Free Radical Damage and its Control

UK

Chapter 1

Chemistry of iron and copper in radical reactions

W.H. KOPPENOL,     Departments of Chemistry and Biochemistry, and Biodynamics Institute, Louisiana State University, Baton Rouge, LA 70803, USA

Abbreviations

adp adenosinediphosphate

amp adenosinemonophosphate

atp adenosinetriphosphate

dtpa diethylenetriamine-N, N, N′, N″, N″-pentaacetate

edda ethylenediamine-N, N′-diacetate

edta ethylenediamine-N, N, N′ N′-tetraacetate

gtp guanosine triphosphate

hedta (N-hydroxyethyl)ethylenediamine-N, N″, N″-triacetate

nta nitrilotriacetate

phen 1,10-phenanthroline

PQ·+ paraquat radical

utp uridine-5′-triphosphate

1 Introduction

In a comparison of several elements it was shown by George [1] in 1965 that oxygen is unique because its reduction by organic compounds is favourable and because the reaction product, water, is not toxic. As such oxygen is the best element on which to base life. Yet oxygen also plays an important role in free radical biology in that it is also essential in the initiation of, and greatly amplifies, damage to biomolecules. Oxyradicals have been implicated in numerous diseases and disorders.

Iron and copper catalyse the formation of oxyradicals. Three reactions are relevant in this context: (1) Autoxidation of metal complexes may yield the superoxide radical which by itself is not very reactive, but is a precursor of more reactive radical species. (2) The one-electron reduction of hydrogen peroxide – the Fenton reaction – results in hydroxyl radicals via a higher oxidation state of iron [2]. (3) A similar reaction with organic peroxides leads to alkoxyl radicals, although a recent report alleges that hydroxyl radicals are also formed [3]. There is a fourth radical, the formation of which does not require mediation by a metal complex. This is the alkyldioxyl radical, ROO•, which is formed at a nearly diffusion-controlled rate from an alkyl radical and dioxygen. As shown in Table 1, the hydroxyl, the alkoxyl and the alkyldioxyl radical are oxidizing species. For a quantitative description one needs to know rate constants for the formation of these radicals and for their subsequent reactions in order to determine which reaction will dominate under physiological conditions. This requires inter alia knowledge of the precise chemical composition of the cell; we are still a long way from this goal.

TABLE 1

Reduction potentials of oxyradicalsa

aData for H- and O-containing radicals were taken from a recent compilation [4]. The values for the carbon containing radicals are estimates derived from bond-dissociation enthalpies [5]. The value for E°′(ROO•/ROOH) has recently been confirmed experimentally [6].

In principle, there are three mechanism for damage: a single reaction, a chain reaction, or a branching mechanism. A single reaction is not likely to lead to extensive damage. However, when a radical like the hydroxyl radical reacts with a biomolecule, another radical is created. A chain reaction ensues that stops only when a radical reacts with another radical or with a transition-metal ion. For extensive damage to occur it might be necessary that branching occurs. For instance, superoxide production may lead to lipid peroxidation; alkenals formed as products of that process are substrates for xanthine oxidase [7]; more superoxide is produced, and a new chain reaction is started. Similarly, during ischaemia atp is converted to hypoxanthine [8]. Any iron that was tightly bound to atp is now bound elsewhere, possibly in a more open complex. Since rate constants increase with a decrease in coordination number (see below), such an iron complex is likely to be more reactive.

Given the concentration of reaction sites in vivo and the magnitude of the relevant rate constants it is not possible to intercept effectively the hydroxyl, alkoxyl or alkyldioxyl radicals [9]. For less reactive compounds, such as hydrogen peroxide and superoxide, nature has developed enzymes to dispose of them. The strategy adopted by nature is threefold: (1) interception with superoxide dismutase and proteins such as catalase and glutathione peroxidase that react with hydrogen peroxide and small alkyl hydroperoxides; (2) repair of water-soluble biomolecules with glutathione, and (3) inhibition of lipid peroxidation with vitamin E, which might [10], or might not [11], be regenerated by vitamin C. Part of the defence mechanism may be that radicals are interconverted to superoxide via the glutathione radical; superoxide, acting as a radical sink, is subsequently scavenged by superoxide dismutase [12]. The overall energetics of these reactions are extremely favourable [13].

