Transient Kinetics of the One‐Electron Transfer Reaction between Reduced Flavocytochrome b2 and Oxidized Cytochrome c

Abstract

The 1-electron transfer reaction from reduced flavocytochrome b2 (fully reduced by 3-electron equivalents) to ferricytochrome c, both purified from the yeast Hansenula anomala, was studied using stopped-flow spectrophotometry in the course of single turnover, for reactants initially mixed in a heme M ratio equal to 1. The cytochrome [Cyt] c reduction proceeded to completion through an apparently 1st-order process. Depending on the experimental conditions (concentrations and/or ionic strength), the reduction is of 2nd-order or 1st-order character. To interpret these kinetic results, computer simulation studies were performed based on a kinetic scheme involving, besides the formation of a complex before the electron transfer step, intramolecular electron transfer steps within flavocytochrome b2 to maintain the concentration of the specific electron donor center, the reduced Cyt b2. As far as the Cyt c reduction rate constant, ka, and its variations were concerned, the simulated data showed that this complicated scheme could approximate a mechanism which is by far the simplest, involving only the 2 former steps. Such a scheme accounts for the hyperbolic dependence of the rate of reduction of Cyt c, ka, upon reductant concentrations which had provided clear evidence for the kinetic existence of a complex in the reaction pathway. At 5.degree. C, the rate constant for the electron transfer is 380 s-1 with an activation energy of 13.8 kJ mol-1 (3.3 kcal mol-1). It predicts the observed variations of ka with ionic strength and provides estimates of the rate constants of the binding step.