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J. Biol. Chem., Vol. 282, Issue 39, 28557-28565, September 28, 2007

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Oxygen Metabolism by Endothelial Nitric-oxide Synthase^*

Ying Tong Gao, Linda J. Roman, Pavel Martásek, Satya Prakash Panda, Yuzuru Ishimura^¶, and Bettie Sue S. Masters¹

From the Department of Biochemistry, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3900, the Department of Pediatrics, Charles University School of Medicine I, 128 08 Prague, Czech Republic, and the ^¶Department of Biochemistry and Integrated Biology, School of Medicine, Keio University, Tokyo 160-8582, Japan

Nitric-oxide synthase (NOS) catalyzes both coupled and uncoupled reactions that generate nitric oxide and reactive oxygen species. Oxygen is often the overlooked substrate, and the oxygen metabolism catalyzed by NOS has been poorly defined. In this paper we focus on the oxygen stoichiometry and effects of substrate/cofactor binding on the endothelial NOS isoform (eNOS). In the presence of both L-arginine and tetrahydrobiopterin, eNOS is highly coupled (>90%), and the measured stoichiometry of O₂/NADPH is very close to the theoretical value. We report for the first time that the presence of L-arginine stimulates oxygen uptake by eNOS. The fact that nonhydrolyzable L-arginine analogs are not stimulatory indicates that the occurrence of the coupled reaction, rather than the accelerated uncoupled reaction, is responsible for the L-arginine-dependent stimulation. The presence of 5,6,7,8-tetrahydrobiopterin quenched the uncoupled reactions and resulted in much less reactive oxygen species formation, whereas the presence of redox-incompetent 7,8-dihydrobiopterin demonstrates little quenching effect. These results reveal different mechanisms for oxygen metabolism for eNOS as opposed to nNOS and, perhaps, partially explain their functional differences.

Received for publication, June 13, 2007 , and in revised form, August 10, 2007.

^* This work was supported in part by National Institutes of Health Grants GM52419 and HL30050 (to B. S. S. M.), Grant AQ-0012 for the Robert A. Welch Foundation Distinguished Chair in Chemistry (to B. S. S. M.), Grant Agency of Czech Republic (GACR) Grant 303/05/0336, and Ministry of Education, Youth and Sports (MSMT) Grant 1M0520 (to P. M.). 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.

¹ To whom correspondence should be addressed: Dept. of Biochemistry, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78229. Tel.: 210-567-6627; E-mail: masters{at}uthscsa.edu.

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