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

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

Spectral intermediates in the reaction of oxygen with purified liver microsomal cytochrome P-450

Stopped flow spectrophotometry has shown the occurrence of two distinct spectral intermediates in the reaction of oxygen with the reduced form of highly purified cytochrome P-450 from liver microsomes. As indicated by difference spectra, Complex I (with maxima at 430 and 450 nm) is rapidly formed and then decays to form Complex II (with a broad maximum at 440 nm), which resembles the intermediate seen in steady state experiments. In the reaction sequence, P-450LMredComplex I-->Complex II-->P-450LMox the last step is rate-limiting. The rate of that step is inadequate to account for the known turnover number of the enzyme in benzphetamine hydroxylation unless NADPH-cytochrome P-450 reductase or cytochrome is added. The latter protein does not appear to function as an electron carrier in this process.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/21753/1/0000147.pd

1,3-Butadiene: linking metabolism, dosimetry, and mutation induction.

There is increasing concern for the potential adverse health effects of human exposures to chemical mixtures. To better understand the complex interactions of chemicals within a mixture, it is essential to develop a research strategy which provides the basis for extrapolating data from single chemicals to their behavior within the chemical mixture. 1,3-Butadiene (BD) represents an interesting case study in which new data are emerging that are critical for understanding interspecies differences in carcinogenic/genotoxic response to BD. Knowledge regarding mechanisms of BD-induced carcinogenicity provides the basis for assessing the potential effects of mixtures containing BD. BD is a multisite carcinogen in B6C3F1 mice and Sprague-Dawley rats. Mice exhibit high sensitivity relative to the rat to BD-induced tumorigenesis. Since it is likely that BD requires metabolic activation to mutagenic reactive epoxides that ultimately play a role in carcinogenicity of the chemical, a quantitative understanding of the balance of activation and inactivation is essential for improving our understanding and assessment of human risk following exposure to BD and chemical mixtures containing BD. Transgenic mice exposed to 625 ppm BD for 6 hr/day for 5 days exhibited significant mutagenicity in the lung, a target organ for the carcinogenic effect of BD in mice. In vitro studies designed to assess interspecies differences in the activation of BD and inactivation of BD epoxides reveal that significant differences exist among mice, rats, and humans. In general, the overall activation/detoxication ratio for BD metabolism was approximately 10-fold higher in mice compared to rats or humans.(ABSTRACT TRUNCATED AT 250 WORDS