A stopped-flow investigation of the reduction of excess cytochrome c peroxidase compound I by limiting amounts of horseheart cytochrome c at high ionic strength
Nuevo, Marites Roque
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The kinetics of the reduction of excess CcP Compound I concentration by a limiting amount of horseheart ferro- cytochrome c at 25°C has been studied using stopped-flow techniques. The reaction was monitored at 437 nm using 200 mM ionic strength potassium phosphate buffer, pH 7.5. A fast phase related to the electron transfer from ferro- cytochrome c to the radical site of CcP Compound I to form an inter- mediate, CcP Compound IIF, was observed. The pseudo-first order rate constant for this reduction step increased with CcP Compound I concentration in a hyperbolic manner and was independent of the initial ferrocytochrome c concentration. The observed intracomplex electron transfer rate associated with this phase was 602 + 4 s'1. Comparison of the spectrum of the intermediate in the Soret region with that of CcP Compound I confirms that the reduction of the radical rather than the oxyferryl heme occurs in the first reduction step at this ionic strength. The rate of reduction of CcP Compound I by excess ferrocytochrome c was also measured under the same conditions. The reaction was biphasic with the radical being reduced in the fast phase and the oxyferryl heme reduced in the slow phase. The intracomplex electron transfer rate for the first reduction step was 652 + 1 s'1. The structure of the intermediate formed in the fast phase was also determined based on the spectrum in the Soret region. Intermediate ionic strength buffers (10, 40, 60, and 100 mM) were also used to determine the rate of reduction at conditions where the amount of CcP Compound I is in excess over the ferrocytochrome c concentration. Reduction of the oxyferryl heme of CcP Compound I was observed in the fast phase using these buffers. The change in the initial site of reduction in CcP Compound I by ferrocytochrome c from the Fe(IV) to the radical site probably occurs between 100 mM and 200 mM ionic strength.