title: Regulation of interprotein electron transfer by Trp 191 of cytochrome c peroxidase.
abstract: Cytochrome c peroxidase (CcP) reacts with peroxide to form compound I, an intermediate that has an oxy-ferryl iron center and a stable indolyl radical at Trp 191. During the normal catalytic cycle, the oxy-ferryl heme and the Trp 191 radical are reduced by sequential electron transfers from ferrous cytochrome c (Cc). To investigate the role of protein structure in these electron transfer reactions, mutagenesis was used to replace Trp 191 with Phe. The Trp 191-->Phe enzyme [CcP(MI,F191)] reacts with peroxide to form an oxy-ferryl iron center and a transient porphyrin radical. The reaction of Cc from horse and yeast with peroxide-oxidized CcP(MI,F191) was characterized under transient and steady-state conditions. The rate of ET from Cc to the oxy-ferryl heme of CcP(MI,F191) was decreased by at least 10,000-fold relative to the CcP(MI) parent. This effect was observed at 20 and 100 mM ionic strength, with both yeast and horse cytochrome c as the substrate. Thus, Trp 191 is a critical component of all pathways that permit rapid reduction of the oxy-ferryl heme by Cc under these conditions. The reaction of the porphyrin radical with Cc was difficult to characterize, owing to the short half-life of this intermediate. The oxidation of Cc by this intermediate had a maximum rate constant of 32 s-1 at pH 6.0, 25 degrees C. Circumstantial evidence suggests that the porphyrin radical is not directly reduced by Cc, but is instead reduced via a protein-based radical intermediate. The steady-state activity of the mutant enzyme was 300-600-fold lower than the CcP(MI) parent, but kcat is 7-20 times greater than the rate constant for reduction of the oxy-ferryl heme under all conditions examined. Thus, the oxy-ferryl heme is not reduced to the ferric state under steady-state conditions. Transient changes in the absorption spectrum further indicate that steady-state oxidation of Cc2+ by CcP(MI,F191) occurs via reaction of peroxide with the oxy-ferryl enzyme.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla 92093-0317, USA.
authors.forename: M A
authors.initials: MA
authors.lastname: Miller
authors.forename: L
authors.initials: L
authors.lastname: Vitello
authors.forename: J E
authors.initials: JE
authors.lastname: Erman
mesh_terms: D000818:Animals; D003574:Cytochrome c Group / Q000378:metabolism; D003578:Cytochrome-c Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D045304:Cytochromes c; D004579:Electron Transport; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006736:Horses; D066298:In Vitro Techniques; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D014364:Tryptophan / Q000737:chemistry
chemical_list: C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D029701:Saccharomyces cerevisiae Proteins; D014364:Tryptophan; D045304:Cytochromes c; D003578:Cytochrome-c Peroxidase