkali cations. The examples in Table 4.3a indi- cate that stability constants as well as selectivity of complexation are low’’, Table 4.3a. Stability constants (log K) for Glymes (MeOH, 25°C) Stability constant of Structure Na* complex K* complex oo Oo [ 1.28 1.72 A oo oO 1.47 2.20 A_R_A oe yo f 1.60 2.55 ‘o._9__o. Sw Oo f 1.67 2.87 A_A_A_A, (iii) Macrocyciic Polyethers or Crown Ethers The first crown ether, dibenzo [18] crown-6 was observed to be quite insoluble in methanol, but readily dissolved on the addition of sodium salts. This obser- vation led to the discovery of the complexing ability of the crown compounds and to the synthesis of a large range of other macrocyclic polyether. The cyclic polyethers, which were first synthesized by Pederson are most widely studied, Some of these compounds are known to facilitate the 34,3588 Bioinorganic Chemistry transport of alkali metal ions through membrane. A review of these polyethers has been published**, Table 4.4 contains a small list of polyethers and their names. The nomenclature is straight forward. For example, in the name, [18] crown-6, [18] defines the number of atoms in the macrocyclic ring and 6 indicates the number of oxygen donor atoms in the ring. Similarly for [21] crown-7 and so on. The size of the ring varies from 9 to 60 atoms with 3 to 20 oxygen atoms in the ring. Table 4.4, Nomenclature of some macrocyclic polyethers {CH,CH,O) 8.No. "We Name of the ligand (CH,CH,O)n x Y m a 1 CoH, CH 2 1 Perhydrobenzo-[15]-cwron-5* 2 CH, CH, 2 2 [18] orown-6 3 C:H, SoHo 2 2 Perhydrobenzo-{18}-crown-6* 4 CH, CH 2 2 Dibenzo-{18}-crawn-6 5 CH gH 2 2 Perhydrodibenzo-[18)-crown-6” 6 CH, CH, = 2 3 [21}-crown-7 7 CoH, Coty 2 3 Dibenzo[21}-crown-7 8 CH, CH, 3 3 (24}-crown-8 9 CoH, CH, 3 3 Dibenzo-24-crown-8 10 CoH, CH 4 4 Dibenzo-30-crown-10 W Coty Ges 9 9 Dibenzo-60-crown-20 a. Pethydrobenzo is also known as cyciohexyl. b. Perhydrodibenzo is also called as dicyclohexyl. Most of the crown ethers give 1:1 complexes. In addition, 1:2 and 2:3 (M:L) complexes were also observed when the cavity of the ring was too small to fit the metal ion. The 1:2 (M:L) complexes are likely to have ‘sandwich’ structure. When the cavity is large compared to the ion, a considerable change takes place in the polyether structure and a 1:1 complex is formed. The structures of some alkali metal cyclic polyether complexes have been determined by X-ray crystallographic studies. A typical complex, RbSCN—dibenzo-18-crown-6, shows the localization of the cation in the center of the ring (Fig. 4.10). Note that the cation is in contact with the anion and the six oxygen atoms of the polyether lie in the same plane and the metal ion lies slightly out of the plane. ‘The crystal structure of NaBr (dibenzo-18-crown-6).2H,0 is given in Fig. 4.11. The distances between the metal ion and six oxygen atoms are about the same for cach complex. A similar structure was observed for Nal (benzo-15-crown- 5). It should be noted that the metal ions of these complexes are in direct interaction with the anions or water molecules. When the ligand is large ring system, the metal ion is enclosed in a coordination sphere. The potassium complex of dibenzo-30-crown-10 shows that the potassium ion is surrounded by a sphere which is composed of 10 ether oxygen atoms. For example, the structure of KI (dibenzo-30-crown-10) complex indicates that unlike the previous complexes, the hydration sphere of alkali metal ion is completely replaced by the polyether ligand (Fig. 4.12).The Alkali Metal and Alkaline Earth Cations 89 Fig. 4.10 Structure of Rb(SCN) dibenzo-[18] crown-6 complex. Fig. 4.11 The asymmetric unit of NaBr(dibenzo-18-crown 6)2H,0 dotted lines indicate hy- drogen bonds between H,O and Br. The 4th H,O which is not shown can be visualized as behind Br of the complex A or in front of the nearer HO of complex B [From M.A. Bush, M.A. Truter, Chem. Commun., 1970, 1430]. Fig. 4.12 The structure of KI (dibenzo-[30]-crown-10) complex (From M.A. Busch, M.R. Truter, Chem. Commun., 1979, 1430).90 Bioinorganic Chemistry The stability constants of 1:1 complexes are given in Table 4.5. The small ligand shows a higher stability constant for Na* than for K*. The larger cycles show the reverse selectivity. It has been recognized that since beginning of crown ether chemistry that the cavity diameter (hole) discriminates between different cations. This