Essential role of the Arg112 residue of cytochrome P450cam for electron transfer from reduced putidaredoxin

• Published in: FEBS letters Vol. 331; no. 1-2; pp. 109 - 113
• Main Authors: Koga, Hideo, Sagara, Yasuhiro, Yaoi, Tsuyoshi, Tsujimura, Mitsushi, Nakamura, Kazuhide, Sekimizu, Kazuhisa, Makino, Ryu, Shimada, Hideo, Ishimura, Yuzuru, Yura, Kei, Go, Mitiko, Ikeguchi, Masamichi, Horiuchi, Tadao
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier 27.09.1993
• Subjects: Amino acid substitution; Arg112, arginine residue at the position 112 of P450cam; Arg112Cys, a mutant in which Arg112 has been changed by Cys; Arginine - chemistry; Bacterial Proteins - chemistry; Bacteriology; Binding of P450cam with putidaredoxin; Biological and medical sciences; Camphor 5-Monooxygenase; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - genetics; Cytochrome P450cam; Electron transfer; Electron Transport; Ferredoxins - chemistry; Fundamental and applied biological sciences. Psychology; Metabolism. Enzymes; Microbiology; Mixed Function Oxygenases - chemistry; Mixed Function Oxygenases - genetics; Mutagenesis; Oxidation-Reduction; P450cam, cytochrome P450cam; Pd, putidaredoxin; PdR, putidaredoxin reductase; Protein Conformation; Pseudomonas putida - enzymology; Putidaredoxin; Random mutagenesis; SDS-PAGE, sodium dodecyl sulfate/polyacrylamide gel electrophoresis; Pseudomonadales; Hemoprotein; Cytochrome P450; Electron transfer; Bacteria; Pseudomonadaceae; Pseudomonas putida; Structure function relationship
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(93)80307-G

Cytochrome P450cam (CYP101) of Pseudomonas putida PpGl 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.