Highly Purified Liver Microsomal Cytochrome P450: Properties and Catalytic Mechanism

Recent studies in this laboratory on two forms of cytochrome P450 purified to homogeneity from rabbit liver microsomes are reviewed. The two forms, phenobarbital-inducible P450LM2 and 5,6-benzoflavone-inducible P450LM4, differ in subunit molecular weight, identity of the C-terminal amino acid, optical and EPR spectra, and other properties. As isolated, oxidized P450LM2 is in the low spin state, whereas P450LM4 is largely, but non entirely, in the high spin state. Mechanistic studies have shown the following: (a) P450LM2 may accept two electrons, calculated per heme, from dithionite or NADPH in the presence of catalytic amounts of the reductase, and may donate two electrons to various oxidizing agents, including molecular oxygen. (b) Hydrogen peroxide is formed in the reconstituted system in the presence of NADPH and oxygen, and the amount varies with the substrate added. (c) Hydrogen peroxide and other hydroperoxides apparently donate the oxygen atom inserted into substrate during hydroxylation in the absence of 0 2 and an external donor. (d) Stopped flow spectrophotometry has provided evidence for two distinct oxygenated complexes of the reduced cytochrome. The reductase and cytochrome b5 may play an effector role in increasing the rate of decomposition of the second complex during oxygen insertion into substrate. A scheme is proposed for the mechanism of action of purified P450LM2, based on these and other findings