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Journal of Inorganic Biochemistry 103 (2009) 8793

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Journal of Inorganic Biochemistry


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Dopamine complexes of iron in the etiology and pathogenesis of Parkinsons disease


Shelly Arreguin, Paul Nelson, Shelby Padway, Matthew Shirazi, Cortlandt Pierpont *
Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215, USA

a r t i c l e

i n f o

a b s t r a c t
Parkinsons disease (PD) is the second most common neurodegenerative disease after Alzheimers. The main pathological hallmark of Parkinsons is the deterioration and death of neurons that produce the neurotransmitter dopamine. Much of the neuronal damage takes place in the substantia nigra, a small region of the midbrain that contains the cell bodies of neurons that produce dopamine. The deterioration and death of dopaminergic neurons are directly associated with misfolding and aggregation of proteins, principally a-synuclein, that are natively unfolded. Present also in the substantia nigra is an unusually high concentration of vestigial iron. Protein misfolding in non-genetic (sporadic) cases of PD has been associated with reactive oxygen species formed as products of O2 reduction by the combination of dopamine and iron. Combinations of Fe3+, dopamine hydrochloride (DAH+Cl), and various ancillary ligands have been studied as a function of pH in aqueous solution to determine the optimum pH for complex formation. With ancillary ligands (L4) derived from nitrilotriacetic acid and ethylenediamine diacetic acid spectral changes are consistent with the formation of L4Fe(DAH+) species that reach a maximum concentration at pH 7.2. With edta as the ancillary ligand, spectral features at pH 7 resemble those of Fe3+ catecholate complexes that contain catecholate ligands bonded through a single oxygen. This demonstrates the ability of the dopamine catechol functionality to penetrate the coordination sphere of even exceptionally stable iron chelates. 2008 Elsevier Inc. All rights reserved.

Article history: Received 14 May 2008 Received in revised form 16 September 2008 Accepted 18 September 2008 Available online 26 September 2008 Keywords: Dopamine Iron Parkinsons disease Electronic spectra Electrochemistry

1. Introduction Iron is an essential component of enzymes involved with normal neuronal function in the brain, but there is increasing evidence that defects in regulation and iron storage contribute to neurodegenerative diseases [1,2]. Parkinsons disease (PD) is the second most common neurodegenerative disease after Alzheimers [3,4]. Parkinsons aficts roughly 1 million people in the US and 4 million people worldwide. These gures are expected to double by 2040 as the worlds elderly population is expected to grow. Indeed, neurodegenerative disorders, including Alzheimers and amyotropic lateral sclerosis, are expected to exceed cancer as the leading cause of death. Contrary to popular perception, Parkinsons is not entirely a disease of the aged. While roughly half of the Parkinsons patients are over 55 years of age, a signicant number acquire the disease at a much earlier age [3]. Origins of the disease have been traced to a genetic predisposition that appears responsible for 510% of all cases, and, particularly those of early onset. Cases that lack an inherited genetic connection, sporadic cases, are far more common and typically occur later in life. One of the main pathological hallmarks of Parkinsons is the deterioration and death of neurons that produce the neurotransmitter dopamine (DA). Much of the
* Corresponding author. Tel.: +1 303 492 8420; fax: +1 303 492 5894. E-mail address: pierpont@colorado.edu (C. Pierpont). 0162-0134/$ - see front matter 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2008.09.007

neuronal damage takes place in the substantia nigra (SN), a small region of the midbrain that contains the cell bodies of neurons that produce dopamine. The axons of those neurons extend into the striatum, a section of the brain associated with motor function. The death of dopaminergic neurons is directly associated with misfolding and brilar aggregation of proteins, principally a-synuclein, that are natively unfolded [5,6]. It is signicant that also present in the SN is an unusually high concentration of vestigial iron. Iron concentrations in the SN of normal subjects are higher than concentrations in the liver, averaging to 18.5 mg/100 g of wet tissue [7,8]. Most of the iron is bound to neuromelanin (NM), a cysteinyl-dopamine-derived polymeric material that is known to sequester metal ions in the SN [9,10]. EPR, IR, and extended X-ray ne-structure absorption studies have shown that the iron is octahedral high-spin Fe(III) coordinated by the phenolic oxygens of NM dopamine functionalities [1114]. Mssbauer spectra indicate that the NM-bound iron is associated with a ferritinlike oxyhydroxide cluster of Fe(III) [1517]. NM has been proposed to regulate the concentrations of metal ions, including iron, and it is known that NM concentrations drop with the onset of PD with a consequent loss in regulation [18]. Protein misfolding in sporadic cases of PD has been associated with oxygen radicals formed as products of O2 reduction by the irondopamine combination [2,19]. It has long been known that catecholate ligands have a high afnity for complexation with

