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Essential role of the Arg 112 residue of cytochrome P450cam for electron transfer from reduced putidaredoxin
Author(s) -
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
Publication year - 1993
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(93)80307-g
Subject(s) - chemistry , electron transfer , cytochrome , mutant , electron transport chain , enzyme , stereochemistry , heme , pseudomonas putida , wild type , biochemistry , photochemistry , gene
Cytochrome P450cam (CYP101) of Pseudomonas putida PpGl in which Arg 112 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 Arg 112 , 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.