Regulative mechanisms in NADH- and NADPH-supported N-oxidation of 4-chloroaniline catalyzed by cytochrome b5-enriched rabbit liver microsomal fractions.
Incorporation into rabbit liver microsomal membranes of detergent-solubilized cytochrome b5 stimulates NADH-supported electron flow to ferric cytochrome P-450, but impairs NADPH-dependent reduction of the pigment such as to make the rates of both reactions equivalent; yet, in the enriched preparations NADPH-driven N-oxidation of 4-chloroaniline proceeds at considerably higher rate than does the NADH-supported process. Analysis of transfer of the second electron to oxyferrous cytochrome P-450, as assessed by measuring substrate-induced reoxidation of ferrous cytochrome b5, reveals faster flow with NADH than with NADPH as the source of reducing equivalents. Quantification of the pools of cytochrome P-450 active in attack on the amine substrate in the presence of either reduced pyridine nucleotide, as well as measurements of maximum arylamine turnover suggest that the cofactor-dependent discrepancy in N-oxidase activity reflects differences in the rates of breakdown of the intermediary enzyme complexes. The NADH- and NADPH-supported pathway of N-oxidation in the cytochrome b5-supplemented microsomal fractions thus probably involves distinct forms of cytochrome P-450. Alternatively, functional linkage of the cofactor-specific electron-transfer chains to a single cytochrome P-450 species might yield aggregates of differing conformational state and catalytic capacity. The latter concept receives support from experiments with individually reconstituted enzyme systems.
D000814:Aniline Compounds / Q000378:metabolism*; D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D003579:Cytochrome Reductases / Q000378:metabolism*; D003573:Cytochrome b Group / Q000378:metabolism*; D042966:Cytochrome-B(5) Reductase; D015786:Cytochromes b5; D004579:Electron Transport; D008862:Microsomes, Liver / Q000201:enzymology*; D008954:Models, Biological; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D010084:Oxidation-Reduction; D011817:Rabbits
D000814:Aniline Compounds; D003573:Cytochrome b Group; D009243:NAD; D009249:NADP; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase; D009251:NADPH-Ferrihemoprotein Reductase; C004658:4-chloroaniline