Excess amounts of transition metals, in particular iron and copper, are toxic. For instance, it has recently been suggested that excess iron plays a role in heart disease [14,15]. Transition-metal ions are sequestered by proteins: iron in ferritin and transferrins [16], and copper in caeruloplasmin. However, a small concentration of low-molecular-weight complexes is likely to be present at all times because of transfer of metals from storage proteins to metalloproteins, and from the turnover of these proteins. Under oxidative stress this pool of iron is increased due to reductive mobilisation and destruction of ferritin [17–25]. Desferrioxamine, being a stronger complexing agent than the naturally occurring ligands, chelates iron and prevents oxidative injury in hepatocytes [26]. The precise nature of the low-molecular-weight complexes present in vivo is not known with certainty. Evidence has been presented that iron bound to atp, amp [27], gtp [28] and possibly citrate [29] may be present in tissues in the micromolar range [21]. No information is available on copper.

Some metal- (especially copper) complexes catalyse the dismutation of superoxide at rates that compare favourably with catalysis by superoxide dismutase. One could therefore argue that the presence of such complexes in vivo might be beneficial. There are, however, additional considerations: (1) such metal complexes may also reduce hydrogen peroxide, which could result in the formation of hydroxyl radicals, and (2) it is extremely likely that the metal will be displaced from its ligands (even when those ligands are present in excess), and becomes bound to a biomolecule, thereby becoming less active as a superoxide dismutase mimic. As an example, copper binds well to DNA and catalyses the formation of hydroxyl radicals in the presence of hydrogen peroxide and ascorbate [30].

Both the reduction of superoxide and that of hydrogen peroxide appear to be inner-sphere reactions; that is, a ligand of the metal ion has to be replaced by superoxide or hydrogen peroxide for the reaction to take place. For superoxide this involves overlap between a metal d-orbital and its own accessible π* orbital. Reduction of hydrogen peroxide involves electron transfer to an empty σ* orbital which is not very accessible [31]. Thus, reductions of hydrogen peroxide are generally slower than those of superoxide. The reductions of alkylhydroperoxides are even slower, due to steric hindrance [32,33].

This review is concerned with the quantitative aspects of metal-catalysed oxyradical reactions. As such one will find discussions of structures of metal complexes, rate constants and reduction potentials, not unlike our review of 1985 [34]. Two areas related to the role of transition metals in radical chemistry and biology have been reviewed recently; these are the metal-ion-catalysed oxidation of proteins [35] and the role of iron in oxygen-mediated toxicities [36]. These topics will not be discussed in detail in this review. Related to this work is a review on the role of transition metals in autoxidation reactions [37]. Additional information can be obtained from Afanas’ev’s two volumes on superoxide [38,39]. This subject is also treated in a more general and less quantitative manner by Halliwell and Gutteridge [40].

2 Autoxidation reactions

2.1 Oxygen

It is well known that oxygen does not react directly with organic molecules because of spin restrictions: ground-state oxygen is a triplet molecule, and most organic molecules are in the singlet state (see ref. , which because of its extremely short lifetime is biologically not relevant, the electrons move in the same plane with paired spins, while in the ¹Δg state both oxygen with radical species.

Fig. 1 Spin-orbital diagram of the different states of oxygen. The x and y refer to the two perpendicular π antibonding orbitals of oxygen. On the right this diagram depicts the real wave-functions for the lowest electronic states. From ref. [43].

2.2 Thermodynamics

At low pH the iron(II) ion is stable with respect to oxidation, due to the high value of the reduction potential of the Fe³+/Fe²+ couple, 0.77 V versus the normal hydrogen electrode. Above pH 2.1, Fe(III), but not Fe(II), hydrolyses, which results in a reduction potential that decreases with 59 mV per pH unit to a value of 0.48 V at pH 7. This applies only to very dilute solutions, since iron(III) hydroxide precipitates above pH 3. Complexation by aminopolycarboxylates, such as edta, which provide mainly oxygen as donor atoms, also reduces the reduction potential, generally to a value near 0.1 V [34]. The standard reduction potential of the oxygen/superoxide couple is −0.33 V (see Fig. 2), independent of pH [44,45]. Although in such an instance the one-electron reduction of oxygen by such metal complexes is thermodynamically unfavourable by approximately 10 kcal/mol, the reaction proceeds because the product, superoxide, disappears by disproportionation. In contrast, reduction by two electrons to hydrogen peroxide is favourable: the Gibbs energy change is −8.1 kcal per two moles of Fe(II) edta, as calculated from E°′(O2/H2O2) = 0.305 V at pH 7 [44] and the reduction potential of the Fe(III)-/Fe(II)-edta couple of 0.12 V [46].

