ESSENTIAL ROLE OF THE ARG112 RESIDUE OF CYTOCHROME-P450CAM FOR ELECTRON-TRANSFER FROM REDUCED PUTIDAREDOXIN

Cytochrome P450 cam (CYP101) of Pseudomonas putida PpG1 in which Arg11 2 is substituted by Cys was isolated by in vitro random mutagenesis of the camC gene DNA coding for P450cam. The absorption spectra of the p urified mutant enzyme were similar to those of the wild type enzyme, b ut 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 o f the mutant P450cam, measured on an anaerobic stopped flow apparatus, was 1/400 of that of the wild type enzyme and the dissociation consta nt of the mutant P450cam for oxidized Pd was several fold higher than that of the wild type enzyme. A considerable decrease in mid-point pot ential of the mutant enzyme was also noted. We conclude that Arg112, w hich is located on the surface of the P450cam molecule and hydrogen-bo nded to one of the heme propionate chains, plays an essential role in the electron transfer from Pd.