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Fe(III) and this carries over to the catechol functionality of dopamine [2022]. Potentiometric speciation studies on Fe(III)dopamine mixtures in aqueous solution have demonstrated the formation of mono, bis and tris complexes [23]. The key to understanding sporadic forms of PD may lie in the relationship between irondopamine coordination chemistry, oxidative stress associated with the formation of reactive oxygen species, and protein oxidation that leads to aggregation. In this report we describe studies on dopamine coordination with iron(III) in aqueous solutions containing additional multidentate ancillary ligands. Ancillary ligands used include the anions of nitrilotriacetic acid (nta), ethylenediaminediacetic acid (edda), and ethylenediaminetetraacetic acid (edta). They all contain a mix of amine nitrogen and carboxylate oxygen donors to model potential protein binding sites. The edta ligand was chosen to investigate DA addition to a ferric complex containing a particularly stable coordination environment for the metal.

100 mL of water to give a colorless solution of pH 3.3. The pH of the solution was increased slowly, under N2, by the addition of aqueous NaOH. 2.1.5. [Cu(DAH+)2] Cu(CH3CO2)2 H2O (0.200 g, 1.0 mmol), Na3(nta) (0.275 g, 1.0 mmol), and DAH+Cl (0.190 g, 1.0 mmol) were dissolved in 100 mL of water to give a pale blue solution of pH 6.3. The pH of the solution was increased slowly, under N2, by the addition of aqueous NaOH. Electronic spectra were recorded at intervals during the titration. 2.2. Physical measurements Electronic spectra were recorded on a Perkin Elmer Lambda 19 UVvisNIR spectrophotometer in aqueous solution. A linear correlation between absorbance and concentration data was determined for absorbance values between 0.1 and 4.0 using a standard dye. Cyclic voltammetry measurements were carried out in aqueous solutions with a BASi-Epsilon using a conventional electrochemical cell with Pt wire working and reference electrodes. The reference electrode was Ag/AgCl (1.0 M KCl) (0.222 V versus SHE), and 0.1 M sodium triuoroacetate was used as the supporting electrolyte. The electrochemical potentials of dopamine at pH 7 (Ea = 0.42 V) and pH 10 (Ea = 0.18 V) were used as an additional reference to convert potentials to NHE [24,25]. pH measurements were made with a Corning 320 digital pH meter. 3. Results The general approach followed in this investigation involved monitoring spectral changes that take place upon the addition of base to aqueous solutions containing Fe3+, dopamine hydrochloride (DAH+Cl), and an ancillary ligand (Ln) [26]. Studies on catecholate complexes of Fe3+ that may model the active site of catecholate dioxygenase enzymes have been carried out with a wide variety of tetradentate ancillary ligands, L4FeIII(Cat) [27]. In some cases these complexes have been observed to activate molecular oxygen with oxidation of the bound catecholate in reactions that have been described as functional models. All of the L4FeIII(Cat) complexes used in these studies have as a common unique spectral feature two transitions in the visible region that vary slightly in position with changes in catecholate substituents and ancillary donor groups [28]. Spectra of these complexes typically consist of a strong broad absorption in the 600 nm region and a weaker band near 400 nm. 3.1. (nta)Fe(DA) The aqueous solution formed initially by combining H3nta, DAH+Cl, and Fe3+ with concentrations of 1 103 M of each species gave an initial pH value of 2.4. The solution was nearly colorless and exhibited no strong spectral features in the range between 350 and 1000 nm (Fig. 1). Solution pH was increased with addition of an aqueous NaOH solution. At pH 4.2 the solution acquired a faint blue color, and by pH 7 the solution was dark blue due to absorptions at 390 (e 2.03 103 M1 cm1) and 605 nm (2.72 103 M1 cm1). Maximum intensity for these bands appeared at pH 7.2. Above pH 7.2 band intensities decreased to pH 8.6 (Fig. 2), and then increased again to give a similar two-band spectrum that maximized in intensity at pH 9.1. At this pH the bands appeared at 570 and 385 nm. Upon increasing pH further to 10.2 the two-band prole collapsed to give a single broad band at 500 nm (e 3.6 103 M1 cm1) and a dark purple color for the solution. Spectra were recorded on solutions prepared in air and under a N2 atmosphere with identical results. However, solutions

