Kinetics of reduction by free flavin semiquinones of the components of the cytochrome c-cytochrome c peroxidase complex and intracomplex electron transfer

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Abstract
The kinetics of reduction by free flavin semiquinones of the individual components of 1:1 complexes of yeast ferric and ferryl cytochrome c peroxidase and the cytochromes c of horse, tuna, and yeast (iso-2) have been studied. Complex formation decreases the rate constant for reduction of ferric peroxidase by 44%. On the basis of a computer model of the complex structure [Poulos, T. L., and Finzel, B. C. (1984) Pept. Protein Rev. 4, 115-171], this decrease cannot be accounted for by steric effects and suggests a decrease in the dynamic motions of the peroxidase at the peroxide access channel caused by complexation. The orientations of the cytochromes within the complex are not equivalent. This is shown by differential decreases in the rate constants for reduction by neutral flavin semiquinones upon complexation, which are in the order tuna .mchgt. horse > yeast iso-2. Further support for differences in orientation is provided by the observation that, with the negatively charged reductant FMNH., the electrostatic environments near the horse and tuna cytochrome electron-transfer sites within their respective complexes with peroxidase are of opposite C sign. For the horse and tuna cytochrome c complexes we have also observed nonlinear concentration dependencies of the reduction rate constants with FMNH.. This is interpreted in terms of dynamic motion at the protein-protein interface. We have directly measured the physiologically significant intracomplex one electron transfer rate constants from the three ferrous cytochromes c to the peroxide-oxidized species of the peroxidase. At low ionic strength these rate constants are 920, 730, and 150 s-1 for tuna, horse, and yeast cytochromes c, respectively. These results are also consistent with the contention that the orientations of the three cytochromes within the complex with CcP are not the same. The effect on the intracomplex electron-transfer rate constant of the peroxidase amino acid side chain(s) that is (are) oxidized by the reduction of peroxide was determined to be relatively small. Thus, the rate constant for reduction by horse cytochrome C of the peroxidase species in which only the heme iron atom is oxidized was decreased by only 38%, indicating that this oxidized side-chain group is not tightly coupled to the ferryl peroxidase heme iron. Finally, it was found that, in the absence of cytochrome c, neither of the ferryl peroxidase species could be rapidly reduced by flavin semiquinones. This is consistent with a facilitation of electron transfer by cytochrome c as proposed in the Poulos and Finzel model, as well as suggestive of a change in the dynamics of the peroxidase structure following the binding and reduction of H2O2.