One-electron reactions in biochemical systems as studied by pulse radiolysis. VI. Stages in the reduction of ferricytochrome c

When ferricytochrome c at pH about 9 is reduced by hydrated electrons and/or CO₂^?, it gives rise to an unstable form of ferrocytochrome c whose absorption spectrum, particularly in the Soret region, differs from that of normal ferrocytochrome c. This form changes intramolecularly (life-time about 0.1 s at ambient temperature) to yield normal ferrocytochrome c, and by 0.5 s the change in absorption spectrum in the range 225–600 nm produced by e^?_aq and/or CO₂^? is identical to the final change produced by reduction with an equivalent amount of sodium dithionite. This shows that both e^?_aq and CO^?₂ reduce cytochrome c with practically 100% efficiency. In the range 600–800 nm the spectrum of the unstable form is the same as that of normal ferrocytochrome c, both having small absorptions at 695 nm as compared with ferricytochrome c. As the unstable form disappears however a further loss of absorption at 695 nm occurs. This is taken to imply that the unstable form decays to a second unstable form which then rapidly donates an electron to the unchanged neutral form of ferricytochrome c, so reducing absorption in the 695 nm band. Subsequent to this process the absorption in the 695 nm band increases over a period of minutes owing to re-equilibration between the neutral and alkaline formes of ferricytochrome c. Between pH 7 and 10 the effect of pH on the absorption changes is consistent with the hypothesis of a second unstable form of ferrocytochrome c. Additional phenomena arise in more alkaline solutions. The rates of the various unimolecular processes are thought to be determined by the rates of change of conformation of the protein parts of the molecule following the change in oxidation state.     

One-electron reduction of hepatic NADH-cytochrome b5 reductase as studied by pulse radiolysis

The reduction of flavin in hepatic NADH-cytochrome b5 reductase by the hydrated electron (eaq-) was investigated by pulse radiolysis. The eaq- reduced the flavin of NADH-cytochrome b5 reductase to form the red semiquinone between pH 5 and 9. The spectrum of the red semiquinone differs from that of enzyme reduced by dithionite in the presence of NAD+. After the first phase of the reduction, conversion of the red to blue semiquinone was observed at acidic pH. Resulting products are the blue (neutral) or red (anionic) semiquinone or a mixture of the two forms. The pK value for this flavin radical was approximately 6.3. Subsequently, the semiquinone form reacted by dismutation to form the oxidized and the fully reduced forms of the enzyme with a rate constant of 1 x 10(3) M-1 s-1 at pH 7.1. In the presence of NAD+, eaq- reacted with NAD+ to yield NAD(.). Subsequently, NAD. transferred an electron to NAD+-bound oxidized enzyme to form the blue and red semiquinone or mixture of the two forms of the enzyme, where pK value of this flavin radical was approximately 6.3. The blue semiquinone obtained at acidic pH was found to convert to the red semiquinone with a first order rate constant of 90 s-1, where the rates were not affected by pH or the concentration of NAD+. The final product is NAD+-bound red semiquinone of the enzyme.     

The mechanism of reduction of cytochrome c as studied by pulse radiolysis.

1. The reaction of hydrated electrons with ferricytochrome c was studied using the pulse-radiolysis technique. 2. In 3.3 mM phosphate-buffer (pH 7.2), 100 mM methanol and at a concentration of cytochrome c of less than 20 muM the reduction kinetics of ferricytochrome c by hydrated electrons is a bimolecular process with a rate constant of 4.5-10-10 M-1-S-1 (21 degrees C). 3. At a concentration of cytochrome c of more than 20 muM the apparent order of the reaction of hydrated electrons with ferricytochrome c measured at 650 nm decreases due to the occurrence of a rate-determining first-order process with an estimated rate constant of 5-10-6s-1 (pH 7.2, 21 degrees C). 4. At high concentration of cytochrome c the reaction-time courses measured at 580 and 695 nm appear to be biphasic. A rapid initial phase (75% and 30% of total absorbance change at 580 and 695 nm, respectively), corresponding to the reduction reaction, is followed by a first-order change in absorbance with a rate constant of 1.3-10-5 S-1 (pH 7.2, 21 degrees C). 5. The results are interpreted in a scheme in which first a transient complex between cytochrome c and the hydrated electron is formed, after which the heme iron is reduced and followed by relaxation of the protein from its oxidized to its reduced conformation. 6. It is calculated that one of each three encounters of the hydrated electron and ferricytochrome c results in a reduction of the heme iron. This high reaction probability is discussed in terms of charge and solvent interactions. 7. A reduction mechanism for cytochrome c is favored in which the reduction equivalent from the hydrated electron is transmitted through a specific pathway from the surface of the molecule to the heme iron.     