2. Experimental 2.1. Preparation of compounds 2.1.1. Starting materials Fe(NO3)3 9H2O (Mallinckrodt), dopamine hydrochloride (3hydroxytyramine hydrochloride, Acros Organics), trisodium nitrilotriacetate monohydrate (Aldrich), ethylenediamine-N,N0 -diacetic acid (Aldrich), disodium ethylenediaminetetraacetate (J. T. Baker), and tetraethylammonium bromide (Aldrich) were analytical reagent grade and were used as received. Deionized water was used through out the experiments. 2.1.2. (NEt4)[Fe(nta)(DAH+)] 2H2O Procedure 1. Fe(NO3)3 9H2O (0.404 g, 1.0 mmol), Na3(nta) (0.275 g, 1.0 mmol), and (Et4N)Br (0.210 g, 1.0 mmol) were combined in 30 mL of 50% methanol and the pH was adjusted to $7.0. This resulted in the formation of a brown precipitate. To this mixture dopamine hydrochloride (DAH+Cl) (0.190 g, 1.0 mmol) dissolved in 20 mL methanol was added dropwise over the period of several minutes. As the DAH+Cl solution was added the pH was observed to drop, the brown precipitate dissolved, and the solution became dark blue-black in color. The pH of the solution was adjusted to $7.0 with the addition of NaOH, under N2, and a black precipitate of (NEt4)[Fe(nta)(DAH+)] separated from the solution. The dark blue-black precipitate was isolated by ltration, dissolved in a minimal quantity of water and recrystallized by the addition of ethanol to the aqueous solution. A dark blue microcrystalline product was obtained in 61% yield and dried under vacuum. Anal. Found: C, 46.8; H, 7.14; N, 7.31. Calc. for FeC22H39N3O10 (fw = 560.8): C, 47.1; H, 7.0; N, 7.5. Procedure 2. Fe(NO3)3 9H2O (0.404 g, 1.0 mmol), Na3(nta) (0.275 g, 1.0 mmol), (Et4 N)Br (0.210 g, 1.0 mmol), and DAH+Cl (0.190 g, 1.0 mmol) were dissolved in 100 mL of water to give a pale yellow solution of pH 2.4. The pH of the solution was increased slowly, under N2, by the addition of aqueous NaOH. Electronic spectra were recorded at intervals during the titration. 2.1.3. [Fe(edda)(DAH+)] Fe(NO3)3 9H2O (0.404 g, 1.0 mmol), edda (0.176 g, 1.0 mmol), and DAH+Cl (0.190 g, 1.0 mmol) were dissolved in 100 mL of water to give a nearly colorless solution of pH 2.8. The pH of the solution was increased slowly, under N2, by the addition of aqueous NaOH. 2.1.4. Na[Fe(Hedta)(DAH+)] Fe(NO3)3 9H2O (0.404 g, 1.0 mmol), Na2(edta) (0.372 g, 1.0 mmol), and DAH+Cl (0.190 g, 1.0 mmol) were dissolved in

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Fig. 1. Electronic spectra of an aqueous solution containing H3nta, DAH+Cl, and Fe3+, each of concentration 1.0 103 M, with pH adjusted by the addition of NaOH solution.

dopaminochrome or other molecular products that may result from intramolecular condensation reactions of oxidized dopamine. The black material formed at higher pH values upon aerobic oxidation of either the iron complexes, or dopamine alone, appears from its insolubility to be polymeric. A synthetic route to [(nta)Fe(DAH+)] was devised by rst preparing a solution of [(nta)Fe(H2O)2] at pH 7. The combination of stoichiometric equivalents of Fe3+ and Na3(nta) in water with adjustment of the pH to 7.0 gave a brown precipitate that is, presumably, an oxo-hydroxo polymer of (nta)Fe. We noted at the outset of this investigation that the sodium salt of [(nta)Fe(DAH+)] was insoluble in all common solvents other than water. Consequently, synthesis of the dopamine complex was carried out in a 50% methanol/water solution containing a stoichiometric equivalent of dopamine hydrochloride and (NEt4)Br to provide a counter cation for crystallization. The pH of the solution was adjusted to pH 7.0 and a dark blue product was isolated by ltration. Samples of (NEt4)[(nta)Fe(DAH+)] obtained following this procedure are observed to have a low solubility, even in water, and are insoluble in all other common solvents. The electronic spectrum obtained for the solid and for a dilute aqueous solution was identical with the pH 7.2 spectrum obtained for the Fe3+:nta:DAH+Cl mixture of 1:1:1 stoichiometry in aqueous solution. While this procedure is a useful route to the [(nta)Fe(DAH+)] anion, its low solubility makes the approach of using the stoichiometric mixture of Fe3+, nta, and DAH+ most useful for investigating the pH-dependent properties of the complex in solution. 3.2. (edde)Fe(DA) The procedure described above for [(nta)Fe(DAH+)] was carried out using ethylenediamine diacetic acid (H2edda) in place of H3nta. The interest in this procedure involved potential isolation of [(edda)Fe(DAH+)] as a neutral complex product, and the opportunity to study spectral pH dependence and band shifts for a tetradentate N2O2 chelating ligand. The result of this study closely followed the observations for nta. The Fe3+:H2edda:DAH+Cl mixture of 1:1:1 stoichiometry was initially strongly acidic and nearly colorless. Adjustment of pH to neutrality gave a dark blue solution with the two-band spectrum shown in Fig. 3. Intensity of this spectrum maximized at pH 7.1 with bands at 410 and 615 nm. Further addition of base led to a collapse in this spectrum with the appearance

