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Wegerich et al. (J. Biol. Inorg. Chem. 18:429-440, 2013), working with singly modified human cytochromes c, claim to have found a new mechanism for the reduction of iron(III) cytochrome c by superoxide. I show that electron transfer by way of the solvent-accessible haem edge-a mechanism not considered by Wegerich et al.-is still the correct mechanism. Furthermore, several deficiencies in this work preclude any comparisons with other publications on this topic.

Oxidation of cytochrome c, a key protein in mitochondrial electron transport and a mediator of apoptotic cell death, by reactive halogen species (HOX, X2), i.e., metabolites of activated neutrophils, was investigated by stopped-flow. The fast initial reactions between FeIIIcytc and HOX species, with rate constants (at pH 7.6) of k > 3 x 10(6) M(-1) s(-1) for HOBr, k > 3 x 10(5) M(-1) s(-1) for HOCl, and k = (6.1+/-0.3) x 10(2) M(-1) s(-1) for HOI, are followed by slower intramolecular processes. All HOX species lead to a blue shift of the Soret absorption band and loss of the 695-nm absorption band, which is an indicator for the intact iron to Met-80 bond, and of the reducibility of FeIIIcytc. All HOX species do, in fact, persistently impair the ability of FeIIIcytc to act as electron acceptor, e.g., in reaction with ascorbate or O2*-. I2 selectively oxidizes the iron center of FeIIcytc, with a stoichiometry of 2 per I2, and with k(FeIIcytc + I2) approximately 4.6 x 10(4) M(-1) s(-1) and k(FeIIcytc + I2*-) = (2.9+/-0.4) x 10(8) M(-1) s(-1). Oxidation of FeIIcytc by HOX species is not selectively directed toward the iron center; HOBr and HOCl are considered to react primarily by N-halogenation of side chain amino groups, and HOI mainly by sulfoxidation. There is some evidence for the generation of HO* radicals upon reaction of HOCl with FeIIcytc. Chloramines (e.g., NH2Cl), bromamine (NH2Br), and cyclo-Gly2 chloramide oxidize FeIIcytc slowly and unselectively, but iodide efficiently catalyzes reactions of these N-halogens to yield fast selective oxidation of the iron center; this is due to generation of I2 by reaction of I- with the N-halogen and recycling of I- by reaction of I2 with FeIIcytc. Iodide also catalyzes methionine sulfoxidation and thiol oxidation by NH2Cl. The possible biological relevance of these findings is discussed.

1. The hydrated electron reacts with ferrocytochrome c to form an unstable intermediate. This intermediate decays in a first-order manner to give, in the first instance, a product which has a similar absorption spectrum in the range 400-610 nm as normal ferricytochrome c. 2. At 21 degrees C the rate constant for the reaction of hydrated electrons with ferrocytochrome c at pH 7.4 (2 mM phosphate buffer) is (3.0 +/- 0.3) = 10(10) M-1 - S-1. As the pH is increased above pH 8.0 the rate constant steadily decreases. The dependence of the rate constant on pH can be explained if ferrocytochrome c has a pK of around 9.2. 3. At 21 degrees C and pH 7.4, the rate constant for the decay of the intermediate is (1.40 +/- 0.15) - 10(5) S-1. This reaction shows no pH dependence in the range 6-2-11.0. 4. A mechanism is proposed whereby the central metal atom of the ferrocytochrome c is oxidased and a thioether bond is reduced. The resulting ferricytochrome c species then slowly develops an absorbance at 606 nm due to the attack of the sulfhydryl group on the haem.

The kinetics of the reaction of OH radicals with ferricytochrome c was studied in the time range 1 microsecond to 1 s by means of pulse radiolysis. The OH radicals reduce ferricytochrome c by 40% +/- 10%. The time course of the reduction is explained by a mechanism whereby a radical formed after hydrogen has been abstracted from the outer surface of the protein reduces the iron by electron tunnelling. We have calculated that the reducing electron in the radical is bound with an energy of at least 1.75 eV and that the frequency factor of the tunnelling process is v=10(11.5)s-1. This model accounts for the observed absorbance change in time range 5 . 10(-6)--10(-1)s. The time course of the reduction of ferricytochrome c by H radicals (Lichtin, N.N., Shafferman A. and Stein, G. (1974) Biochim. Biophys. Acta 357, 386--398) is explained by the same model.