is shown in Table 4.6. Table 4.5. Stability constants of 1:1 alkali meta! ion complexes with macrocyclic polyether ligands (in methanol at 25°C) Ligand Nat Kt Cat ad Cs Dicyclohexyl-14- 218 1.30 = — = ‘crown-4 Dicyclohexyl-18- 4.08 6.01 - - - ‘crown-6 (in water) (1.21) (2.02) 3.24 3.57 Dibenzo-18 crown-6 4.36 5.00 Dibenz0-21-crown-7 2.40 4.50 Dibenzo-30-crown-10 20 4.60 18-Crown-6 4.32 6.10 3.88 <55 7.04 (in water) (0.80) (2.03) 272 3.78 2,6-dioxo-18-crown-6 25 2.79 _ = 34 1,4-Dioxo-19-crown-6 18 2.55 - - 11.44 Table 4.6. Correlation between ‘ing size and cation selectivity Macrocycle lonic diameter A Cavity diameter A Selectivity Perhydrodibenzo-14-crown-4 156 u- Perhydrobenzo-15-crown-5 1.96 Na * Perhydrobenzo-18-crown-6 266 kt Dibenzo-21- crown-7 2.98 Rb * Dibenzo-24- crown-8 330 = cs* ‘These relationships seem to be limited when the cavity is large and the ligand is flexible enough that maximum metal-ligand binding energy can be achieved by conformational change. For example dibenzo-30-crown-10 shows selectivity pattem K* ~ Rb* > Cs* >> Na* >> Lit 6 The size of the ring is one factor that can influence the stability of macrocyclic. metal complex. Other factors such as solvation energy, donor atoms may operate. A fundamental result is the large increase in stability constant for the complexes obtained with macrocyclic ligands as compared to open chain ligands by a factor of 10-10°. This enhancement is called macrocyclic effect. In addition, a solvent effect on the binding of cyclic polyethers to alkali cation has been reported’’. The selectivity of dimethyl dibenzo-18-crown-6 with respect to alkali cation in tetrahydrofuran (THF) was found to be Na* >> K* > Cs* > Li* but this sequence changes to K* selective in oxetane. Frensdroff nicely demonstrated the effect of donor atoms on the stability, He substituted nitrogen or sulphur atoms for oxygen atoms in the [18]-crown-6 and dibenzo [18]-crown-6 in the manner shown in structures (4.1) and (4.2) and Table 4.7.The Alkali Metal and Alkaline Earth Cations 91 Ayr) rN ae, has (4.1) (4.2) Table 4.7. Effect of donor atoms on the stability constant of potassium complex of 18-crown- 6 derivatives in methanol at 25 °C Donor atoms in the structure Structural type A B Log K (a) ° ° 6.10 (4.1) NH o 3.90 (41) NH NH 2.04 (4.4) s s 1.18 (42) ° ° 5.00 (42) NR ° 4.10 (42) NH ° 3.20 (42) NH NH 1.83 The stability of the potassium complexes decreases in the order of decreas- ing electronegativity, namely O > NR > NH > S. In other words, as the electronegativity of the ligand decreases, the electrostatic attraction between the metal ion and the ligand diminishes. (iv) Cryptands (Macrobicyclic Ligands) This class of ligand was synthesized in late 60's*. They form very stable complexes with alkali and alkaline earth cations. The ligands (Fig. 4.13) have an increasing cavity size and form a complex with a large number of cations. These ligands contain a molecular cav- ity (or crypt). The complexes formed are called cryptates. The formation of a row -! cryptate complex can be represented by 1 the equilibrium shown in Fig. 4.14 Wa where K, is the formation of the com- plex. These ligands form stable and se- lective complexes with spherical cations. ‘The complexes may be designated [M™* CL] where C is the mathematical sym- bol for inclusion and L is the ligand. The cryptands form complexes with suitable alkali and alkaline earth cati- ons which are several orders of magni- tude more stable than those of natural macrocycles. For example [K* C 2.2.2] is about 10* times more thermodynamically stable than [K*C valinomycin]. pt, m =n=0 Fig. 4.13 Crypiands.92 _ Bioinorganic Chemistry gi Fig. 4.14 Formation equilibrium of a crypiate inclusion complex between the macrocyclic ligand {2.2.2} and metat cation: K, is the formation constant of the complex. The large increase in stability constant observed on going from a macrocyclic ligand to a macrobicyclic ligand is called as the macrobicyclic or cryptate effect’. The X-ray crystal structure of Rb* 2.2.2 cryptate is given in Figure 4.15. The cation is held in the centre of the cavity of the ligand. Fig. 4.15 Rb* 2.2.2 cryptate. (v) Spherands This class of compounds designated by Cram“ occupies a special position as complexing agents for alkali and alkaline earth cations. These are based on several types of units. The most investigated, macrocyclic oligomer of methoxytoluene and its derivatives are given in Fig. 4.16. These macrocycles are obtained by skilful synthesis and represent the highest sophistication in cation receptor design. Cram’s idea is simply to build the receptor in such a way that the coordination sites are strictly in the required position for complexation without any degree of freedom. As a result the stability constants observed are exceptionally high (10!'