Oxygen Metabolism by Endothelial Nitric-oxide Synthase

• Published in: The Journal of biological chemistry Vol. 282; no. 39; pp. 28557 - 28565
• Main Authors: Gao, Ying Tong, Roman, Linda J., Martásek, Pavel, Panda, Satya Prakash, Ishimura, Yuzuru, Masters, Bettie Sue S.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 28.09.2007; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Arginine - chemistry; Arginine - metabolism; Biopterins - analogs & derivatives; Biopterins - chemistry; Biopterins - metabolism; Catalysis; Humans; Nitric Oxide - chemistry; Nitric Oxide - metabolism; Nitric Oxide Synthase Type I - chemistry; Nitric Oxide Synthase Type I - metabolism; Nitric Oxide Synthase Type III - chemistry; Nitric Oxide Synthase Type III - metabolism; Nitrosamines - chemistry; Nitrosamines - metabolism; Oxygen - chemistry; Oxygen - metabolism; Reactive Oxygen Species - chemistry; Reactive Oxygen Species - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M704890200

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.