Cytochrome P-450 as a microsomal peroxidase utilizing a lipid peroxide substrate

Abstract

Linoleic acid hydroperoxide (LAHPO) when incubated with heme compounds or liver microsomes is rapidly decomposed, presumably by a free-radical mechanism, to yield a complex range of products. In this study, a spectrophotometric method has been developed for investigating the peroxidase reaction using N,N,N′,N′-tetramethyl- p-phenylenediamine (TMPD) as hydrogen donor and LAHPO as substrate. An intracellular distribution study showed that the mitochondrial and microsomal fractions from rat liver exhibited the highest peroxidase activity per milligram protein. The microsomal peroxidase activity had a pH optimum of 4.7, was inhibited 50% by 1 m m cyanide, and was heat labile. Compounds that can form type I and type II spectra with cytochrome P-450 inhibited the peroxidase activity. Microsomes from phenobarbital-injected rats exhibited a 2.5-fold higher specific P-450 content and showed a similarly enhanced peroxidase activity. The peroxidase activity of microsomes was enhanced 2–8-fold by reagents that converted cytochrome P-450 to P-420 (e.g., lysolecithin, p-hydroxymercuribenzoate, N-bromosuccinimide, iodine, trypsin, deoxycholate). Isolation of the microsomal cytochromes showed that cytochrome b 5 had low peroxidase activity whereas microsomal “P-450 particles” containing P-450 as the sole protoheme constituent were very active. In the absence of a hydrogen donor, LAHPO destroyed cytochrome P-450 rapidly and inhibited demethylation activity in liver microsomes but did not affect cytochrome b 5. It was concluded that cytochrome P-450 was responsible for most of the peroxidase activity of liver microsomes. A mechanism for the microsomal peroxidase activity is proposed in which LAHPO oxidizes the P-450 thiol ligand to form high spin P-420. The latter then acts as a peroxidase and decomposes LAHPO.