Fig. 2 Oxidation state diagram of oxygen at pH 7 at otherwise standard conditions (1 molal concentrations, 1 atm for gases). The x-axis gives the oxidation state, the y-axis the product of reduction potential and oxidation state. As such the slope represents the reduction potential. A compound that lies above a line joining its neighbours is unstable with respect to disproportionation, as is the case for superoxide and hydrogen peroxide. The line from hydrogen peroxide to the middle of the water–hydroxyl line represents the one-electron reduction potential of the couple H2O2/•OH, H2O. Adapted from ref. [4].

It has been suggested that a (dioxygen)iron(II), or perferryl¹ complex, a likely intermediate in the autoxidation of iron(II), could abstract an allylic hydrogen and initiate lipid peroxidation [48]. Such complexes are weak oxidants at best, as has been shown before [49] and, with the exception of iron(II) edta [50], have not been observed. Constraints on the reduction potential E°′(HLFeII O2/HLFeIII, H2O2) come from the following thermodynamic cycle and considerations. If such a complex were to be an initiator of oxyradical damage, one might expect that approximately 1% of the low-molecular-weight iron(II) be complexed to oxygen at a cellular oxygen tension of, say, 0.01 atm. This requires a standard Gibbs energy change of 0 kcal/mol. In the following sequence of reactions HL represents a ligand with a covalently bound hydrogen:

(1)

(2)

(3)

(4)

The reduction potential of 0.2 V for Reaction (4) at pH 7 depends very much on the reduction potential of the Fe(III)/Fe(II) couple, Reaction (3). The 0.1 V assumed here for that half-reaction is close to that of various iron–aminopolycarboxylate complexes. The uncertainty in our reduction potential for Reaction (4) is estimated at 0.2 V The abstraction of a doubly allylic hydrogen is estimated to require a reduction potential of 0.6 V (see Table 1), which makes the reaction with a (dioxygen)iron(II) complex unfavourable by approximately 10 kcal/mol.

A variation on the (dioxygen)iron(II) complex, an FeIIO2 FeIII intermediate, was proposed by Aust and coworkers as the instigator of oxyradical damage [37,51]. There is no thermodynamic data available that allows one to calculate how oxidising such a complex would be. It is conceivable that an equal mixture of iron(II) and iron(III) compounds imposes a reduction potential on the system that is favourable for catalysis of lipid peroxidation.

Not many reduction potentials are known for copper complexes. That of the Cu²+/Cu+ couple is 0.16 V. Since E°(Cu+/Cu°) is 0.52 V, the disproportionation of Cu+ to Cu⁰ and Cu²+ is favourable. This reaction does indeed occur, which makes is impossible to study stable copper(I) solutions. Reduction potentials of copper(II)-/copper(I)-(1,10-phenanthroline)2 and a few derivatives have been calculated from a kinetic analysis of appropriate rate constants: values range from 108 mV for the 5-methyl-1, 10-phenanthroline complex to 219 mV for the complex with a nitro group at the 5 position [52]. Values of 0.17 V and 0.12 V are given by Phillips and Williams [53] for the phenanthroline and bipyridine complexes, respectively. Such complexes can thermodynamically catalyse both the superoxide dismutation and the one-electron reduction of hydrogen peroxide (see below).

