Reduction of cytochrome b5 by NADPH–cytochrome P450 reductase

Abstract

The reduction of mammalian cytochrome b 5 ( b 5) by NADPH–cytochrome P450 (P450) reductase is involved in a number of biological reactions. The kinetics of the process have received limited consideration previously, and a combination of pre-steady-state (stopped-flow) and steady-state approaches was used to investigate the mechanism of b 5 reduction. In the absence of detergent or lipid, a reductase– b 5 complex is formed and rearranges slowly to an active form. Electron transfer to b 5 is rapid within this complex (>30 s −1 at 23 °C), as fast as to cytochrome c. With excess b 5 present, a burst of reduction is observed, consistent with rapid electron transfer to one or two b 5 molecules per reductase, followed by a subsequent rate-limiting event. In detergent vesicles, the reductase and b 5 interact rapidly but electron transfer is slower (∼3 s −1 at 23 °C). Experiments with dimyristyl lecithin vesicles yielded results intermediate between the non-vesicle and detergent systems. These steady-state and pre-steady-state kinetics provide views of the different natures of the reduction of b 5 by the reductase in the absence and presence of vesicles. Without vesicles, the encounter of the reductase and b 5 is rapid, followed by a slow reorganization of the initial complex (∼0.07 s −1), very fast reduction, and dissociation. In vesicles, encounter is rapid and the slow step (∼3 s −1) is reduction within a complex less favorable for reduction than in the non-vesicle systems.