Microsomal iron-dependent NADPH oxidation: Evidence for the involvement of membrane-bound nonheme iron in NADPH oxidation by rat heart microsomes

Abstract

Rat heart microsomes were found to contain nonheme iron and two lines of evidence suggested that this iron was involved in NADPH oxidation. As first evidence, pretreatment of rats with iron gluconate increased microsomal iron content and NADPH oxidation. As second evidence, treatment of microsomes with nonionic detergent Triton N-101 decreased membrane iron content and NADPH oxidation. Triton N-101-solubilized nonheme iron was nondialyzable and ammonium sulfate-precipitable, indicative of association with protein(s). This protein-bound iron per se did not oxidize NADPH but its addition to detergent-treated microsomes restored very high rates of NADPH oxidation, that were abolished by inhibiting NADPH-cytochrome P450 reductase with p-hydroxymercuribenzoate. Since heart microsomes did not contain cytochrome P450, these results suggested that stimulation of NADPH oxidation was mediated by direct electron transfer from reductase to iron. Purified rat heart ferritin and hemosiderin did not stimulate NADPH oxidation and the stimulation observed with detergent-solubilized microsomal iron was much higher than that observed with EDTA-Fe 3+, a very effective electron acceptor for the reductase. This suggested that (i) microsomal iron was different from other intracellular iron-storage proteins, and (ii) microsomal iron was unusually permissive to one-electron transfer from reductase.