Effects of cytochrome b5 on cytochrome P-450-catalyzed reactions. Studies with manganese-substituted cytochrome b5.
The effects of cytochrome b5 with manganese-protoporphyrin IX substituted for heme were compared with those of native cytochrome b5 and the apoenzyme on the oxygenation of substrates in the reconstituted system containing liver microsomal cytochrome P-450, NADPH-cytochrome P-450 reductase, and phosphatidylcholine. Mn-b5, unlike b5, remains essentially fully oxidized in the presence of NADPH and NADPH-cytochrome P-450 reductase under aerobic conditions. The effects of various concentrations of b5 and its derivatives were determined at constant P-450 and reductase concentrations. Cytochrome b5 inhibits benzphetamine demethylation by isozyme 2, the effect increasing up to the highest concentrations tested, and stimulates 7-ethoxycoumarin deethylation by isozyme 2 and acetanilide p-hydroxylation by isozyme 4, the optimal b5:P-450 molar ratio being about 2. In contrast, Mn-b5 inhibits all three reactions and apo-b5 is either inactive or slightly inhibitory. The activities of the three substrates as well as testosterone were determined with P-450 isozymes 2, 3b, 3c, and 4 in the reconstituted system with no additions or with b5 or Mn-b5 present. Cytochrome b5 is stimulatory, inhibitory, or without any effect, the result depending on both the substrate and P-450 isozyme present, whereas Mn-b5 is inhibitory in most instances. Both b5 and its manganese derivative alter the rates of testosterone 6 beta- or 16 alpha-hydroxylation by most of the P-450 cytochromes. The activities are influenced by the molar ratio of reductase to P-450. The Km values of benzphetamine, ethoxycoumarin, and acetanilide are, with one exception, significantly decreased in the presence of b5 or Mn-b5. We conclude that some of the effects of b5 on the oxygenase system are not accounted for by its role as an electron donor to cytochrome P-450.
D000818:Animals; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003573:Cytochrome b Group / Q000378:metabolism*; D045303:Cytochromes b; D015786:Cytochromes b5; D006418:Heme / Q000378:metabolism; D006427:Hemin / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D011524:Protoporphyrins / Q000378:metabolism; D011817:Rabbits; D039181:Steroid 16-alpha-Hydroxylase; D013329:Structure-Activity Relationship
D003573:Cytochrome b Group; D011524:Protoporphyrins; D006418:Heme; D006427:Hemin; D045303:Cytochromes b; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D039181:Steroid 16-alpha-Hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase; D008274:Magnesium