Publications

• NCB5OR is a novel soluble NAD(P)H reductase localized in the endoplasmic reticulum.

  Zhu H., Larade K., Jackson T.A., Xie J., Ladoux A., Acker H., Berchner-Pfannschmidt U., Fandrey J., Cross A.R., Lukat-Rodgers G.S., Rodgers K.R., Bunn H.F.

  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 re ... >> More

  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. << Less

  J. Biol. Chem. 279:30316-30325(2004) [PubMed] [EuropePMC]

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

  Zhu H., Qiu H., Yoon H.-W., Huang S., Bunn H.F.

  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) cont ... >> More

  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. << Less

  Proc. Natl. Acad. Sci. U.S.A. 96:14742-14747(1999) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Crystal structures of the naturally fused CS and cytochrome b₅ reductase (b₅R) domains of Ncb5or reveal an expanded CS fold, extensive CS-b₅R interactions and productive binding of the NAD(P)⁺ nicotinamide ring.

  Benson D.R., Lovell S., Mehzabeen N., Galeva N., Cooper A., Gao P., Battaile K.P., Zhu H.

  Ncb5or (NADH-cytochrome b₅ oxidoreductase), a cytosolic ferric reductase implicated in diabetes and neurological diseases, comprises three distinct domains, cytochrome b₅ (b₅) and cytochrome b₅ reductase (b₅R) domains separated by a CHORD-Sgt1 ... >> More

  Ncb5or (NADH-cytochrome b₅ oxidoreductase), a cytosolic ferric reductase implicated in diabetes and neurological diseases, comprises three distinct domains, cytochrome b₅ (b₅) and cytochrome b₅ reductase (b₅R) domains separated by a CHORD-Sgt1 (CS) domain, and a novel 50-residue N-terminal region. Understanding how interdomain interactions in Ncb5or facilitate the shuttling of electrons from NAD(P)H to heme, and how the process compares with the microsomal b₅ (Cyb5A) and b₅R (Cyb5R3) system, is of interest. A high-resolution structure of the b₅ domain (PDB entry 3lf5) has previously been reported, which exhibits substantial differences in comparison to Cyb5A. The structural characterization of a construct comprising the naturally fused CS and b₅R domains with bound FAD and NAD⁺ (PDB entry 6mv1) or NADP⁺ (PDB entry 6mv2) is now reported. The structures reveal that the linker between the CS and b₅R cores is more ordered than predicted, with much of it extending the β-sandwich motif of the CS domain. This limits the flexibility between the two domains, which recognize one another via a short β-sheet motif and a network of conserved side-chain hydrogen bonds, salt bridges and cation-π interactions. Notable differences in FAD-protein interactions in Ncb5or and Cyb5R3 provide insight into the selectivity for docking of their respective b₅ redox partners. The structures also afford a structural explanation for the unusual ability of Ncb5or to utilize both NADH and NADPH, and represent the first examples of native, fully oxidized b₅R family members in which the nicotinamide ring of NAD(P)⁺ resides in the active site. Finally, the structures, together with sequence alignments, show that the b₅R domain is more closely related to single-domain Cyb5R proteins from plants, fungi and some protists than to Cyb5R3 from animals. << Less

  Acta Crystallogr D Struct Biol 75:628-638(2019) [PubMed] [EuropePMC]