KINETICS OF FERRIC CYTOCHROME-P450 REDUCTION BY NADPH-CYTOCHROME P450REDUCTASE - RAPID REDUCTION IN THE ABSENCE OF SUBSTRATE AND VARIATIONS AMONG CYTOCHROME-P450 SYSTEMS

The reduction of ferric cytochrome P450 (P450) to ferrous is the first chemical step in almost all P450 reactions, and many characteristics of this step have been reported. Reduction kinetics of rabbit and huma n P450s were measured in a variety of systems. As reported earlier, P4 50 reduction is biphasic in microsomes and some purified P450 systems, However, this is not an inherent property of P450s, and some low-and high-spin iron P450s were reduced with single-exponential kinetics. Co ntrary to a generalized view, the presence of substrate is not necessa ry for rapid reduction of all P450s. Also, low-spin heme can be reduce d as rapidly as high-spin in several P450s. P450s varied considerably in their reduction behavior, and even a single P450 showed remarkably different reduction kinetics when placed in various environments, P450 3A4 reduction was examined in liver microsomes, a reconstituted syste m, a fusion protein in which it was linked to NADPH-P450 reductase, an d baculovirus and bacterial membranes in which P450 3A4 and NADPH-P450 reductase were coexpressed; the systems differed considerably in term s of the need for the substrate testosterone and cytochrome b(5) (b(5) ) for reduction and as to whether reduction was rate-limiting in the o verall catalytic cycle, When b(5) was included in reconstituted system s, its reduction kinetics were linked with those of some P450s. This b ehavior could be simulated in kinetic models in which electrons flowed from the ferrous P450 CO complex to oxidized b(5). Overall, the kinet ics of ferric P450 reduction cannot be generalized among different P45 0s in various systems, and concepts regarding influence of substrate, reaction sequence, and a rate-limiting step are not very universal.