Unno M, Shimada H, Toba Y, Makino R, Ishimura Y
Department of Biochemistry, School of Medicine, Keio University, Shinanomachi, Shinjuku-ku, Tokyo 160, Japan.
J Biol Chem. 1996 Jul 26;271(30):17869-74. doi: 10.1074/jbc.271.30.17869.
The mechanism for the reduction of ferric cytochrome P450cam by reduced putidaredoxin, the physiological electron donor for the cytochrome, has been studied by using site-directed mutants of cytochrome P450cam, in which Arg112, an amino acid residue at the presumed binding site for putidaredoxin, was changed to several other amino acid residues. The affinity of reduced putidaredoxin for ferric cytochrome P450cam to form a diprotein complex was decreased greatly by changing Arg112 to a neutral amino acid such as Cys, Met, or Tyr. The rate of intracomplex electron transfer from putidaredoxin to cytochrome P450cam also diminished upon replacing the basic residue with neutral ones, being 42, 18, 4.0, 1.3, and 0. 16 s-1 for Arg (wild type), Lys, Cys, Met, and Tyr enzymes, respectively. Furthermore, the oxidation-reduction potential of cytochrome P450cam (Fe3+/Fe2+ couple) decreased in a similar way to the decrease in the rate of electron transfer upon amino 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 112 affects the oxidation-reduction potential of the heme iron in cytochrome P450cam, thereby diminishing the rate of electron transfer between the two metal centers. The rate of electron transfer from putidaredoxin to oxyferrous cytochrome P450cam also diminished upon substitution of Arg112 with a neutral amino acid.