At ionic strengths below 0.1 M the oxidation of horse ferrocytochrome c by tris(1,10-phenanthroline)cobalt (III) and tris(2,2'-bipyridine)cobalt(III) proceeds by a pathway which is independent of the transition metal complex concentration. Formation of an activated form of the protein appears to be rate limiting. The rate of oxidation decreases as the ionic strength increases. This dependence of the reaction rate on inert electrolyte concentration indicates that electrostatic association of anions under physiological ionic strength confers stability to the protein. The activated form of the protein, which reacts at least 10(4) times as fast as the predominant form, is thought to be a conformation of the reduced protein with an open heme crevice. Binding of the open form of ferrocytochrome c with the redox-inactive cationic transition metal complexes hexamminecobalt(III) and tris(1,10-phenanthroline)chromium(III) inhibits the oxidation by tris(1,10-phenanthroline)cobalt(III). Reactions of tris(1,10-phenanthroline)cobalt(III) with 4-carboxy-2,5-dinitrophenyllysine 13 and 72 ferrocytochromes c show no dependence on ionic strength. NMR studies at pH 7 demonstrate that ferricytochrome c is partly (15%) in the open conformation at low ionic strength. Furthermore, the interaction of redox-inert tris (1,10-phenanthroline)chromium(III) with ferricytochrome c under conditions identical to those of the kinetic studies demonstrates that the transition metal complex binds only to the open form of the protein. Titration with increasing amounts of tris(1,10-phenanthroline) chromium(III) shows changes in the NMR spectrum that are inconsistent with a single binding site.

The electric potential field around native horse cytochrome c and 12 singly modified 4-carboxy-2,4-dinitrophenyl- (CDNP) lysine cytochromes c is asymmetric, mainly because of the inhomogeneous distribution of negative charges. Dipole moments of 325 and 308 debye, (1.08.10(-27) and 1.03.10(-27) coulomb.meter), respectively, were calculated for horse ferri- and ferrocytochrome c. The angle between the heme plane and the dipole vector of horse ferricytochrome c is 33 degrees and increases 1 degree upon reduction to the ferrous form. Dipole moments of the CDNP-lysine cytochromes c differ from that of native cytochrome c by as much as 140 debye in magnitude and 45 degrees in direction. It is proposed that its dipole moment causes cytochrome c to orient itself in the electric fields of its redox partners, and that the CDNP-lysine cytochromes c, which have different dipole moments, do not form a productive complex. Reorientation to the correct position for electron transfer increases the activation energy and lowers the rate of reaction. This model describes quantitatively the relative activities of those CDNP-lysine cytochromes c which are modified outside of the interaction domain and it allows correction of the activities of those modified inside the domain, on the front surface of the molecule, for the change in dipole moment. The interaction domain for the reaction with cytochrome c reductase includes in decreasing order of involvement lysines 13, 72, 86, 27, and 87. That for the reaction with cytochrome c oxidase is slightly smaller, with lysines 13, 72, 86, and 27. The cytochrome c peroxidase domain is the largest of all and is defined by lysines 72, 86, 13, 87, 27,, and 73. All refined interaction domains encompass the exposed heme edge and are to a large extent overlapping, indicating that electron transfer takes place at or close to this prosthetic group and that cytochrome c must move on the outer surface of the inner mitochondrial membrane during electron transport between reductase and oxidase. For a quantitative description of the electrostatic interaction of cytochrome c with other molecules, it is essential to take into account the totality of its charge configuration.

An analysis of the effect of electrostatic properties of 4-carboxy-2,6-dinitrophenyllysine (CDNP-lysine) cytochromes c on their reactions with strongly and weakly binding redox partners is given. For strongly binding systems (cytochrome-c oxidase, cytochrome-c reductase, sulphite oxidase and yeast cytochrome-c peroxidase) the magnitude of the dipole moments of the CDNP cytochromes c determines their relative reactivities. For weakly binding redox agents, such as hexacyanoferrate(III), cobalt(III)tris(1,10-phenanthroline), azurin and plastocyanin, the electrostatic potential at the haem edge accounts for the greater part of the relative activities. Relative rate data were obtained from the literature. It is concluded that the dipole moment of native cytochromes c may account for an approx. 50-fold increase in the efficiency of its physiological activity towards membrane-bound enzymes. A correction on a formula to describe the contribution of a molecular dipole moment to the ionic strength dependence of a bimolecular rate constant (Koppenol, W. H. (1980) Biophys. J. 29, 493-508) leads to an equation nearly identical to that obtained by Van Leeuwen et al. (Van Leeuwen, J.W., Mofers, F.J.M. and Verrman, E.C.I. (1981) Biochim. Biophys. Acta 635, 434-439).

A criticism of a recent paper by M. Fragata and F. Bellemare (Biophys. Chem. 15 (1982) 111) is presented. These authors developed a model of polarity-dependent ferrocytochrome c oxidation which is shown to be incorrect. It fails to show that the use of the 'overal dipole moment' is likely to be unreliable, and that reactivity is best explained by a polarity effect on the dipole of the haem of cytochrome c.

The reaction between hydrogen peroxide and ferrous EDTA generates an oxidizing intermediate (I1) which is not the hydroxyl radical. It oxidizes ferrocytochrome c and also reacts with hydrogen peroxide (k5 = 3.2 X 10(3) M-1 S-1) to form a second oxidizing transient (I2). I1 is not scavenged by t-butyl alcohol whereas I2 is. I1 is found to be significantly less reactive than the hydroxyl radical toward benzoate ion, t-butyl alcohol, acetate ion, arginine, and serine, but is scavenged by compounds with readily oxidizable functional groups such as ethanol and isopropyl alcohol. This indicates that I1 does not undergo the characteristic reactions of the hydroxyl radical but shows a pattern of reactivity more associated with a metal ion oxidant like a ferryl (FeO2+)-EDTA complex.

