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 number of CcP(MI) mutants with native and ruthenium-labeled horse ferrocytochrome c have been studied by stopped-flow spectroscopy and laser flash photolysis. At 100 mM ionic strength, pH 7.5, native horse ferrocytochrome c reduces the radical on the indole group of Trp-191 in cytochrome c peroxidase compound I (CMPI) with a second-order rate constant of 1.3 x 10(8) M-1 s-1. Ferrocytochrome c then reduces the oxyferryl 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 independent 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 the electron transfer equilibrium between the radical and the oxyferryl heme Fe(IV) in the transient form of CMPII. The second-order rate constants for reduction of the radical and the oxyferryl heme in the mutants Y39F, Y42F, H181G, W223F, and Y229F are nearly the same as for wild-type CcP(MI). The intracomplex rate constants for reduction of the radical in these mutants by the ruthenium-labeled cytochrome c derivatives are also similar to that for CcP(MI). This rules out a direct role for these aromatic residues in electron transfer.(ABSTRACT TRUNCATED AT 250 WORDS)
Hahm
S
S
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville 72701.
Miller
M A
MA
Geren
L
L
Kraut
J
J
Durham
B
B
Millett
F
F
D000818:Animals; D003574:Cytochrome c Group / Q000378:metabolism*; D003578:Cytochrome-c Peroxidase / Q000378:metabolism*; D005609:Free Radicals; D006418:Heme / Q000378:metabolism*; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D010782:Photolysis; D011994:Recombinant Proteins / Q000378:metabolism; D014364:Tryptophan / Q000378:metabolism
D003574:Cytochrome c Group; D005609:Free Radicals; D011994:Recombinant Proteins; D006418:Heme; D014364:Tryptophan; D003578:Cytochrome-c Peroxidase