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J. Biol. Chem., Vol. 279, Issue 29, 30316-30325, July 16, 2004

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NCB5OR Is a Novel Soluble NAD(P)H Reductase Localized in the Endoplasmic Reticulum^*

Hao Zhu, Kevin Larade, Timothy A. Jackson¶, Jianxin Xie, Annie Ladoux||, Helmut Acker**, Utta Berchner-Pfannschmidt**, Joachim Fandrey, Andrew R. Cross, Gudrun S. Lukat-Rodgers¶¶, Kenton R. Rodgers¶¶, and H. Franklin Bunn||||

From the Hematology Division, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115 ^**Labor fuer optische Systemphysiologie, Max-Planck-Institut fuer molekulare Physiologie, 44227 Dortmund, Germany Universitätsklinikum Essen, Institut fuer Physiologie, 45122 Essen, Germany Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, and ^¶¶Department of Chemistry, North Dakota State University, Fargo, North Dakota 58105-5516

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: k_cat = 0.05 and 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.

Received for publication, March 9, 2004 , and in revised form, April 28, 2004.

^* This work was supported by National Institutes of Health Grants RO1 DK56050 (to H. F. B), K01 DK59901 (to H. Z.), and RO1 AI24838 (to A. R. C.); Juvenile Diabetes Research Foundation International Innovative Grant 5-2003-589 (to H. F. B.); United States Department of Agriculture Grant 97-5305-5158 (to G. S. L.-R. and K. R. R.); Hermann Frasch Foundation Grant 466-HF97 (to G. S. L.-R. and K. R. R.); Germany Bundesministerium für Bildung und Forschung Grant 13N7447/5 (to H. A.); Deutsche Forschungsgemeinschaft Grant FA 225/19-1 (to J. F.), and NSERC Canada (K. L.) and France INSERM (A. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Supplementary Fig. 1.

^¶ Present address: Anesthesia Department, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114.

^|| Present address: Centre de Biochimie, CNRS UMR-6543, Parc Valrose, 06108 Nice Cedex 2, France.

To whom correspondence may be addressed: Hematology Division, Brigham and Women's Hospital, Harvard Medical School, 221 Longwood Ave., Boston, MA 02115. Tel.: 617-278-0870; Fax: 617-739-0748; E-mail: haozhu{at}rics.bwh.harvard.edu. |||| To whom correspondence may be addressed: Hematology Division, Brigham and Women's Hospital, Harvard Medical School, 221 Longwood Ave., Boston, MA 02115. Tel.: 617-732-5841; Fax: 617-739-0748; E-mail: HFBunn{at}rics.bwh.harvard.edu.

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