Quinone and nitrofurantoin redox cycling by recombinant cytochrome b5 reductase

Abstract

NADH cytochrome b₅ reductase mediates electron transfer from NADH to cytochrome b₅ utilizing flavin adenine dinucleotide as a redox cofactor. Reduced cytochrome b₅ is an important cofactor in many metabolic reactions including cytochrome P450-mediated xenobiotic metabolism, steroid biosynthesis and fatty acid metabolism, hemoglobin reduction, and methionine and plasmalogen synthesis. Using recombinant human enzyme, we discovered that cytochrome b5 reductase mediates redox cycling of a variety of quinones generating superoxide anion, hydrogen peroxide, and, in the presence of transition metals, hydroxyl radicals. Redox cycling activity was oxygen-dependent and preferentially utilized NADH as a co-substrate; NADH was 5-10 times more active than NADPH in supporting redox cycling. Redox cycling activity was greatest for 9,10-phenanthrenequinone and 1,2-naphthoquinone, followed by 1,4-naphthoquinone and 2-methyl-1,4-naphthoquinone (menadione), nitrofurantoin and 2-hydroxyestradiol. Using menadione as the substrate, quinone redox cycling was found to inhibit reduction of cytochrome b₅ by cytochrome b₅ reductase, as measured by heme spectral changes in cytochrome b₅. Under anaerobic conditions where redox cycling is inhibited, menadione had no effect on the reduction of cytochrome b₅. Chemical redox cycling by cytochrome b₅ reductase may be important in generating cytotoxic reactive oxygen species in target tissues. This activity, together with the inhibition of cytochrome b₅ reduction by redox-active chemicals and consequent deficiencies in available cellular cytochrome b₅, are likely to contribute to tissue injury following exposure to quinones and related redox active chemicals.

Keywords: Cytochrome b5; Free radicals; Reactive oxygen species; Redox cycling.