Direct evidence for electron transfer from ferrous cytochrome b5 to the oxyferrous intermediate of liver microsomal cytochrome P-450 LM2.
Interaction and electron transfer between highly purified microsomal cytochrome P-450 from phenobarbital-induced rabbits and cytochrome b5 from uninduced rabbits was investigated by difference and stopped-flow spectrophotometry. Formation of a 1:1 complex between ferric P-450 and b5, demonstrated by difference spectrophotometry, was observed only when both cytochromes were incorporated into micelles of phosphatidylcholine. The dissociation constant (Kd) of the complex was decreased from 2.3 microM to 0.4 microM in the presence of 1 mM benzphetamine. The apparent Kd of benzphetamine was reduced from 220 microM to 50 microM upon addition of b5. The influence of ferrous b5 on the autooxidation of the oxyferrous intermediate of P-450 in the presence and in the absence of substrate was investigated by stopped-flow spectrophotometry. Both cytochromes were reduced photochemically, so that experiments could be carried out in the absence of the corresponding reductases and reduced pyridine nucleotides. Kinetic analysis of the data showed that formation of a 1:1 complex between ferrous P-450 and b5 was a prerequisite for electron transfer between the cytochromes. Here again, incorporation of both cytochromes into micelles was absolutely required for this process. Kd was decreased from 7.5 microM to 2.2 microM in the presence of 1 mM benzphetamine. The rate of electron transfer from b5 to oxyferrous P-450, derived from the kinetics of reoxidation of either b5 or P-450, was increased from 2.5 s-1 to about 4 to 7 s-1 in the presence of 1 mM benzphetamine. These results provide the first quantitative data on the electron transfer between the b5 and P-450, whose rate constant is compatible with the observations made on the effect of b5 on hydroxylation reactions catalyzed by the P-450 enzyme system.
D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003580:Cytochromes / Q000378:metabolism*; D015786:Cytochromes b5; D004579:Electron Transport; D007700:Kinetics; D008823:Micelles; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010634:Phenobarbital / Q000494:pharmacology; D011817:Rabbits; D013053:Spectrophotometry
D003580:Cytochromes; D008823:Micelles; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D010634:Phenobarbital