— 10!%)*14?, 4.4 IONTRANSPORT The structure of cell is given in Chapter 1. The cell is surrounded by a mem-‘The Alkali Metal and Alkaline Eerth Cations 93 Fig. 4.16 Spherands. brane which separates its aqueous interior from the plasma. Biological mem- brane function as barriers that prevents the uncontrolled access of ions and polar substances to the cell. These membranes have structures that selectively admits ions, nutrients and regulatory substances. Frequently these specialised structures are themselves subject to regulation by hormones, ions and effectors adsorbed from the extra-cellular environment. The cell membrane thus plays an important role in coordinating biological functions. In general, a biological membrane is composed of protein and lipid and in most cases is believed to be about 70 A thick. The lipids and proteins differ quantitatively from one membrane to another. The ratio of lipid to protein and the thickness of the membrane depend on the type of membrane. For example, red blood cell (ghosts) contains about 60% protein and 40% lipid by weight. OF the latter, about 70% by weight is phospholipid and 30% cholesterol? On the other hand, myelin of human brain contains about 20% protein and 80% lipid. About 20% lipid is cholesterol. A good review on the composition of lipid in membrane is available**. The transport of alkali metal ions by antibiotics and synthetic polyether through model membranes may provide considerable insight into the physicochemical mechanisms responsible for the function of the natural mem- brane. These model membranes can be formed with a variety of amphiphatic molecules such as phospholipids, dissolved in appropriate solvents**“**, These membranes are found to be 40-100 A®° thick and arranged with polar head of94 Bioinorganic Chemistry the phospholipids facing the aqueous phase and with hydrophobic interior. This hydrophobic interior is believed to be typical for most, if not all, biologi- cal membranes, There can be carrier-assisted passive transfer of material independent of an energy source, the so-called facilitated transport. Or the cell can accumulate cations by working against concentration gradient. The second process requires energy and is known as ‘active transport.’ 4.4.1 Facilitated Transport The conductance observed in the artifical bilayer membrane is very low (107 - 10° Q~ cm)". It is due to the inability of most ions to partition into low-dielectic constant hydrocarbon. Blcrodes However upon addition of about 10% 4 5 M to 107 M a macrocyclic antibiotic, such as valinomycin or macrotetrolides f fl either one or both sides of the mem- Mine brane (Figure 4.17) and 0.1 M KCI, the ‘Membrane conductance increases to around 107 \w &' cm. It is believed that the large abba ‘ “ Fig, 4.17 Schematic diagram of appara- increase in conductance is a conse- tis Used for measuring slecat: quence of an increased ion permeabil- cal properties and permeability ity of the membrane. This antibiotic of lipid membranes. induced ion transport has also been re- ported in mitochondria® *'. In addition, these antibiotics exhibit striking selec- tivity in the transport of monovalent cations. A similar capacity to distinguish between sodium and potassium ions also occur in biological membranes. Facilitated transport involves three possible mechanisms, namely (i) pores, (ii) carrier and (iii) channel (Figure 4.18). Before considering active transport, these mechanisms are briefly discussed below. Aqueous phase Membrane. jueous phase Mech: Pore . s Carrier e ° Carrier Relay e ° COMBE Channel @ Alkali Metal on @Aikali Metal ton-antibiotic Complex SUncomplexed Antibiotics Fig. 4.18 Illustration of possible modes of passage induced by modifiers.