Examining the mechanism of stimulation of cytochrome P450 by cytochrome b5: the effect of cytochrome b5 on the interaction between cytochrome P450 2B4 and P450 reductase.
Dissociation constants K(d) for cytochrome P450 reductase (reductase) and cytochrome P450 2B4 are measured in the presence of various substrates. Aminopyrine increases the dissociation constant for binding of the two proteins. Furthermore, cytochrome b(5) (b(5)) stimulates metabolism of this substrate and dramatically decreases the substrate-related K(d) values. Experiments are performed to test if the b(5)-mediated stimulation is effected through a conformational change of P450. The effects of a redox-inactive analogue of b(5) (Mn b(5)) on product formation and reaction stoichiometry are determined. Variations in the concentration of Mn b(5) stock solution that have been shown to effect the aggregation state of the protein alter the rate of P450-mediated NADPH oxidation but have no effect on the rate of product formation. Thus, the electron transfer capability of b(5) is necessary for stimulation of metabolism. Furthermore, stopped flow spectrometry measurements of the rate of first electron reduction of the P450 by reductase indicate that the coupling of P450 2B4-mediated metabolism improves, in the presence of Mn b(5), with slower delivery of the first electron of the catalytic cycle by the reductase. These results are consistent with a model involving the regulation of the P450 catalytic cycle by conformational changes of the P450 enzyme. We propose that the conformational change(s) necessary for progression of the catalytic cycle is inhibited when reduced, but not oxidized, reductase is bound to the P450.
D000693:Anaerobiosis; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D015786:Cytochromes b5 / Q000378:metabolism*; D007700:Kinetics; D008110:Liver Extracts; D008297:Male; D008345:Manganese / Q000494:pharmacology; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D051381:Rats; D020318:Rats, Long-Evans
D008110:Liver Extracts; D008345:Manganese; D015786:Cytochromes b5; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit); D009251:NADPH-Ferrihemoprotein Reductase