Identification of a cytochrome b-type NAD(P)H oxidoreductase ubiquitously expressed in human cells

Cytochrome 6-type NAD(P)H oxidoreductases are involved in many physiologica l processes, including iron uptake in yeast, the respiratory burst, and per haps oxygen sensing in mammals. We have identified a cytosolic cytochrome b -type NAD(P)H oxidoreductase in mammals, a flavohemoprotein (b5+b5R) contai ning cytochrome b5 (b5) and b5 reductase (b5R) domains, A genetic approach, using BLAST searches against DBEST for FAD-, NAD(P)H-binding sequences fol lowed by reverse transcription-PCR, was used to clone the complete cDNA seq uence of human b5+b5R from the hepatoma cell line Hep 3B. Compared with the classical single-domain b5 and b5R proteins localized on endoplasmic retic ulum membrane, b5+b5R also has binding motifs for heme, FAD, and NAD(P)H pr osthetic: groups but no membrane anchor. The human b5+b5R transcript was ex pressed at similar levels in all tissues and cell lines that were tested. T he two functional domains b5* and b5R* are linked by an approximately 100-a a-long hinge bearing no sequence homology to any known proteins. When human b5+b5R was expressed as c-myc adduct in COS-7 cells, confocal microscopy r evealed a cytosolic localization at the perinuclear space. The recombinant b5+b5R protein can be reduced by NAD(P)H, generating spectrum typical of re duced cytochrome b with alpha, beta, and Soret peaks at 557, 527, and 425 n m, respectively. Human b5+b5R flavohemoprotein is a NAD(P)H oxidoreductase, demonstrated by superoxide production in the presence of air and excess NA D(P)H and by cytochrome c reduction in vitro. The properties of this protei n make it a plausible candidate oxygen sensor.