Fig. 2. pH-dependent spectral changes for the solution described in Fig. 1 in the basic pH range.

in the higher pH range, above 8, were observed to react over the period of several minutes in air to produce a black precipitate. Under N2 the solutions were stable, even at higher pH, and the spectral changes recorded in Figs. 1 and 2 could be reproduced by the addition of aqueous HNO3 to purple solutions at pH 10.5. It was of interest to see if Fe3+ or Fe2+ was released upon formation of the black precipitate in the higher pH range in the presence of air. Spot tests for these metal ions revealed that most of the iron was contained in the solution over the precipitate as Fe3+, although more quantitative experiments will be required to determine that all of the initial iron is contained in the solution. We view the appearance of the two-band spectra as an indication that titration from the initial acidic pH leads to formation of [(nta)Fe(DAH+)] to pH 7.2. Above 7.2 additional base leads to deprotonation of the amine nitrogen of the dopamine ligand leading to the [(nta)Fe(DA)]2 dianion at pH 9.1. The structures of these complex anions would resemble the [(nta)Fe(3,5-DBCat)]2 ion described by Que and coworkers [27]. In this case the two-band spectrum appears with transitions at 408 and 622 nm in CH3CN [28]. In these experiments we failed to see evidence for the formation of

Fig. 3. Electronic spectra of an aqueous solution containing H2edda, DAH+Cl, and Fe3+, each of concentration 1.0 103 M, at pH values that gave maximum band intensity for [(edda)Fe(DAH+)] (pH 7.1) and [(edda)Fe(DA)] (pH 9.1).

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of a new, but similar, spectrum at pH 9.1 which we believe to be the spectrum of the [(edda)Fe(DA)] anion with the dopamine ligand fully deprotonated. Spectral bands for this product are also shown in Fig. 3. As with nta, at higher pH the solution became more air sensitive, leading to formation of a dark black-brown solid. Chemical analytical data indicated that [(edda)Fe(DAH+)] could be formed by using a rational synthetic procedure, but the dark blue product obtained from aqueous solution was of low solubility in all solvents. 3.3. (edta)Fe(DA) Titrations carried out on aqueous solutions containing 1.0 103 M concentrations of Fe3+:Na2H2edta:DAH+Cl gave quite different spectral results. First, the appearance of a charge transfer band indicating coordination of the dopamine catechol with Fe3+ did not appear until a pH value of 6.7 (Fig. 4). Upon increasing pH the band grew in intensity and shifted slightly to higher energy as the solution became dark purple. It appeared with maximum intensity at pH 9.3 with kmax at 510 nm (3.6 103 M1 cm1). This result is clearly different from the spectra obtained with nta and edda which indicated a chelated monocatecholate structure. Complexes of phenoxo ligands and monodentate catecholate ligands bound to Fe3+ typically have bands in this location with similar intensity [29,30]. We speculate that the dopamine ligand is able to displace one of the edta carboxylate arms, bonding with the iron through one oxygen as a phenoxo ligand (I). The remaining DA oxygen may be linked to the displaced carboxylate through a hydrogen bond. A similar interaction was observed between tetrachlorocatecholate ligands of Fe(OPPh3)3(HCl4Cat)(Cl4Cat) [31].
O O O N N O O NH3+

Fig. 4. Electronic spectra of an aqueous solution containing Na2H2edta, DAH+Cl, and Fe3+, each of concentration 1.0 103 M, with pH adjusted by the addition of NaOH solution.

in oxidation potential carries over to pH 10 [24,25]. We have duplicated the electrochemical characterization on dopamine hydrochloride at pH values of 7.0 and 10.0 and used the reported oxidation potentials as a reference in converting the electrochemical events observed for the iron complexes from the Ag/AgCl reference potential to the NHE reference. 3.4.1. [(nta)Fe(DAH+)] CV scans were recorded on aqueous solutions containing [(nta)Fe(DAH+)], prepared by combining H3nta, Fe3+, and DAH+ in equal concentrations and adjusting pH by addition of base. Over the full scan range between +1.0 and 1.0 (NHE) at pH 7.2, the pH value at which all three components are associated as [(nta)Fe(DAH+)] from spectral measurements, the CV consists of an irreversible oxidation at 0.77 V, an irreversible reduction at 0.55 V that only appears after scanning positively past the oxidation, and coupled oxidation and reduction peaks centered at +0.35 V and separated by 82 mV (Fig. 5). Scans of 400 mV centered about 0.00 V were used to provide clear resolution of this quasireversible couple that we assign as the SQ/Cat couple for the coordinated dopamine ligand at +0.35 V (NHE). This agrees with the value of +0.36 V reported for the dopamine ligand of [Ru(edta) (DA)]3 at pH 7.0 [25]. The pH of the solution was increased to 9.3 and then to 10.3; CV scans were recorded over the full range.

