Electrochemical measurement of second-order electron transfer rate constants for the reaction between cytochrome b5 and cytochrome c
The second-order electron transfer reaction between cytochrome b5 and cytochrome c has been studied by cyclic voltammetry utilizing a gold electrode modified with β-mercaptopropionate. When cyclic voltammetry is performed on a solution containing a mixture of cytochrome b5, cytochrome c and polylysine, cytochrome b5 undergoes reversible electrochemistry at the electrode surface while cytochrome c discriminates against the electrode surface. The selectivity of the modified electrode for negatively charged proteins makes it possible to selectively reduce a protein possessing a net negative charge and a relatively low reduction potential (outer mitochondrial membrane cytochrome b5, E° = -102 mV; microsomal cytochrome b5, E° = 3 mV) in the presence of another protein possessing a net positive charge and a relatively high reduction potential (cytochrome c, E° = 265 mV). The electrochemical reduction of ferricytochrome b5 at the electrode surface is followed by a second-order electron transfer reaction between ferrocytochrome b5 and ferricytochrome c that yields ferricytochrome b5 and ferrocytochrome c. This fast homogeneous electron transfer reaction which is preceded by a heterogeneous electron transfer reaction results in a characteristic cyclic voltammogram containing a pre-peak to the reduction current. The second- order rate constant for the homogeneous reaction was obtained by invoking the above reaction scheme for digital simulation of a cyclic voltammogram which was subsequently fitted to the experimental data. Second-order rate constants obtained with this method are 2.9 x 108 and 8.9 x 108 for the homogeneous electron transfer reactions between rat liver outer mitochondrial membrane (OM) ferrocytochrome b5 and beef liver microsomal ferrocytochrome b5, with horse heart ferricytochrome c, respectively. These values are in good agreement with second-order rate constants obtained for the same protein systems by flash photolysis [Meyer, T. E., Rivera, M., Walker, F. A., Mauk, M. R., Mauk, A. G., Cusanovich, M. A., and Tollin, G. (1993) Biochemistry' 32, 622-627].
Publication Source (Journal or Book title)
Seetharaman, R., White, S., & Rivera, M. (1996). Electrochemical measurement of second-order electron transfer rate constants for the reaction between cytochrome b5 and cytochrome c. Biochemistry, 35 (38), 12455-12463. https://doi.org/10.1021/bi9529723