The mechanism for the reduction of ferric cytochrome P450_cam by reduced putidaredoxin, the physiological electron donor for the cytochrome, has been studied by using site-directed mutants of cytochrome P450_cam, 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 P450_cam 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 P450_cam 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 P450_cam (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 P450_cam, thereby diminishing the rate of electron transfer between the two metal centers. The rate of electron transfer from putidaredoxin to oxyferrous cytochrome P450_cam also diminished upon substitution of Arg¹¹² with a neutral amino acid.