‘The Alkali Metal and Alkaline Earth Cations 95 (i) Pores Certain antibiotics are known to function as ‘hole punchers’ on the membrane and make them ‘leaky’ to ions and small solute. The examples for this class include nystatin and amphotericin B. They are cyclical molecules containing carboxyl and amino-sugar groups. These antibiotics are amphipathic molecule that are not soluble in hydrocarbon. This property makes them unlikely to act as carriers. A requirement for their action is for the membrane to contain cholesterol or some other sterol. When the micromolar amount of nystatin or amphotericin B is added to the aqueous phase on both sides of the membrane, conductance increases from 10° to 10 2" cm™ or higher. The conductance increases with the increase of the antibiotic concentration. The pore is believed to be formed by the interaction of several antibiotic molecules with the cholesterol molecules of the membrane causing a structural arrangement of a region of the membrane. The radius of the pore is smaller than 4 A because glucose, with a Stokes-Einstein radius of 4 A is impermeable. The conductance observed in the presence of nystatin or amphotericin B has a very high negative temperature coefficient. A 10° rise in temperature results in about a 10* decrease in conductance. If the antibiotics are removed from the aqueous phases, the membrane conductance gradually decrease and eventually returns to the unmodified state. (ii) Carriers A carrier molecule is required to encapsulate the cation, thus providing an organic, lipid-soluble surface to the membrane. A suitable ligand can provide an ‘organic overcoat’ for the cation. The compounds which are believed to function as cation carrier in transport are valinomycin, macrotetrolides, cyclic polyethers and nigercin groups. The cation-antibotic complex (which is lipid soluble) is then diffused through the membrane and partitioned out the mem- brane into the solution. Model studies with liquid membrane systems have been carried out using the U-tube technique. A system is constructed using a U-tube consisting of two aqueous layers separated by a semi-permeable medium or membrane. Chloro- form (CHCI,) is selected because its dielectric constant is almost equal to that of the membrane. A coloured alkali-metal salt (eg. potassium-4-nitrophenolate) is introduced on one side of the barrier and this is set aside as a control experiment (Fig. 4.19 a). SS = (a) ©) Fig. 4.19 The U-tube experiment: The shaded portions represent the colour imparted to the solvent phases by the potassium o-nitrophenolate, The right hand columns contain water {From Chem. Soc. Rev., 6 (1977) 325}.96 Bioinorganic Chemistry A second tube is prepared with a carrier ligand in CHC1, layer. The move- ment of colour on transport by the ligand carrier can then be noted. If the colour transfers rapidly into the organic layer, but no further, the ligand is acting as an ion receptor (Fig. 4.19b). If the colour is readily transferred through CHCI, layer and into the second aqueous layer the added molecule is acting as an ion carrier (Fig. 4.19¢). The difference in behaviour is related to the stability constant of the com- plex. A high stability constant leads to ‘ion reception’ and a medium value to ‘ion carriage’. A best carrier for ion transport is a ligand which gives a moder- ately stable rather than very stable complex. A very stable complex can’t re- lease the ion efficiently from the complex. For example, the cyclic dodecadepsipeptide valinomycin has a high ion selectivity of Kt ion versus Na*. Enniatin B is a cyclohexadepsipeptide which has a reduced K*/Na* selectivity relative to valinomycin. This is ascribed to the greater flexibility of latter ligand. The structure of the 1:1 enniatin B ~ Ki complex is given in Fig. 4.7. (iii) Channel The antibiotics which are believed to proceed by the channel mechanism are the neutral linear peptides such as gramicidin A. It has been shown that gramicidin A dimerizes in a solvent with a medium dielectric constant like dioxane™. The gramicidin is known to accelerate the self diffusion efflux rate of alkali metal ions of phospho lipid vesicles, cardiolipin vesicles, electroplax membranes and bilayers. The strongest evidence for channel mechanism is the discrete step changes in conductance observed for membranes from glycerol monooleate or lecithin in n-decane in the presence of gramicidin A® and 1 M NaCl or KCl. These conductance changes are quite large and are dependent on electrolyte concentration and applied potential. The net transfer of charge per conducting channel was found to be as high as 2 x 10” ions sec™'. This