Characterization of recombinant adrenodoxin reductase homologue (Arh1p) from yeast -: Implication in in vitro cytochrome P45011β monooxygenase system

The mammalian electron transfer chain of mitochondrial cytochrome P450 forms involved in steroidogenesis includes very specific proteins, namely adrenodoxin reductase and adrenodoxin. Adrenodoxin reductase transfers electrons from NADPH to adrenodoxin, which subsequently donates them to the cytochrome P450 forms. The Saccharomyces cerevisiae ARH1 gene product (Arh1p) presents homology to mammalian adreanodoxin reductase. We demonstrate the capacity of recombinant Arh1p, made in Escherichia coli, to substitute for its mammalian homologue in ferricyanide, cytochrome b: reduction, and, more importantly, in vitro 11 beta-hydroxylase assays. Electrons could be transfer-red from NADPH and NADH as measured in the cytochrome c reduction assay. Apparent K-m values were determined tea be 0.5, 0.6, and 0.1 mu M for NADPH, NADH, and bovine adrenodoxin, respectively, These values differ slightly from those of mammalian adrenodoxin reductase, except for NADH, which is a very poor electron donor to the mammalian protein. Subcellular fractionation studies have localized Arh1p to the inner membrane of yeast mitochondria. The biological function of Arh1p remains unknown, and to date, no mitochondrial cytochrome P450 has been identified. ARH1 is, however, essential for yeast viability because ale ARH1 gene disruption is lethal not only in aerobic growth conditions but also, surprisingly enough, during fermentation.