Constitutive NADPH-Dependent Electron Transferase Activity of the Nox4 Dehydrogenase Domain

• Type of Material: Article
• Authors: Yukio Nisimoto, Emory University; Heather M. Jackson, Emory University; Hisamitsu Ogawa, Fujita Health University; Tsukasa Kawahara, Emory University; John Lambeth, Emory University
• Language: English
• Date: 2010-03-23
• Publisher: American Chemical Society
• Publication Version: Final Published Version
• Copyright Statement: © 2010 American Chemical Society. This is an open-access article distributed under the ACS AuthorChoice Terms & Conditions. Any use of this article, must conform to the terms of that license which are available at http://pubs.acs.org.
• Final Published Version (URL): http://dx.doi.org/10.1021/bi9022285
• Title of Journal or Parent Work: Biochemistry
• Volume: 49
• Issue: 11
• Start Page: 2433
• End Page: 2442
• Grant/Funding Information: This work was supported by National Institutes of Health Grant CA105116 and American Heart Association Grant 0815088E.
• Keywords: DIAPHORASE ACTIVITY; P67(PHOX); SMOOTH-MUSCLE-CELLS; REACTIVE OXYGEN GENERATION; SUPEROXIDE-PRODUCTION; ACTIVATION DOMAIN; Life Sciences & Biomedicine; NAD(P)H OXIDASE; COLON EPITHELIAL-CELLS; FLAVOCYTOCHROME B(558); Science & Technology; Biochemistry & Molecular Biology; CYTOCHROME-B
• Research Categories: Chemistry, Biochemistry; Health Sciences, Pathology

Abstract

NADPH oxidase 4 (Nox4) is constitutively active, while Nox2 requires the cytosolic regulatory subunits p47(phox) and p67(phox) and activated Rac with activation by phorbol 12-myristate 13-acetate (PMA). This study was undertaken to identify the domain on Nox4 that confers constitutive activity. Lysates from Nox4-expressing cells exhibited constitutive NADPH- but not NADH-dependent hydrogen peroxide production with a K(m) for NADPH of 55 +/- 10 microM. The concentration of Nox4 in cell lysates was estimated using Western blotting and allowed calculation of a turnover of approximately 200 mol of H(2)O(2) min(-1) (mol of Nox4)(-1). A chimeric protein (Nox2/4) consisting of the Nox2 transmembrane (TM) domain and the Nox4 dehydrogenase (DH) domain showed H(2)O(2) production in the absence of cytosolic regulatory subunits. In contrast, chimera Nox4/2, consisting of the Nox4 TM and Nox2 DH domains, exhibited PMA-dependent activation that required coexpression of regulatory subunits. Nox DH domains from several Nox isoforms were purified and evaluated for their electron transferase activities. Nox1 DH, Nox2 DH, and Nox5 DH domains exhibited barely detectable activities toward artificial electron acceptors, while the Nox4 DH domain exhibited significant rates of reduction of cytochrome c (160 min(-1), largely superoxide dismutase-independent), ferricyanide (470 min(-1)), and other electron acceptors (artificial dyes and cytochrome b(5)). Rates were similar to those observed for H(2)O(2) production by the Nox4 holoenzyme in cell lysates. The activity required added FAD and was seen with NADPH but not NADH. These results indicate that the Nox4 DH domain exists in an intrinsically activated state and that electron transfer from NADPH to FAD is likely to be rate-limiting in the NADPH-dependent reduction of oxygen by holo-Nox4.