The peroxidase properties of cytochrome P-450.

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. The reaction was found to be first-order with respect to catalyst concentration and first-order with respect to LAHPO concentration. -- The mitochondrial and microsomal fractions from rat liver exhibited the highest peroxidase activity per mg protein. The microsomal peroxidase activity had a pH optimum of 4.7, was inhibited 50% by 1 mM cyanide and was unaffected by EDTA. The peroxidase activity of microsomes was enhanced 2- to 8-fold by reagents that converted cytochrome P-450 to P-420 (e.g., lysolecithin, p-hydroxymercuribenzoate, N-bromosuccinimide, iodine, trypsin, deoxycholate). Microsomes from phenobarbital-injected rats exhibited a 2.5-fold higher specific P-450 content and showed a similarly enhanced peroxidase activity. -- LAHPO destroyed cytochrome P-450 rapidly and inhibited demethylation activity in liver microsomes. Cytochrome b₅ had low peroxidase activity whereas microsomal “P-450 particles” containing P-450 as the sole prothoheme constituent were very active. It was concluded that cytochrome P-450 is 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 which then acts as a peroxidase. -- The peroxidase properties of cytochrome P-420, in its “high spin” and “low spin” forms, were next investigated using a preparation of P-420 from hepatic microsomal “P-450 particles”. The peroxidase activity of “high spin” P-420 was inhibited 50% by 1 mM cyanide, was completely abolished by boiling and was unaffected by EDTA. The cyanide difference spectrum of oxidized “high spin” p-420 revealed a peak at 426 nm, a trough at 403 nm and a binding constant for cyanide of about 1 mM. “high spin” and “low spin” cytochromes P-420 were rapidly destroyed when incubated with LAHPO. -- A comparison of the effectiveness of various heme compounds in catalyzing the peroxidase reaction showed that “high spin” P-420 was the most effective catalyst. Other high spin heme compounds such as hematin and methemoglobin were also very effective catalysts whereas low spin hemoproteins such as “low spin” P-420, “low spin” P-450, cytochrome b₅, and oxyhemoglobin exhibited much lower catalytic activities. “High spin” P-420 and thyroid peroxidase showed remarkably similar spectral properties and it is suggested that the two peroxidases may be identical hemoproteins.