Reduction of cytochrome b5 by NADPH-cytochrome P450 reductase

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 degrees 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 b5 molecules per reductase, followed by a subsequent rate-limiting event. In detergent vesicles, the reductase and b5 interact rapidly but electron transfer is slower (similar to 3 s(-1) at 23 degrees 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 b5 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 (similar to 0.07 s(-1)), very fast reduction, and dissociation. In vesicles, encounter is rapid and the slow step (similar to 3 s(-1)) is reduction within a complex less favorable for reduction than in the non-vesicle systems. (c) 2005 Elsevier Inc. All rights reserved.