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

Abstract

SUMMARY1. 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 #M the reduction kinetics of ferricytochrome c by hydrated electrons is a bimolecular process with a rate constant of 4.5 • 101 o M-t "s-1 (21 °C).3. At a concentration of cytochrome c of more than 20/~M 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 • 106 s-1 (pH 7.2, 21 °C).4. At high concentrations 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 • l0 s s -1 (pH 7.2, 21 °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.

Changes in conformation of reduced cytochrome c in neutral aqueous solution

“…The variation in AE.o is consistent with radicals formed by hydroxyl attack on propan-2-ol, ethanol and methanol being able to reduce cytochrome c, at various rates and/or efficiencies, whereas the radicals formed from 2-methylpropan-2-ol do not do so. This agrees with work with ethanol and 2-methylpropan-2-ol but disagrees with work with methanol (Wilting et al, 1975). We have no explanation of the earlier results with methanol.…”

Pulse Radiolysis Studies for Mechanism in Biochemical Redox Reactions

“…Interestingly, the reaction of e aq − with Fe 3+ cytochrome c proceeded efficiently with a reduction yield of 85%. 49 The species e aq − is a nonspecific reductant and reacts with several amino acid residues, including cysteine, cysteine, and histidine. 50 The heme group in cytochrome c is deeply buried, and only 1−2% of the surface of cytochrome c allows direct contact between the heme and the solvent.…”

“…The kinetics of the reactions of e aq – with a number of redox proteins have been studied mainly with a view to elucidating the protein redox properties. Interestingly, the reaction of e aq – with Fe 3+ cytochrome c proceeded efficiently with a reduction yield of 85% . The species e aq – is a nonspecific reductant and reacts with several amino acid residues, including cysteine, cysteine, and histidine .…”

Structural Determination of a Filamentous Chaperone to Fabricate Electronically Conductive Metalloprotein Nanowires

“…28 The Soret peak shifted from 409 to 418 nm after addition of a reducing agent, sodium borohydride, which is representative of electron transfer to the iron core of the heme, as changes in bond energy within the porphyrin ligand introduce variations in the absorption spectrum. 32 Correspondingly, α and β bands, at 522 and 551 nm, respectively, were observed upon reduction and are characteristic features of low-energy transitions in the porphyrin under reduced state of the heme. 33 Charge Transport Measurements of the Metalloprotein Nanowires.…”

“…In their air-oxidized state, the absorption spectrum of the cytc3-containing nanowires displayed a dominant Soret peak at 409 nm, characteristic of ferric heme configurations of cytc3 when Fe­(III) ions are chelated to the porphyrin molecule . The Soret peak shifted from 409 to 418 nm after addition of a reducing agent, sodium borohydride, which is representative of electron transfer to the iron core of the heme, as changes in bond energy within the porphyrin ligand introduce variations in the absorption spectrum . Correspondingly, α and β bands, at 522 and 551 nm, respectively, were observed upon reduction and are characteristic features of low-energy transitions in the porphyrin under reduced state of the heme …”