Interaction and electron transfer between cytochrome b5 and cytochrome P-450 in the reconstituted p-nitroanisole O-demethylase system.
The interaction and electron transfer between cytochrome b5 and cytochrome P-450B1 were investigated using the reconstituted p-nitroanisole O-demethylase system. Apocytochrome b5 was prepared from detergent-solubilized cytochrome b5 by the acid-butanone method. The apocytochrome b5 thus obtained has been substituted with several metalloporphyrin derivatives. The reconstituted system containing cytochrome b5 substituted with heme derivatives such as proto-, meso-, and deuteroheme exhibited demethylation activity at the maximum turnover rates of 94, 58, 30%, respectively, compared to that containing the native cytochrome b5, while neither apocytochrome b5 nor cobaltic protoporphyrin-cytochrome b5 displayed the activity. Kinetic analysis showed the formation of a 1:1 complex between cytochrome P-450B1 and each of these substituted cytochrome b5's, except for cobaltic protoporphyrin-cytochrome b5; the affinities differed with the cytochrome b5 species used. The synergistic effect with the addition of the NADH-linked electron transport system was more remarkable at the lower reduction levels of cytochrome b5 in the steady state. Interaction between the components involved in NADH- and NADPH-linked electron transport systems was modulated by the existence of Triton X-100. The optimal concentration in the reconstituted system for the demethylation was observed at around 0.03% of Triton X-100, where the reduction rates for cytochrome b5 and cytochrome P-450B1 by the respective reductases were maximal. These results indicate that the two electron transport systems are closely coupled and exhibit the demethylase activity.
D000818:Animals; D001051:Apoenzymes / Q000302:isolation & purification; D055598:Chemical Phenomena; D002621:Chemistry; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003573:Cytochrome b Group / Q000378:metabolism*; D015786:Cytochromes b5; D004579:Electron Transport; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009575:Nitroanisole O-Demethylase / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism*; D011817:Rabbits
D001051:Apoenzymes; D003573:Cytochrome b Group; D009243:NAD; D009249:NADP; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D009575:Nitroanisole O-Demethylase; D010088:Oxidoreductases