suggests that the effective ionic mobility across the membrane is little less than for the inorganic ion in water, and consequently a channel, rather than carrier mecha- nism is indicated. Another kind of evidence which implies a channel mecha- nism, is the high power dependence of conductance on gramicidin concentration. This is consistent with the tendency of gramicidin to self-associate. Eisenman and co-workers™ have designed an experimental approach to dis- tinguish between carrier and channel mechanisms by freezing and melting lipid layers. A carrier requires a liquid like membrane interior through which it can diffuse freely to mediate the conductance. Thus freezing the membrane will result in a decrease in conductance when carrier mechanism is involved. In contrast, when a channel mechanism operates, freezing the membrane will result in essentially no effect on conductance. The intricacies involved in the distinction between these mechanisms are discussed earlier. 4.4.2 Active Transport of Cations A constant expenditure of metabolic energy is required for the transport of alkali cation against its electrochemical potential gradient. Since this processThe Alkali Meta! and Alkaline Earth Cations 97 requires energy it is called active transport of cations. It opposes the passive leakage of Na* and K* toward equilibrium. Just as water pump requires energy for charging the overhead tank, this kind of transport also requires energy for the maintenance of ionic gradient. Hence this mode of transport is popularly called “sodium pump”. The hydrolysis of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and inorganic phosphate is believed to provide the energy source for this process. Large cells such as the giant axon of the squid and easily available human erythrocytes have been used to develop concepts of general applicability. Ghost cells are prepared by exposure of red cells to hypertonic media for a short time, during which hemoglobin and cytoplasmic contents leak out though the tempo- rarily porous membrane and other substances may be allowed to diffuse into the membrane. Restoration of tonicity reseals the membrane. The ability to manipulate the intracellular contents of giant axons of erythro- cyte ghosts has been most useful in transport studies. Gardos® demonstrated that ATP in resealed ghosts could support active transport of cations. Later, Cadwell® and his co-workers showed that ATP introduced into the squid axon could support the efflux of isotopic Na*, Thus, it is clear that the transport function is located in membrane. This transport function presumably hydro- lyses ATP as the energy source for the active transport of cations. The trans- ports of Na* and K* in these tissues are coupled since, under most conditions, the efflux of labeled Na* requires the presence of external K*. The cardiac glycosides, of which ‘ouabain’ is perhaps the most widely used, inhibit the fluxes of both ions simultaneously. Great interest was generated by Skou’s™” observation that the fluxes of both Na* and K* ions are inhibited. Skou has suggested that the enzyme (ATPase) provides a means for the transformation of the metabolic energy into osmotic work. In resealed ghosts, the hydrolysis of intracellular ATP was stimulated by Na* or K* only when the latter were present intracellularly and extracellularly, respectively: 59, Quabain, a highly specific inhibitor of both (Na* + Kt) — ATPase and active transport, is effective only at the extracellular surfaces of red cells and squid axons®', The enzyme is thus clearly vectorial, oriented asymmetrically in the membrane and so organized that the ions which activate it are the ions transported. The enzyme is obtainable in many tissues and occurs at levels which may vary over about 2000-fold. It is most concentrated in the tissues of kidney, brain and the electric organ of the eel, Electrophorus electrophorus. The possibility that the ion-dependent hydrolysis of ATP and the concomi- tant structural changes that are associated with translocation of ion might be observable in a cell-free preparation has led to numerous efforts to purify the enzyme and a great deal of work on its mechanism of action. This has been reviewed extensively*®, The vectorial characteristic of the enzyme alluded to above would suggest a system of allosterically interacting proteins rather than a simple protein”.