ROLE OF ARG(112) OF CYTOCHROME P450(CAM) IN THE ELECTRON-TRANSFER FROM REDUCED PUTIDAREDOXIN - ANALYSES WITH SITE-DIRECTED MUTANTS

The mechanism for the reduction of ferric cytochrome P450(cam) by redu ced putidaredoxin, the physiological electron donor for the cytochrome , has been studied by using site-directed mutants of cytochrome P450(c am), in which Arg(112), an amino acid residue at the presumed binding site for putidaredoxin, was changed to several other amino acid residu es. The affinity of reduced putidaredoxin for ferric cytochrome P450(c am) to form a diprotein complex was decreased greatly by changing Arg( 112) to a neutral amino acid such as Cys, Met, or Tyr. The rate of int racomplex electron transfer from putidaredoxin to cytochrome P450(cam) also diminished upon replacing the basic residue with neutral ones, b eing 42, 18, 4.0, 1.3, and 0.16 s(-1) for Arg (wild type), Lys, Cys, M et, and Tyr enzymes, respectively. Furthermore, the oxidation-reductio n potential of cytochrome P450(cam) (Fe3+/Fe2+ couple) decreased in a similar way to the decrease in the rate of electron transfer upon amin o acid substitution; the values were -138, -162, -182, -200, and -195 mV for Arg (wild type), Lys, Cys, Met, and Tyr enzymes, respectively. These results indicate that the amino acid substitution at position 11 2 affects the oxidation-reduction potential of the heme iron in cytoch rome P450(cam), thereby diminishing the rate of electron transfer betw een the two metal centers. The rate of electron transfer from putidare doxin to oxyferrous cytochrome P450(cam) also diminished upon substitu tion of Arg(112) with a neutral amino acid.