Fe
O O O H

(I)

As in the case of the complexes prepared with nta and edda, spectral changes may be reversed by adding dilute HNO3 to the solution at pH 10. Above pH 10 the solution became sensitive to air, decomposing to give the black precipitate observed with other ancillary ligands. The spectral changes shown in Fig. 4 were observed for a solution in air; identical results were obtained for a titration carried out under a N2 atmosphere. 3.4. Electrochemical characterization on [(nta)Fe(DAH+)] and [(edta)Fe(DAH+)] The observation that air sensitivity of the irondopamine complexes in aqueous solution increases with increasing pH may appear as a negative shift in the electrochemical potential associated with the catecholate/semiquinonate oxidation of the chelated dopamine ligand. Early electrochemical characterization on solutions of dopamine showed a linear dependence for the Cat/SQ oxidation potential over pH values on the acidic side of neutrality [32]. It is also known that basic solutions of dopamine, as is true generally for most catechols, have an extreme oxygen sensitivity, and recent CV studies have shown that the negative shift

Fig. 5. CV on a 1 103 M aqueous solution of [(nta)Fe(DA)]2 at pH 10.3.

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The irreversible oxidation was observed to be shifted negatively to +0.66 V (NHE), but the irreversible reduction appeared essentially unchanged at 0.50 V (NHE). The SQ/Cat couple appeared at +0.13 V at pH 9.3. These observations are similar to the electrochemical properties in DMF solution reported by Que for a series of Fe(L4)(3,5-DBCat) complexes studied as models for the active site of the catechol1,2-dioxygenase enzymes [33]. They show that the dopamine ligand, in its coordination chemistry with iron, has properties in common with other catecholate ligands. With the characterization by Rein et al. [25] on [Ru(edta)(DA)]3, CV scans show that there is no strong negative shift in the SQ/Cat redox potential with increasing pH that might be associated with the dramatic change in oxygen sensitivity for the dopamine complexes over the pH range from 7.0 to 10.0 [25]. The greatest shift is observed for the irreversible oxidation of the coordinated dopamine ligand that appears at the most positive potential. 3.4.2. [(edta)Fe(DAH+)] CV scans recorded over the range 1.0 V (NHE) on aqueous solutions of Na2(H2edta), DAH+, and Fe3+ at pH values of 7.3 and 9.4 produced results that were quite similar to the scans obtained using nta as the ancillary ligand despite the structural difference inferred from the spectral properties. At pH 7.3, the irreversible oxidation appeared at 0.93 V, the irreversible reduction appeared at 0.16 V and the quasi-reversible SQ/Cat couple is centered about 0.38 V. At pH 9.4 the SQ/Cat couple appeared at 0.20 V, shifted negatively upon addition of base. The irreversible oxidation appeared at 0.42 V and the reduction at 0.60 V, again, shifted negatively as observed for [(nta)Fe(DAH+)]. The similarity in the electrochemistry of [(nta)Fe(DAH+)] and [(edta)Fe(DAH+)] was unexpected given the difference in electronic spectra and mode of coordination of the catecholate ring of the dopamine ligand. However, Palaniandavar and coworkers have found that there is little difference in the SQ/Cat couple for [FeL(3,5-DBCat)Cl] and [FeL(3,5-DBCat)] (L = N,N-dimethyl-N-(2hydroxy-3,5-dimethylbenzyl)-N-(pyrid-2-ylmethyl)ethylenediamine) where the catecholate ligand is bound monodentate in the former and chelated in the latter [30]. For both [(nta)Fe(DAH+)] and [(edta)Fe(DAH+)] the shift in the SQ/Cat potential was roughly 0.2 V with the shift in pH leading to deprotonation of the DAH+ amine proton. The shifts in the irreversible oxidation and reduction potentials for [(edta)Fe(DAH+)] were more than 500 mV over the pH range 7.39.4, while for [(nta)Fe(DAH+)] shifts in these potentials were less than 100 mV.
Fig. 6. Electronic spectra of an aqueous solution containing H3nta, DAH+Cl, and Cu2+, each of concentration 1.0 103 M, with pH adjusted by the addition of NaOH solution.