2.3 Kinetics and mechanisms

The rate of autoxidation can be calculated if the Gibbs energy of reaction and the rate constant for the reverse reaction, the reduction of the metal complex by superoxide, are known. The reduction potential of the Fe(III)-/Fe(II)–edta complex is 0.12 V at pH 7 [46], and that of the oxygen/superoxide couple is −0.33 V (see above), which results in a Δrxn G°′ of −10.6 kcal/mole at pH 7 for the reduction of Fe(III) edta by superoxide. This reaction proceeds with a rate constant of ∼5 × 10⁶ M−1 s−1 [54,55]. A rate constant of ∼30 M−1 s−1 for the oxidation of iron(II) edta by oxygen at pH 7 is calculated from the Gibbs energy change and the rate constant. It follows from the pH dependence of the reaction of iron(III) edta with superoxide [55] that the autoxidation would be faster at lower pH. Near neutral pH, rate constants of 600 M−1 s−1 [56] and 270 M−1 s−1 [57] have been reported, much faster than the 30 M−1 s−1 estimated above. We observed that the autoxidation of dilute (micromolar) iron(II) edta solutions is first-order in iron(II) complex and in oxygen, with a rate constant of 110 M−1 s−1 at neutral pH, in better agreement with the thermodynamically predicted value (Koppenol and Rush, unpublished).

The first study of the kinetics of autoxidation reactions of a number of iron(II) salts was published in 1901 by McBain [58]. It established that the reaction is first order in oxygen pressure and second order in iron(II). In contrast, a similar study on iron(II)hydrogencarbonate, published in 1907 by Just [59] showed that the reaction is first order in iron(II) and in oxygen. This work is also noteworthy for another reason, for it mentions for the first time the superoxide anion. Most other studies report a second-order dependence in Fe(II) [60–66]. A reaction mechanism was proposed by Weiss [67] in 1935 which covers Just’s observations [59]. It involves first the formation of superoxide, which after protonation oxidises another Fe(II):

(5)

(6)

(7)

These reactions are followed by the reduction of hydrogen peroxide which consumes two more iron(II) ions. According to Reaction (5), the autoxidation of iron would be first-order in iron(II). However, for most other anions the autoxidations are second-order in iron(II), and for that reason this mechanism was criticised by George [62]. He studied the oxidation of iron(II)perchlorate solutions at higher oxygen pressures and showed that this reaction is second-order in iron(II) and first-order in oxygen [62]. His mechanism involves formation of an intermediate dioxygen–iron(II) complex, Reaction (8), followed by rate-limiting reaction with a second iron(II), Reaction (9):

(8)

(9)

being 0.94 V at pH 7 [44]. Such an oxidation would have to be fast to compete with the dismutation reaction.

The oxidation of iron(II) by dioxygen is pH dependent: at pH 7.03 the half-life of this process, 2700s, is ∼10 times greater than that at pH 7.45 [68]. The presence of chelating agents drastically decreases the halflife. In the case of edta the halflife is 10s near neutral pH. No rate constants were reported. The autoxidations of iron(II) aminopolycarboxylates proceed with rate constants of 270, 100, 80 and 7 M−1 s−1 for the ligands edta, hedta, nta and dtpa, respectively [57], and a relationship between the rate constant for the oxidation and the ratio of the stability constants of the iron(III) chelate to that of iron(II) was established. Similar autoxidation rates were obtained by another group [69]. An intermediate iron(II)edta–oxygen complex has been postulated [50]. This complex is believed to become protonated at lower pH to form the hydrodioxyl radical, as proposed for unchelated iron [67], and reacts at higher pH’s with excess iron(II)edta to form hydrogen peroxide. The protein apotransferrin was also shown to increase the rate of autoxidation [70], probably by removing iron(III) from equilibrium. At pH 7.0, phosphate increases the rate of oxygen consumption in iron(II)-solutions, and this process is slowed by dtpa [71]. Recently the curious observation was made that phosphate slows the rate of autoxidation, although no rate constants were given [72]. It can be concluded from this overview that the autoxidation of physiologically relevant iron(II) complexes has not been well characterised, and that conflicting reports exist about the effect of phosphate on the autoxidation reaction.

