REACTION OF HORSE CYTOCHROME-C WITH THE RADICAL AND THE OXYFERRYL HEME IN CYTOCHROME-C PEROXIDASE COMPOUND-I

The reactions of recombinant cytochrome c peroxidase [CcP(MI)] and a n umber of CcP(MI) mutants with native and ruthenium-labeled horse ferro cytochrome c have been studied by stopped-flow spectroscopy and laser flash photolysis. At 100 mM ionic strength, pH 7.5, native horse ferro cytochrome c reduces the radical on the indole group of Trp-191 in cyt ochrome c peroxidase compound I (CMPI) with a second-order rate consta nt of 1.3 x 10(8) M(-1) s(-1). Ferrocytochrome c then reduces the oxyf erryl heme Fe(IV) in CMPII with a rate constant of 2.0 x 10(6) M(-1) s (-1). The rate constant for the reduction of the radical is nearly ind ependent of pH from 5 to 8, but the rate constant for reduction of the oxyferryl heme Fe(IV) increases 33-fold as the pH is decreased from 8 to 5. This increase in rate is correlated with the pH dependence of t he electron transfer equilibrium between the radical and the oxyferryl heme Fe(IV) in the transient form of CMPII. The second-order rate con stants for reduction of the radical and the oxyferryl heme in the muta nts Y39F, Y42F, H181G, W223F, and Y229F are nearly the same as for wil d-type CcP(MI). The intracomplex rate constants for reduction of the r adical in these mutants by the ruthenium-labeled cytochrome c derivati ves are also similar to that for CcP(MI). This rules out a direct role for these aromatic residues in electron transfer. These results suppo rt reduction of both the radical at Trp-191 and the oxyferryl heme Fe( IV) by the pathway recently proposed by Pelletier and Kraut [(1992) Sc ience 258, 1748-1755] on the basis of the crystal structure of the com plex between yeast CcP(MI) and yeast isol-cytochrome c.