the charge transfer bands observed for the FeDA complexes, but consistent with bands at this energy, and with this intensity, reported for bis-catecholate complexes of Cu2+. In fact, bis-catecholate complexes of Cu2+ are reported to show two bands, one in the 400 nm region with a molar extinction coefcient of 200 250 M1 cm1, and a second in the 650 nm region with e $ 35 M1 cm1 [35]. Since the mole ration of Cu2+ to DAH+ present in solution initially was 1:1, the band at 640 nm in Fig. 6 is probably a superposition of a low-energy band from [Cu(DA)2]2 and a Cu-nta species, in the higher pH range. The results of this titration are consistent with the conclusion that Cu2+ is a weaker Lewis acid than Fe3+, and has a much weaker afnity for the catecholate functionality of dopamine than iron. In the presence of a much higher concentration of iron it is unlikely that a Cu2+DAH+ complex, at physiological pH, contributes significantly to the formation of reactive oxygen species leading to asynuclein oxidation.

5. Discussion Recent nano-imaging experiments using synchrotron-based Xray uorescence has been used to investigate the distribution of iron in dopamine producing neurons with a special resolution of 90 nm [36,37]. A tyrosine hydroxylase inhibitor was used to suppress dopamine synthesis with the observation that concentrations of iron and dopamine are strongly coupled; in the absence of dopamine, cells failed to accumulate iron. At the subcellular level, iron and dopamine were found to co-locate in dopamine neurovesicles typically 200 nm in size. The observation that dopamine and iron form a coordination complex at physiological pH provides strong evidence for the formation of intracellular dopamineiron(III) complexes. The irondopamine complexes formed in solutions containing 1:1 Fe3+ to DAH+ rations appear to closely resemble L4Fe(Cat) species studied in conjunction with interests in the catechol dioxygenase enzymes and the properties of these complexes may carry over to the dopamine analogs. Most signicantly, the ability to reductively activate molecular oxygen may contribute to the formation of reactive oxygen species that are associated with Parkinsons disease. The mechanism may follow the steps described by Que and co-workers with the formation of an iron-bound peroxosemiquinone species (II) [27].

4. Dopamine coordination with Cu2+ Concentrations of copper in the SN are 16 ng/mg of wet tissue, signicantly lower than the iron concentration, but of interest with respect to the potential activity of a copperDA complex in peroxidation chemistry as a pathogenic factor in PD [34]. The aqueous solution formed initially by combining Na3nta, DAH+Cl, and Cu2+ with concentrations of 1 103 M of each species gave an initial pH value of 6.3. The electronic spectrum of this solution, Fig. 6, showed a ligand eld transition for Cu2+ at 650 nm and a second band of lower intensity at 390 nm. The solution was pale bluegreen in color. Upon addition of NaOH solution to increase pH, under an atmosphere of N2 to prevent the formation of dopamine melanin, the band at 650 nm decreased slightly in intensity to pH 8.0 and moved slightly higher in energy to 640 nm. It remained at this intensity level (e $ 75 M1 cm1) as solution pH was increased further. The band at 390 nm moved to 410 nm and increased markedly in intensity to pH of 11.5. The molar extinction for this band at pH 11.5 is 210 M1 cm1, far less intense than

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O Fe
3+

O + O2
2 H+

(O2 ) Fe

3+

O
2 H+

O
2 H+

HO2C

Fe

3+

Fe
HO2C

3+

H2O2

Fe

3+

HO2

O
+

O
+

O HO
Scheme 1.

NH2 +

NH2 O

O N
Scheme 2.

O O O Fe O
(II)

oxygens. Infrared spectra on the material obtained from the reactions described above, under basic aerobic conditions, show a strong broad absorption in the 1600 cm1 region typical of C, N, and O double bonds. Condensation reactions of this type have proven to be a useful synthetic route to iminoquinone ligands [4446]. Natural neuromelanin may form under similar conditions, but with additional condensation reactions involving sulfur-containing amino acids to give a more heterogenous product containing a high sulfur content [11]. 6. Conclusions Iron and dopamine are present in high concentration in dopaminergic neurons of the substantia nigra. pH-dependent studies on dilute aqueous solutions containing equimolar concentrations of ferric iron, dopamine hydrochloride, and multidentate ancillary ligand show that at physiological pH an irondopamine complex is formed. Earlier research carried out on ironcatecholate complexes as models for catechol dioxygenase enzymes provides a mechanism for the formation of reduced oxygen species. Charge for oxygen reduction is provided by the catecholate ring of dopamine which is released as a benzoquinone. The dopamine benzoquinone may undergo condensation with the sulfur atoms of cysteine and the amine functionalities of other dopamine molecules to form neuromelanin, a polymeric neuronal chelating agent that is thought to be associated with the regulation of intracellular iron concentration. Models for sporadic Parkinsons disease suggest an associated loss of iron regulation leading to the formation of reduced oxygen species, a-synuclein oxidation and misfolded protein. Fibrilar aggregates of misfolded protein are the main constituents of Lewy bodies that are ultimately responsible for the deterioration and death of dopaminergic neurons.

