Identification of a cytochrome b-type NAD(P)H oxidoreductase ubiquitously expressed in human cells.
Cytochrome b-type NAD(P)H oxidoreductases are involved in many physiological processes, including iron uptake in yeast, the respiratory burst, and perhaps oxygen sensing in mammals. We have identified a cytosolic cytochrome b-type NAD(P)H oxidoreductase in mammals, a flavohemoprotein (b5+b5R) containing cytochrome b5 (b5) and b5 reductase (b5R) domains. A genetic approach, using BLAST searches against DBEST for FAD-, NAD(P)H-binding sequences followed by reverse transcription-PCR, was used to clone the complete cDNA sequence of human b5+b5R from the hepatoma cell line Hep 3B. Compared with the classical single-domain b5 and b5R proteins localized on endoplasmic reticulum membrane, b5+b5R also has binding motifs for heme, FAD, and NAD(P)H prosthetic groups but no membrane anchor. The human b5+b5R transcript was expressed at similar levels in all tissues and cell lines that were tested. The two functional domains b5* and b5R* are linked by an approximately 100-aa-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 revealed a cytosolic localization at the perinuclear space. The recombinant b5+b5R protein can be reduced by NAD(P)H, generating spectrum typical of reduced cytochrome b with alpha, beta, and Soret peaks at 557, 527, and 425 nm, respectively. Human b5+b5R flavohemoprotein is a NAD(P)H oxidoreductase, demonstrated by superoxide production in the presence of air and excess NAD(P)H and by cytochrome c reduction in vitro. The properties of this protein make it a plausible candidate oxygen sensor.
Zhu
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Hematology Division, Brigham and Women's Hospital, Harvard Medical School, 221 Longwood Avenue, Boston, MA 02115, USA.
Qiu
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H
Yoon
H W
HW
Huang
S
S
Bunn
H F
HF
D000595:Amino Acid Sequence; D002451:Cell Compartmentation; D003579:Cytochrome Reductases / Q000235:genetics*; D042966:Cytochrome-B(5) Reductase; D015786:Cytochromes b5 / Q000235:genetics*; D003600:Cytosol / Q000201:enzymology; D005420:Flavoproteins / Q000235:genetics; D015870:Gene Expression; D006420:Hemeproteins / Q000235:genetics; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics*; D009363:Neoplasm Proteins / Q000235:genetics; D010100:Oxygen / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014018:Tissue Distribution
D005420:Flavoproteins; D006420:Hemeproteins; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins; D015786:Cytochromes b5; D009247:NADH, NADPH Oxidoreductases; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase; D010100:Oxygen