The reverse reaction, the reduction of iron(III) complexes by superoxide, proceeds with rate constants varying from 1.9 × 10⁶ M−1 s−1 for edta to 7.6 × 10⁵ M−1 s−1 for hedta, to negligible for dtpa [54,55,73]. The same trend as seen for the reaction of iron(II) complexes with hydrogen peroxide (see below) is observed here: The rate constant decreases when the number of ligand atoms provided by the chelating agent increases. Superoxide forms an adduct with iron(II) complexes [54,56]; this complex is also formed from the iron(III) complex with hydrogen peroxide [74]. Recently it was reported that iron(III)citrate undergoes autoreduction [75]. This process is known to be photocatalysed and was described more than 50 years ago [60].

The reduction of oxygen by copper(I) is faster than that of the iron(II) complexes: 5 × 10⁴ M−1 s−1 for CuI(phen)2 [52] and 4 × 10⁴ M−1 s−1 for CuI(histidine)2 [76]. It is this relatively fast autoxidation that limits the usefulness of copper complexes as mimics of superoxide dismutase under conditions of high superoxide concentrations [77]. Copper(II) catalyses the dismutation of superoxide at near diffusion-controlled rates: kcat = 8 × 10⁹ M−1 s−1 [78,79].

3 Fenton reactions

3.1 Introduction

The reaction of iron(II) with hydrogen peroxide is named after H.J.H. Fenton who observed in 1876 [80] that addition of hydrogen peroxide to a mixture of tartaric acid and iron(II) sulfate, followed by addition of base, resulted in a dark purple colour. A full account was published in 1894 [81]. Having first used it as an analytical tool for the assay of tartaric acid, Fenton then employed this type of reaction to study the oxidation of a variety of organic compounds.

The Fenton reaction is generally considered to yield the hydroxyl radical [90], and attacks various small molecules with rates of 10⁸–10¹⁰ M−1 s−1, while its reaction with proteins is diffusion-controlled [91]. In the presence of oxygen the peroxyl radical is formed [92], which can start various chain reactions [93]. The propagation reactions are well understood and, as mentioned above, are responsible for far more damage than the initiating event.

While the concept of the hydroxyl radical as an initiator has received wide support, recent evidence suggests that a higher oxidation state of iron might be involved. This concept is not new: as early as 1932 it was proposed that a higher oxidation state of iron, the ferryl, in alkaline solution [104,105]. Ferryl may be represented as FeII(H2O2), [FeIV=O]²+, FeIII–O−, or FeIV(OH−)2.

3.2 Thermodynamics

The one-electron reduction of hydrogen peroxide is thermodynamically favourable if the reduction potential of the metal complex is 0.32 V or less at pH 7 [4]. This value is based on the reduction potential of the HO•/H2O couple, according to the following equation:

(10)

The reduction potential for the hydroxyl/water couple was not precisely known until recently [106,107]. Based on older values for the hydroxyl radical/water couple one will find higher values of 0.8 V [108] or 0.46 V [34] in earlier papers by the present author. The implication of the value of 0.32 V for the reduction of hydrogen peroxide is that complexes such as tris-1, 10-phenanthroline iron(II) and tris-2, 2′-bipyridine iron(II) are unlikely to reduce hydrogen peroxide since the reduction potentials of the respective iron complexes are in excess of 1 V, which makes the reaction unfavourable by 16 kcal/mol.

3.3 Kinetics

Rate constants have been determined for the reduction of hydrogen peroxide by iron(II) and a number of iron(II) complexes. These rate constants have been compiled in Table 2. It is immediately clear that there is not much agreement between the results of various groups. However, there is a discernable trend: metal complexes with more water-accessible coordination sites react faster. Graf et al. [117] have commented upon the importance of coordinated water molecules for the Fenton reaction. It is also clear that the rate of the Fenton reaction for a chelated complex near neutral pH is much faster than that of aqueous iron(II) at low pH. The use of the low-pH value of 76 M−1 s−1 in a recent calculation [118] of the flux of hydroxyl radicals in a cell gives an estimate that is at least two orders of magnitude too low.

TABLE 2

Rate constants for the Fenton reactiona

aError limits are given in the original papers.

bAbbreviations and remarks:

H, Hardwick [109] (20.2°C, 0.1 N, HClO4); this reference contains a discussion of earlier work. BFA, Borggaard et al. [110] (20°C, pH 6, 0.2 M ionic strength); values apply to unprotonated species and were determined polarographically.

SW, Sutton and Winterbourn [111] (pH 7.4, variable ionic strength).