Support for a structure of this type comes from structural characterization on complexes of Rh, Ir, and Sb [3840], and for the ironbound peroxysemiquinone intermediate formed during ring cleavage by an extradiol dioxygenase enzyme [41]. The peroxosemiquinone may rearrange with ring opening to give a dicarboxylic acid as in the enzymatic reaction (Scheme 1), or, in a protic environment, dissociate to give either semiquinone and superoxide in a one-electron transfer step or benzoquinone and hydrogen peroxide in a two-electron transfer. Reactions of this type have been observed for oxygenated catecholate complexes of copper [42]. Catecholates and the catecholate form of dopamine clearly have the potential to reduce O2 as evidenced by the consequence of exposing a basic catechol solution to air. Brown-black synthetic dopamine melanin is commonly prepared in a basic solution containing Cu2+ [43]. The black polymeric product that we have obtained as the aerobic oxidation product from basic dopamine solutions containing Fe3+ appears to be the same material. From the products obtained by treating basic catecholate solutions containing transition metal ions with amines, we suggest that the synthetic melanin is formed by Schiff base condensation between a benzoquinone (or semiquinonate) oxygen and an amine nitrogen (Scheme 2) with further polymeric extension through other ring

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References
[1] L. Zecca, M.B.H. Youdim, P. Riederer, J.R. Connor, R.R. Crichton, Nat. Rev. Neurosci. 5 (2004) 863873. [2] R.J. Ward, R.R. Crichton, in: A. Sigel, H. Sigel, R.K.O. Sigel (Eds.), Neurodegenerative Diseases and Metal Ions, Metal Ions in Life Sciences, vol. 1, Wiley, Chichester, England, 2006, pp. 227279. [3] M. Vila, S. Przedborski, Nat. Med. 10 (2004) S58S62. [4] H.J. Federoff, R.E. Burke, S. Fahn, G. Fiskum (Eds.), Parkinsons Disease, the Life Cycle of the Dopamine Neuron, Ann. N.Y. Acad. Sci., (2003). [5] V.N. Uversky, J. Neurochem. 103 (2007) 1737. [6] V.N. Uversky, in: V.N. Uversky, A.L. Fink (Eds.), Protein Misfolding, Aggregation and Conformational Diseases, Part B: Molecular Mechanisms of Conformational Diseases, Springer, New York, 2007, pp. 61110. [7] M. Gerlach, K.L. Double, M.E. Gtz, M.B.H. Youdim, P. Riederer, in: A. Sigel, H. Sigel, R.K.O. Sigel (Eds.), Neurodegenerative Diseases and Metal Ions, Metal Ions in Life Sciences, vol. 1, Wiley, Chichester, England, 2006, pp. 125149. [8] M.E. Gtz, K. Double, M. Gerlach, M.B.H. Youdim, P. Riederer, Ann. N. Y. Acad. Sci. 1012 (2004) 193208. [9] L. Zecca, R. Fariello, P. Riederer, D. Sulzer, A. Gatti, D. Tampellini, FEBS Lett. 510 (2002) 216220. [10] H. Fedorow, F. Tribl, G. Halliday, M. Gerlach, P. Riederer, K.L. Double, Prog. Neurobiol. 75 (2005) 109124. [11] L. Zecca, D. Tampellini, M. Gerlach, P. Riederer, R.G. Fariello, D. Sulzer, J. Clin. Pathol: Mol. Pa. 54 (2001) 414418. [12] T. Shima, T. Sarna, H. Swartz, A. Stroppolo, R. Gerbasi, L. Zecca, Free Radical Biol. Med. 23 (1997) 110119. [13] M.G. Bridelli, D. Tampellini, L. Zecca, FEBS Lett. 457 (1999) 1822. [14] A.J. Kropf, B.A. Bunker, M. Eisner, S.C. Moss, L. Zecca, A. Stroppolo, P.R. Crippa, Biophys. J. 75 (1998) 31353142. [15] L. Zecca, L. Gallorini, V. Schnemann, A.X. Trautwein, M. Gerlach, P. Riederer, P. Vezzoni, D. Tampellini, J. Neurochem. 76 (2001) 17661773. [16] M. Gerlach, A.X. Trautwein, L. Zecca, M.B.H. Youdim, P. Reiderer, J. Neurochem. 65 (1995) 923926. [17] M. Zareba, A. Bober, W. Korytowski, L. Zecca, T. Sarna, Biochim. Biophys. Acta 1138 (1995) 610. [18] B.A. Faucheux, M.-E. Martin, C. Beaumont, J.-J. Hauw, Y. Agid, E.C. Hirsch, J. Neurochem. 86 (2003) 11421148. [19] W. Linert, G.N.L. Jameson, R.F. Jameson, K.A. Jellinger, in: A. Sigel, H. Sigel, R.K.O. Sigel (Eds.), Neurodegenerative Diseases and Metal Ions, Metal Ions in Life Sciences, vol. 1, Wiley, Chichester, England, 2006, pp. 281320. [20] A. Avdeef, S.R. Sofen, T.L. Bregante, K.N. Raymond, J. Am. Chem. Soc. 100 (1978) 53625370.

