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.