Properties of the oxygenated form of liver microsomal cytochrome P-450.
The formation and autoxidative decomposition of the oxygenated forms of two isozymes of rabbit liver microsomal cytochrome P-450 were studied: 5,6-benzoflavone- or isosafrole-inducible P-450LM4, isolated in the high spin state, and P-450LM3b, isolated in the low spin state. When an anaerobic solution of photochemically reduced isozyme 4 was mixed with aerobic buffer in a stopped flow spectrophotometer, the dioxygen complex with absorption maxima at 555 and 418 nm was rapidly formed. The monophasic reaction had a pseudo-first order rate constant of about 58 s-1. Autoxidation of the complex, which was complete in about 20 s, exhibited biphasic first order kinetics at 580 nm with rate constants of about 0.92 and 0.22 s-1. The results obtained with isozyme 3b were similar, except that the decomposition of the ferrous oxy intermediate appeared to be triphasic. Superoxide could not be detected as a product of the autoxidative decomposition of the oxy form of P-450 isozyme 4. Hydrogen peroxide was produced in about 70% yield when oxygen was in excess, whereas in a titration in which small increments of oxygen were added to an excess of the ferrous cytochrome the reduction apparently led to the formation of water. The biphasic kinetics of some of the reactions involving purified mammalian cytochrome P-450 has in some instances been attributed to the formation of aggregates by these hydrophobic proteins. This was ruled out as an explanation of the kinetics observed for the autoxidative decay of ferrous dioxygen P-450LM4, since two phases were also observed with a preparation converted from the usual aggregated state to the monomeric state by exposure to a zwitterionic detergent.
D000693:Anaerobiosis; D000818:Animals; D001571:Benzoflavones / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D007536:Isomerism; D007700:Kinetics; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D010084:Oxidation-Reduction; D011817:Rabbits; D012451:Safrole / Q000494:pharmacology; D013053:Spectrophotometry; D019324:beta-Naphthoflavone
D001571:Benzoflavones; D019324:beta-Naphthoflavone; D003577:Cytochrome P-450 Enzyme System; D012451:Safrole; C015959:isosafrole