[21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34]

[35] [36] [37] [38] [39] [40] [41] [42] [43]

[44] [45] [46]

B. Howlin, A.R. Mohd-Nor, J. Silver, Inorg. Chim. Acta 91 (1984) 153160. C. Gerard, H. Chehhal, R.P. Hugel, Polyhedron 13 (1994) 541547. L.K. Charkoudian, K.J. Franz, Inorg. Chem. 45 (2006) 36573664. X.-L. Wen, Y.-H. Jia, Z.-L. Liu, Talanta 50 (1999) 10271033. F.N. Rein, R.C. Rocha, H.E. Toma, J. Inorg. Biochem. 85 (2001) 155166. The notation DAH+ will be used to refer to the dopamine anion with the amine nitrogen protonated. M. Costas, M.P. Mehn, M.P. Jensen, L. Que Jr., Chem. Rev. 104 (2004) 939986. D.D. Cox, S.J. Benkovic, L.M. Bloom, F.C. Bradley, M.J. Nelson, L. Que Jr., D.E. Wallick, J. Am. Chem. Soc. 110 (1988) 20262032. M. Scarpellini, A. Neves, A.J. Bortoluzzi, I. Vencato, V. Drago, W.A. Ortiz, C. Zucco, J. Chem. Soc. Dalton Trans. (2001) 26162623. R. Mayilmurugan, E. Suresh, M. Palaniandavar, Inorg. Chem. 46 (2007) 6038 6049. D. Zirong, S. Bhattacharya, J.K. McCusker, P.M. Hagen, D.N. Hendrickson, C.G. Pierpont, Inorg. Chem. 31 (1992) 870877. A.W. Sternson, R. McCreery, B. Feinberg, R.N. Adams, Electroanal. Chem. Int. Electrochem. 46 (1973) 313321. L. Que Jr., R.C. Kolanczyk, L.S. White, J. Am. Chem. Soc. 109 (1987) 53735380. L. Zecca, A. Stroppolo, A. Gatti, D. Tampellini, M. Toscani, M. Gallorini, G. Giaveri, P. Arosio, P. Santambrogio, R.G. Fariello, E. Karatekin, M.H. Kleinman, N. Turro, O. Hornykiewicz, F.A. Zucca, Proc. Natl. Acad. Sci. USA 101 (2004) 98439848. M.J. Sever, J.J. Wilker, Dalton Trans. (2004) 10611072. A. Carmona, G. Devs, R. Ortega, Anal. Bioanal. Chem. 390 (2008) 15851594. R. Ortega, P. Cloetens, G. Devs, A. Carmona, S. Bohic, PloS ONE 2 (2007) e925. S. Dutta, S.-M. Peng, S. Bhattacharya, Inorg. Chem. 39 (2000) 22312234. P. Barbaro, C. Bianchini, K. Lynn, C. Mealli, A. Meli, F. Vizza, Inorg. Chim. Acta 198200 (1992) 3156. V.K. Cherkasov, G.A. Abakumov, E.V. Grunova, A.I. Poddelsky, G.K. Fukin, E.V. Baranov, Y.V. Kurskii, L.G. Abakumova, Chem. Eur. J. 12 (2006) 39163927. E.G. Kovaleva, J.D. Lipscomb, Science 316 (2007) 453457. G. Speier, E. Kelemen, Z. Tyeklar, P. Toth, S. Tisza, A. Rockenbauer, A.M. Whalen, N. Alkire, C.G. Pierpont, Inorg. Chem. 40 (2001) 56535659. J. Li, C. Scheller, E. Koutsilieri, F. Grifths, P.M. Beart, L.D. Mercer, G. Halliday, E. Kettle, D. Rowe, P. Riederer, M. Gerlach, M. Rodriguez, K.L. Double, J. Neurochem. 95 (2005) 599608. G. Speier, J. Csihony, A.M. Whalen, C.G. Pierpont, Inorg. Chem. 35 (1996) 3519 3524. K.J. Blackmore, Z.W. Ziller, A.F. Heyduk, Inorg. Chem. 44 (2005) 55595561. P. Chaudhuri, C.N. Verani, E. Bill, E. Bothe, T. Weyhermller, K. Wieghardt, J. Am. Chem. Soc. 123 (2001) 22132221.