REACTIONS OF PHENOXYL RADICALS WITH NADPH-CYTOCHROME P-450 OXIDOREDUCTASE AND NADPH - REDUCTION OF THE RADICALS AND INHIBITION OF THE ENZYME

Phenoxyl radicals are intermediates of one-electron oxidation of pheno lic 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 signa l of the phenoxyl radical of a vitamin E homolog, 2,2,5,7,8-pentamethy l-6-hydroxychromane. The quenching required both reductase and NADPH a nd was not supported by NADH. NADPH quenched directly the EPR signal o f phenoxyl radical of a phenolic antitumor drug, etoposide, in the abs ence of the OR. Quenching of the EPR signal was accompanied by increas ed rate of NADPH oxidation and decreased rate of etoposide oxidation. Phenoxyl radicals of etoposide did not inactivate the OR, In the absen ce of NADPH, OR was inhibited irreversibly when exposed to phenoxyl ra dicals of phenol. The activity of the flavoprotein could not be recove red by dithiothreitol (DTT) but the inhibition was prevented by satura tion of OR with NADP(+) prior to the exposure to phenoxyl radicals. Th e OR was also inhibited by 5,5'-dithionitrobenzoic acid (DTNB). The in hibition was reversible by subsequent addition of DTT. OR pretreated w ith 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 electr ons from NADPH via the FAD/FMN of the OR. Phenoxyl radicals with highe r redox potential, e.g., phenoxyl radicals of etoposide, oxidize NADPH directly. Phenoxyl radicals of phenol can also inactivate OR likely b y oxidation of cysteine 565 in the NADPH binding region of the enzyme.