CHARACTERIZATION OF RECOMBINANT ADRENODOXIN REDUCTASE HOMOLOG (ARH1P)FROM YEAST - IMPLICATION IN IN-VITRO CYTOCHROME P45011-BETA MONOOXYGENASE SYSTEM

The mammalian electron transfer chain of mitochondrial cytochrome P450 forms involved in steroidogenesis includes very specific proteins, na mely adrenodoxin reductase and adrenodoxin. Adrenodoxin reductase tran sfers electrons from NADPH to adrenodoxin, which subsequently donates them to the cytochrome P450 forms. The Saccharomyces cerevisiae ARH1 g ene product (Arh1p) presents homology to mammalian adreanodoxin reduct ase. We demonstrate the capacity of recombinant Arh1p, made in Escheri chia coli, to substitute for its mammalian homologue in ferricyanide, cytochrome b: reduction, and, more importantly, in vitro 11 beta-hydro xylase 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 st udies have localized Arh1p to the inner membrane of yeast mitochondria . The biological function of Arh1p remains unknown, and to date, no mi tochondrial cytochrome P450 has been identified. ARH1 is, however, ess ential for yeast viability because ale ARH1 gene disruption is lethal not only in aerobic growth conditions but also, surprisingly enough, d uring fermentation.