Reactions of phenoxyl radicals with NADPH-cytochrome P-450 oxidoreductase and NADPH: reduction of the radicals and inhibition of the enzyme.

Abstract

Phenoxyl radicals are intermediates of one-electron oxidation of phenolic compounds by various peroxidases. This report describes reactions of phenoxyl radicals with human NADPH-cytochrome P-450 oxidoreductase (OR) and NADPH. Purified truncated OR catalyzed quenching of EPR signal of the phenoxyl radical of a vitamin E homolog, 2,2,5,7,8-pentamethyl-6-hydroxychromane. The quenching required both reductase and NADPH and was not supported by NADH. NADPH quenched directly the EPR signal of phenoxyl radical of a phenolic antitumor drug, etoposide, in the absence of the OR. Quenching of the EPR signal was accompanied by increased rate of NADPH oxidation and decreased rate of etoposide oxidation. Phenoxyl radicals of etoposide did not inactivate the OR. In the absence of NADPH, OR was inhibited irreversibly when exposed to phenoxyl radicals of phenol. The activity of the flavoprotein could not be recovered by dithiothreitol (DTT) but the inhibition was prevented by saturation of OR with NADP+ prior to the exposure to phenoxyl radicals. The OR was also inhibited by 5,5'-dithionitrobenzoic acid (DTNB). The inhibition was reversible by subsequent addition of DTT. OR pretreated with DTNB was protected from inhibition by phenoxyl radicals of phenol. The results indicate that phenoxyl radical of 2,2,5,7,8-pentamethyl-6-hydroxychromane is likely reduced enzymatically by transfer of electrons from NADPH via the FAD/FMN of the OR. Phenoxyl radicals with higher redox potential, e.g., phenoxyl radicals of etoposide, oxidize NADPH directly. Phenoxyl radicals of phenol can also inactivate OR likely by oxidation of cysteine 565 in the NADPH binding region of the enzyme.