NCB5OR is a novel soluble NAD(P)H reductase localized in the endoplasmic reticulum.
The NAD(P)H cytochrome b5 oxidoreductase, Ncb5or (previously named b5+b5R), is widely expressed in human tissues and broadly distributed among the animal kingdom. NCB5OR is the first example of an animal flavohemoprotein containing cytochrome b5 and chrome b5 reductase cytodomains. We initially reported human NCB5OR to be a 487-residue soluble protein that reduces cytochrome c, methemoglobin, ferricyanide, and molecular oxygen in vitro. Bioinformatic analysis of genomic sequences suggested the presence of an upstream start codon. We confirm that endogenous NCB5OR indeed has additional NH2-terminal residues. By performing fractionation of subcellular organelles and confocal microscopy, we show that NCB5OR colocalizes with calreticulin, a marker for endoplasmic reticulum. Recombinant NCB5OR is soluble and has stoichiometric amounts of heme and flavin adenine dinucleotide. Resonance Raman spectroscopy of NCB5OR presents typical signatures of a six-coordinate low-spin heme similar to those found in other cytochrome b5 proteins. Kinetic measurements showed that full-length and truncated NCB5OR reduce cytochrome c actively in vitro. However, both full-length and truncated NCB5OR produce superoxide from oxygen with slow turnover rates: kcat = approximately 0.05 and approximately 1 s(-1), respectively. The redox potential at the heme center of NCB5OR is -108 mV, as determined by potentiometric titrations. Taken together, these data suggest that endogenous NCB5OR is a soluble NAD(P)H reductase preferentially reducing substrate(s) rather than transferring electrons to molecular oxygen and therefore not an NAD(P)H oxidase for superoxide production. The subcellular localization and redox properties of NCB5OR provide important insights into the biology of NCB5OR and the phenotype of the Ncb5or-null mouse.
Zhu
Hao
H
Hematology Division, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA. haozhu@rics.bwh.harvard.edu
Larade
Kevin
K
Jackson
Timothy A
TA
Xie
Jianxin
J
Ladoux
Annie
A
Acker
Helmut
H
Berchner-Pfannschmidt
Utta
U
Fandrey
Joachim
J
Cross
Andrew R
AR
Lukat-Rodgers
Gudrun S
GS
Rodgers
Kenton R
KR
Bunn
H Franklin
HF
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D019556:COS Cells; D037282:Calreticulin / Q000378:metabolism; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D019295:Computational Biology; D042966:Cytochrome-B(5) Reductase / Q000096:biosynthesis* / Q000737:chemistry; D015786:Cytochromes b5 / Q000378:metabolism*; D045304:Cytochromes c / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005260:Female; D005292:Ferricyanides / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008706:Methemoglobin / Q000737:chemistry; D051379:Mice; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010641:Phenotype; D017785:Photons; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012689:Sequence Homology, Nucleic Acid; D013059:Spectrum Analysis, Raman; D013347:Subcellular Fractions / Q000378:metabolism; D013481:Superoxides / Q000737:chemistry; D013997:Time Factors; D014162:Transfection; D014466:Ultraviolet Rays
D037282:Calreticulin; D005292:Ferricyanides; D011994:Recombinant Proteins; D013481:Superoxides; C007931:hexacyanoferrate III; D006418:Heme; D045304:Cytochromes c; D008706:Methemoglobin; D015786:Cytochromes b5; D009247:NADH, NADPH Oxidoreductases; D042966:Cytochrome-B(5) Reductase; D010100:Oxygen