title: Essential role of the Arg112 residue of cytochrome P450cam for electron transfer from reduced putidaredoxin.
abstract: Cytochrome P450cam (CYP101) of Pseudomonas putida PpG1 in which Arg112 is substituted by Cys was isolated by in vitro random mutagenesis of the camC gene DNA coding for P450cam. The absorption spectra of the purified mutant enzyme were similar to those of the wild type enzyme, but its substrate-dependent NADH oxidation activity in the presence of putidaredoxin (Pd) and putidaredoxin reductase (PdR) was extremely low. The rate constant of electron transfer from reduced Pd to the heme of the mutant P450cam, measured on an anaerobic stopped flow apparatus, was 1/400 of that of the wild type enzyme and the dissociation constant of the mutant P450cam for oxidized Pd was several fold higher than that of the wild type enzyme. A considerable decrease in mid-point potential of the mutant enzyme was also noted. We conclude that Arg112, which is located on the surface of the P450cam molecule and hydrogen-bonded to one of the heme propionate chains, plays an essential role in the electron transfer from Pd.
authors.affiliation: Department of Microbiology, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Koga
authors.forename: Y
authors.initials: Y
authors.lastname: Sagara
authors.forename: T
authors.initials: T
authors.lastname: Yaoi
authors.forename: M
authors.initials: M
authors.lastname: Tsujimura
authors.forename: K
authors.initials: K
authors.lastname: Nakamura
authors.forename: K
authors.initials: K
authors.lastname: Sekimizu
authors.forename: R
authors.initials: R
authors.lastname: Makino
authors.forename: H
authors.initials: H
authors.lastname: Shimada
authors.forename: Y
authors.initials: Y
authors.lastname: Ishimura
authors.forename: K
authors.initials: K
authors.lastname: Yura
mesh_terms: D001120:Arginine / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D019475:Camphor 5-Monooxygenase; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics; D004579:Electron Transport; D005288:Ferredoxins / Q000737:chemistry*; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics; D016296:Mutagenesis; D010084:Oxidation-Reduction; D011487:Protein Conformation; D016958:Pseudomonas putida / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D005288:Ferredoxins; C100137:putidaredoxin; D003577:Cytochrome P-450 Enzyme System; D001120:Arginine; D006899:Mixed Function Oxygenases; D019475:Camphor 5-Monooxygenase