title: NCB5OR is a novel soluble NAD(P)H reductase localized in the endoplasmic reticulum.
abstract: 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.
authors.affiliation: Hematology Division, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA. haozhu@rics.bwh.harvard.edu
authors.forename: Hao
authors.initials: H
authors.lastname: Zhu
authors.forename: Kevin
authors.initials: K
authors.lastname: Larade
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Jackson
authors.forename: Jianxin
authors.initials: J
authors.lastname: Xie
authors.forename: Annie
authors.initials: A
authors.lastname: Ladoux
authors.forename: Helmut
authors.initials: H
authors.lastname: Acker
authors.forename: Utta
authors.initials: U
authors.lastname: Berchner-Pfannschmidt
authors.forename: Joachim
authors.initials: J
authors.lastname: Fandrey
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Cross
authors.forename: Gudrun S
authors.initials: GS
authors.lastname: Lukat-Rodgers
authors.forename: Kenton R
authors.initials: KR
authors.lastname: Rodgers
authors.forename: H Franklin
authors.initials: HF
authors.lastname: Bunn
mesh_terms: 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
chemical_list: 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