K, G, Various references (see notes below).

RR, Rahhal and Richter [112] (pH 7.0); rapid mixing study.

WNDF, Wink et al. [113]; UV spectroscopy.

YP, Yamazaki and Piette [114]; ESR-flow study.

cValues for aquo are in k (M−1 s−1).

dRush and Koppenol [96] (25°C, pH 7.2, 38 mM ionic strength); stopped-flow study.

eRush and Koppenol [2]; stopped-flow study.

fRush et al. [32]; stopped-flow study.

gGilbert and Jeff [115], pH 4; ESR flow study.

hCroft et al. [116], pH 7; ESR flow study.

The rate constants for the reduction of hydrogen peroxide by copper(I) phenanthroline and aqueous copper(I) are 1.1 × 10³ M−1 s−1 [119] and 4.1 × 10³ M−1 s−1 [120], respectively.

3.4 Intermediates

It has been argued that at neutral pH the Fenton reaction proceeds via an intermediate. Such an intermediate could be a oxoiron(IV) compound as shown in the following scheme:

Loss of water from the FeIV(OH−)2 species would yield FeO²+, a compound formed in the oxidation of iron(III) haems by hydrogen peroxide (as well as an oxidised porphyrin ligand). One might ask the question whether copper can form a cupryl, or oxocopper(III), species. It has been argued that this is not possible because formation of such a species requires the presence of an empty t2g metal orbital, such that a π-bond between one of these orbitals and a full 2p orbital of oxygen(2–) can be formed [121]. Copper(III) still has 8 d-electrons, and there are no empty t2g orbitals. Thus, if a higher oxidation state of copper is involved, it is copper in the 3+ oxidation state, not a copperoxo(1+) species. However, iron(IV) has only 4 d-electrons, allowing the formation of one such π-metal–oxo bond.

At low pH the reaction of Fe(II)aq with hydrogen peroxide leads to the hydroxyl radical [82,96,122] and spectral evidence for an intermediate has recently been obtained [113]. At neutral pH, in the presence of chelating agents, the situation is more complicated, as reviewed in 1989 [123]. We have investigated four reactions that indicate that an intermediate is involved.

The first is the oxidation of ferrocytochrome c by a mixture of iron(II) edta and hydrogen peroxide [124]. Had the hydroxyl radical been formed, cytochrome c would have been degraded, not just oxidised [125]. The interaction between ferric cytochrome c and the hydroxyl radical is peculiar in that a radical is created on the surface which reduces the haem [126].

The second and third are based on the use of scavengers. When a scavenger reacts with the hydroxyl radical the resulting radical is either reducing, oxidizing or neutral [127]. Thus, when hydrogen peroxide is mixed with excess iron(II) chelate the absorbance change in the UV corresponds to zero Fe(III) formed per hydrogen peroxide consumed (no absorbance change), 2 Fe(III)/H2O2, or 1 Fe(III)/H2O2, respectively. The scavenger tert-butanol reacts with the hydroxyl radical to form a radical that decays by reacting with another tert-butanol radical. If this is the case, then the reaction of an iron(II) complex with hydrogen peroxide should yield one iron(III) per hydrogen peroxide. However, we observed that tert-butanol is unable to prevent the oxidation of a second iron(II) complex [96,128]. This can be explained by Reactions (11) and 12:

(11)

(12)

The experimental observation of 1.7–2.0 mol HLFe(II) oxidised per mol of hydrogen peroxide in the presence of tert-butanol was confirmed by Rahhal and Richter [112]. They showed that the oxidizing species reacted equally fast with Fe²+ -dtpa as with hydrogen peroxide, from which it was concluded that the oxidizing species could not be the hydroxyl radical. If the hydroxyl radical had been formed, the scavenger tert-butanol would have intercepted it and the oxidation of the second iron(II) would have been prevented, because the tert-butanol radical is believed to be inactive. Gilbert and Jeff [115] offered an alternative explanation. They suggested that the hydroxyl radical is formed, and that the tert-butanol radical oxidises the iron(II) chelate. This reaction is followed by a reductive elimination [129]. This is shown in Reactions (13)–(15), in which the chelating agent HL is