The temperature and pH dependence of the reaction of the superoxide radical anion with ferricytochrome c have been measured using the pulse-radiolysis technique. The temperature dependence of the reaction at low ionic strength yields an activation energy of 31 +/- 5 kJ/mol as compared to 14 +/- 3 kJ/mol for the reaction of CO2.(-) under the same conditions. The pH dependence fits the single pK'a of ferricytochrome c of 9.1. The bimolecular rate constant for the reaction of the superoxide anion with ferricytochrome c at pH 7.8, 21 +/- 2 degrees C, in the presence of 50 mM phosphate and 0.1 mM EDTA is (2.6 +/- 0.1) X 10(5) M-1 s-1. Using this value, 1 unit of superoxide dismutase activity (McCord, J. M., and Fridovich, I. (1969) J. Biol. Chem. 244, 6049-6055) is calculated to be 3.6 +/- 0.3 pmol of enzyme if the assay is performed in a total volume of 3.0 ml. Copper ions reduce the yield of the reaction of ferricytochrome c with CO2.(-). The reactivities of native and singly modified 4-carboxy-2,4-dinitrophenyllysine cytochromes c towards the superoxide anion radical are in the order native greater than 4-carboxy-2,4-dinitrophenyllysine 60 greater than lysine 13 greater than lysine 87 greater than lysine 27 greater than lysine 86 greater than lysine 72, indicating that electron transfer takes place at or close to the solvent accessible heme edge. The mechanism of the reaction is discussed in terms of the approach of superoxide anion radicals to the heme edge and the available molecular orbitals of both heme and free radicals.

1. At neutral pH ferricytochrome c is reduced by the superoxide anion radical (O2-), without loss of enzymatic activity, by a second order process in which no intermediates are observed. The yield of ferrocytochrome c (82-104%), as related to the amount of O2- produced, is slightly dependent on the concentration of sodium formate in the matrix solution. 2. The reaction (k1 equals (1.1+/-0.1) - 10(6) M-1 - s-1 at pH 7.2, I equals 4 mM and 21 degrees C) can be inhibited by superoxide dismutase and trace amounts of copper ions. The inhibition by copper ions is removed by EDTA without interference in the O2- reduction reaction. 3. The second-order rate constant for the reaction of O2- with ferricytochrome c depends on the pH of the matrix solution, decreasing rapidly at pH greater than 8. The dependence of the rate constant on the pH can be explained by assuming that only the neutral form of ferricytochrome c reacts with O2- and that the alkaline form of the hemoprotein is unreactive. From studies at pH 8.9, the rate for the transition from the alkaline to the neutral form of ferricytochrome c can be estimated to be 0.3 s-1 (at 21 degrees C and I equals 4 mM). 4. The second-order rate constant for the reaction of O2- with ferricytochrome c is also dependent on the ionic strength of the medium. From a plot of log k1 versus I1/2-(I + alphaI1/2)-1 we determined the effective charge on the ferricytochrome c molecule as +6.3 and the rate constant at I equals 0 as (3.1+/-0.1) - 10(6) M-1 - s-1 (pH 7.1, 21 degrees C). 5. The possibility that singlet oxygen is formed as a product of the reaction of O2- with ferricytochrome c can be ruled out on thermodynamic grounds.

1. The electric potential fields around tuna ferri- and ferrocytochrome c were calculated assuming that (i) all of the lysines and arginines are protonated, (ii) all of the glutamic and aspartic acids and the terminal carboxylic acid are dissociated, and (iii) the haem has a net charge of +1e in the oxidized form. 2. Near the haem crevice high values for the potential (greater than +2.5 kT/e) are found. Consequently, electron transfer via the haem edge is favored if the oxidant or reductant is negatively charged. 3. The inhomogeneous distribution of charges leads to a dipole moment of 244 and 238 debye for oxidized and reduced tuna cytochrome c, respectively. Horse cytochrome c has dipole moments of 303 (oxidized) and 286 (reduced) debye. 4. A line through the positive and negative charge centres, the dipole axis, crosses the tuna cytochrome c surface at Ala 83 (positive part) and Lys 99 (negative part). The direction of the dipole axis of horse cytochrome c is very similar. Since the centre of the domain on the cytochrome c surface, which is involved in the binding to cytochrome c oxidase, is found at the beta-carbon of the Phe 82 in horse cytochrome c (Ferguson-Miller, S., Brautigan, D.L. and Margoliash, E. (1978) J. Biol. Chem. 253, 149--159) it is suggested that the direction of the dipole is of physiological importance. 5. The activity coefficients of horse ferri- and ferrocytochrome c were calculated as a function of ionic strength using a formula derived by Kirkwood (Kirkwood, J.G. (1934) J. Chem. Phys. 2, 351--361). 6. Due to the high net charge at pH 7.5 the influence of the dipole moments of horse ferri- and ferrocytochrome c on the respective activity coefficients can be neglected at I less than or equal to 50 mM. 7. Using the Brønsted relation the effect of ionic strength on reaction rates of horse cytochrome c was calculated. Good agreement is found between theory and experimental results reported in the literature.