Electron transfer in milk xanthine oxidase as studied by pulse radiolysis

Electron transfer within milk xanthine oxidase has been examined by the technique of pulse radiolysis. Radiolytically generated N-methylnicotinamide radical or 5-deazalumiflavin radical has been used to rapidly and selectively introduce reducing equivalents into the enzyme so that subsequent equilibration among the four redox-active centers of the enzyme (a molybdenum center, two iron-sulfur centers, and FAD) could be monitored spectrophotometrically. Experiments have been performed at pH 6 and 8.5, and a comprehensive scheme describing electron equilibration within the enzyme at both pH values has been developed. All rate constants ascribed to equilibration between specific pairs of centers in the enzyme are found to be rapid relative to enzyme turnover under the same conditions. Electron equilibration between the molybdenum center and one of the iron-sulfur centers of the enzyme (tentatively assigned Fe/S I) is particularly rapid, with a pH-independent first-order rate constant of approximately 8.5 x 10(3) s-1. The results unambiguously demonstrate the role of the iron-sulfur centers of xanthine oxidase in mediating electron transfer between the molybdenum and flavin centers of the enzyme.     

Intramolecular electron transfer and binding constants in iron hexacyanide-cytochrome c complexes as studied by pulse radiolysis.

Internal oxidation and reduction rates of horse cytochrome c in the complexes CII . Fe(III)(CN)6(3)- and CIII . Fe(II)(CN)6(4)-, are 4.6 . 10(4)s-1 and 3.3 . 10(2)s-1, respectively. The binding site of the iron hexacyanide ions on either CII or CIII are kinetically almost indistinguishable; binding constants range from 0.87 . 10(3) to 2 . 10(3)M-1. The present pulse radiolytic kinetic data is compared with that from NMR, T-jump and equilibrium dialysis studies.     

Kinetic behavior of the monodehydroascorbate radical studied by pulse radiolysis

The reactions of the monodehydroascorbate radical (As.-) with various biological molecules were investigated by pulse radiolysis. As.- reacted with both fully reduced and semiquinone forms of hepatic NADH-cytochrome b5 reductase with second-order rate constants of 4.3 x 10(6) and 3.7 x 10(5) M-1 s-1, respectively, at pH 7.0. In contrast, no reaction of As.- with ferrous cytochrome b5 could be detected by pulse radiolysis, whereas the oxidation of cytochrome b5 by As.- was observed by ascorbate-ascorbate oxidase method. This suggests that the rate constant of As.- with the ferrous cytochrome b5 must be several orders in magnitude smaller than that of the disproportionation of As.-. On the other hand, As.- reduced Fe3+EDTA with a second-order rate constant of 4.0 x 10(6) M-1 s-1 but did not reduce ferric hemoproteins such as metmyoglobin, methemoglobin, and cytochrome b5 by either the pulse radiolysis or the ascorbate-ascorbate oxidase method.     

Reactivity of semiquinones with ascorbate and the ascorbate radical as studied by pulse radiolysis

Free-radical interactions between hydroquinones (QH2) and ascorbate (AscH-) have a profound impact in many biological situations. Despite the obvious biological significance, not much is known about the kinetics of reactions of QH2 and AscH- with their corresponding free radicals, i.e., semiquinones, Q1.-, and the ascorbate radical, Asc.-. Furthermore, a general approach to reliably measure rate constants for the above reactions is fraught with complications. In this work, the kinetic behavior of Q.- and Asc.-, after pulse radiolytic oxidation of mixtures of a series of alkyl- and methoxysubstituted hydroquinones and ascorbate by azide radicals in aqueous buffer, pH 7.40, was monitored in submillisecond range by time-resolved UV spectroscopy. Rate constants for reactions of Q.- with AscH-(reaction [1]) and Asc.- (reaction [2]) were directly determined by using new kinetic procedures which distinguished between reactions [1] and [2]. The results show that the rate constants for reaction [2] vary only within a narrow range from 1.2 x 10(8) to 2.5 x 10(8) M(-1) s(-1) and do not display any pronounced correlation with Q.- structures. In contrast, the value of k1 for nonsubstituted Q.- was found to be (1.8 +/- 0.2) x 10(5) M(-1) s(-1) and decreases with the number of alkyl and methoxy substituents as well as with the decrease of the one-electron reduction potential E(Q.-/QH2).