Oxidation of yeast iso-1 ferrocytochrome c by yeast cytochrome c peroxidase compounds I and II. Dependence upon ionic strength.

The reduction of cytochrome c peroxidase compound I by excess yeast iso-1 ferrocytochrome c is biphasic. Two pseudo-first-order rate constants can be measured by stopped-flow techniques. The fastest rate process is the reduction of cytochrome c peroxidase compound I to compound II, and the slower process is the reduction of II to the native enzyme. The yeast iso-1 ferrocytochrome c concentration dependence of the reduction of cytochrome c peroxidase compound I to compound II is consistent with a mechanism involving two binding sites for cytochrome c on cytochrome c peroxidase. Electron transfer from cytochrome c bound at the high-affinity binding site to the Fe(IV) site in cytochrome c peroxidase compound I is dependent upon ionic strength, increasing from 15 +/- 6 to 2000 +/- 100 s-1 over the ionic strength range 0.01-0.20 M. The reduction rate of the Fe(IV) site in the 2:1 yeast iso-1 ferrocytochrome c/cytochrome c peroxidase compound I complex is essentially independent of ionic strength with a value of 3800 +/- 300 s-1. The Fe(IV) site in cytochrome c peroxidase compound I is preferentially reduced by yeast ferrocytochrome c between 0.01 and 0.20 M ionic strength while the Trp-191 radical is preferentially reduced above 0.30 M ionic strength. The association rate constant for the binding of yeast iso-1 ferrocytochrome c to cytochrome c peroxidase compound I can be evaluated and varies from a remarkable 1 x 10(10) M-1 s-1 at 0.01 M ionic strength to 1.2 x 10(5) M-1 s-1 at 1.0 M ionic strength. Between 0.01 and 0.20 M ionic strength, the reduction of cytochrome c peroxidase compound II to the native enzyme is anomalous. The reaction is independent of the cytochrome c concentration and directly proportional to the initial cytochrome c peroxidase compound I concentration.