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

FEBS Letters

Ishimura, Yuzuru¹  Horiuchi, Tadao²  Makino, Ryu¹  Shimada, Hideo¹  Tsujimura, Mitsushi³  Yaoi, Tsuyoshi³  Yura, Kei⁴  Ikeguchi, Masamichi²  Sekimizu, Kazuhisa³  Koga, Hideo³  Nakamura, Kazuhide³  Sagara, Yasuhiro³  Go, Mitiko⁴ 

[1] Department of Biochemistry, School of Medicine, Keio University, Tokyo 160, Japan;Department of Bioengineering, Faculty of Engineering, Soka University, Hachioji, Tokyo 192, Japan;Department of Microbiology, Faculty of Pharmaceutical Sciences, Kyushu University 62, Higashi-ku, Fukuoka 812, Japan;Department of Biology, Faculty of Science, Nagoya University, Nagoya 464-01, Japan

关键词: Cytochrome P450cam;    Random mutagenesis;    Amino acid substitution;    Putidaredoxin;    Electron transfer;    Binding of P450cam with putidaredoxin;    P450cam;    cytochrome P450cam;    Pd;    putidaredoxin;    PdR;    putidaredoxin reductase;    SDS-PAGE;    sodium dodecyl sulfate/polyacrylamide gel electrophoresis;    Arg112;    arginine residue at the position 112 of P450cam;    Arg112Cys;    a mutant in which Arg112 has been changed by Cys;   

DOI  :  10.1016/0014-5793(93)80307-G

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown