Reduction Kinetics of Purified Rat Liver Cytochrome P-450. Evidence for a Sequential Reaction Mechanism Dependent on the Hemoprotein Spin State

The anaerobic reduction kinetics of purified rat liver ferric cytochrome P-450 from phenobarbital-treated rat liver microsomes, reconstituted with saturating NADPH-cy-tochrome P-450 reductase, have been investigated and were shown not to be monophasic. From experiments correlating changes in the rate of fast-phase reduction with the spin state of the heme iron existing at preequilibrium, data were obtained consistent with a model for spin-state control of cytochrome P-450 reduction wherein the high-spin form of the hemoprotein is more rapidly reduced than the low-spin form. In addition, the temperature dependence of the reduction process in the presence of the substrate benzphetamine was studied. From the results obtained it is suggested that the endothermic nature of the low- to high-spin transition largely accounts for the apparent activation energy observed for the reduction process, with the actual rate constant for reduction of high-spin cytochrome P-450 being relatively temperature insensitive when compared to the rate constant for reduction of the membrane-bound form of the hemoprotein.