H. Zhu et al., Identification of a cytochrome b-type NAD(P)H oxidoreductase ubiquitously expressed in human cells, P NAS US, 96(26), 1999, pp. 14742-14747
Citations number
36
Categorie Soggetti
Multidisciplinary
Journal title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
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.