Oxygen metabolism by neuronal nitric-oxide synthase.
Nitric-oxide synthases (NOS) catalyze nitric oxide (NO) formation from the amino acid L-arginine. NOS is known to catalyze more than one reaction: the NO-producing reaction is considered to be the coupled reaction, and the uncoupled reactions are those that produce reactive (reduced) oxygen species (ROS), such as superoxide anion (O-2.) and/or hydrogen peroxide (H2O2). As an oxygenase, NOS has been known for more than two decades, yet there is no complete description of oxygen stoichiometry. The present paper is focused on oxygen stoichiometry and the effects of cofactor binding on the neuronal isoform (nNOS) on oxygen uptake and product formation. Products of the uncoupled reactions are analyzed using diacetyldeuteroheme-substituted horseradish peroxidase as a trapping agent for both O-2. and H2O2. The addition of calmodulin not only stimulated the oxygen uptake rate but also changed the product of the uncoupled reaction, supporting the possibility of two different sites for electron leakage to molecular oxygen. Quantitative analysis of the uncoupled (substrate-free) reaction revealed a stoichiometry close to the theoretical value, and adding L-arginine not only initiates the coupled reaction, but also inhibits oxygen uptake. The presence of tetrahydrobiopterin affects oxygen metabolism by lowering the apparent Km value of nNOS for oxygen in the uncoupled reaction.
Gao
Ying Tong
YT
Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229-3900, USA.
Panda
Satya Prakash
SP
Roman
Linda J
LJ
Martásek
Pavel
P
Ishimura
Yuzuru
Y
Masters
Bettie Sue S
BS
D000818:Animals; D000838:Anions; D001120:Arginine / Q000737:chemistry; D002147:Calmodulin / Q000378:metabolism; D002384:Catalysis; D004926:Escherichia coli / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007700:Kinetics; D008956:Models, Chemical; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D010101:Oxygen Consumption; D017382:Reactive Oxygen Species; D013379:Substrate Specificity; D013481:Superoxides / Q000737:chemistry; D013997:Time Factors
D000838:Anions; D002147:Calmodulin; D017382:Reactive Oxygen Species; D013481:Superoxides; D001120:Arginine; D006861:Hydrogen Peroxide; D052248:Nitric Oxide Synthase Type I; D010100:Oxygen