REACTION OF FERROUS CYTOCHROME-C PEROXIDASE WITH DIOXYGEN - SITE-DIRECTED MUTAGENESIS PROVIDES EVIDENCE FOR RAPID REDUCTION OF DIOXYGEN BY INTRAMOLECULAR ELECTRON-TRANSFER FROM THE COMPOUND-I RADICAL SITE

The reaction of dioxygen with the ferrous forms of the cloned cytochrome c peroxidase [CCP(MI)] and mutants of CCP(MI) prepared by site-directed mutagenesis was studied by photolysis of the respective ferrous-CO complexes in the presence of dioxygen. Reaction of ferrous CCP(MI) with dioxygen transiently formed a Fe(II)-O2 complex (bimolecular rate constant = (3.8 +/- 0.3) x 10(4) M-1 s-1 at pH 6.0; 23-degrees-C) that reacted further (first-order rate constant = 4 +/- 1 s-1) to form a product with an absorption spectrum and an EPR radical signal at g = 2.00 that were identical to those of compound I formed by the reaction of CCP(MI)III with peroxide. Thus, the product of the reaction of CCP(MI)II with dioxygen retained three of the four oxidizing equivalents of dioxygen. Gel electrophoresis of the CCP(MI)II + dioxygen reaction products showed that covalent dimeric and trimeric forms of CCP(MI) were produced by the reaction of CCP(MI)II with dioxygen. Photolysis of the CCP(MI)II-CO complex in the presence of ferrous cytochrome c prevented the appearance of the cross-linked forms and resulted in the oxidation of 3 mol of cytochrome c/mol of CCP(MI)II-CO added. The results provide evidence that reaction of CCP(MI)II with dioxygen causes transient oxidation of the enzyme by 1 equiv above the normal compound I oxidation state. Mutations that eliminate the broad EPR signal at g = 2.00 characteristic of the compound I radical also prevented the rapid oxidation of the ferrous enzyme by dioxygen. The Trp 191 --> Phe, Gln, His and Asp 235 --> Asn mutants readily formed a ferrous-dioxygen complex, but the rapid further reaction of this intermediate to an oxyferryl form was not observed. Instead, the Fe(II)-O2 enzymes returned slowly to the ferric state without detectable accumulation of intermediates (apparent rate constant = (1 +/- 0.5) x 10(-4) s-1). Other active site mutations did not prevent the rapid oxidation of the ferrous enzyme to compound I by dioxygen. The dramatic effects of mutations at Trp 191 and Asp 235 are interpreted as an indication that intramolecular electron transfer from the compound I radical site to the coordinated dioxygen ligand promotes the rapid oxidation of CCP(MI)II by dioxygen.