Thermodynamics of Oxidation-Reduction Reactions in Mammalian Nitric-oxide Synthase Isoforms

• Published in: The Journal of biological chemistry Vol. 279; no. 18; pp. 18759 - 18766
• Main Authors: Gao, Ying Tong, Smith, Susan M E, Weinberg, J Brice, Montgomery, Heather J, Newman, Elena, Guillemette, J Guy, Ghosh, Dipak K, Roman, Linda J, Martasek, Pavel, Salerno, John C
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 30.04.2004
• Subjects: Animals; Calmodulin - pharmacology; Cattle; Electrochemistry; Flavins - chemistry; Heme - chemistry; Humans; Hydrogen-Ion Concentration; Isoenzymes - chemistry; Nitric Oxide Synthase - chemistry; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Oxidation-Reduction; Rats; Recombinant Proteins - chemistry; Thermodynamics; Titrimetry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M308936200

The three mammalian nitric-oxide synthases produce NO from arginine in a reaction requiring 3 electrons per NO, which are supplied to the catalytic center from NADPH through reductase domains incorporating FAD and FMN cofactors. The isoforms share a common reaction mechanism and requirements for reducing equivalents but differ in regulation; the endothelial and neuronal isoforms are controlled by calcium/calmodulin modulation of the electron transfer system, while the inducible isoform binds calmodulin at all physiological Ca 2+ concentrations and is always on. The thermodynamics of electron transfer through the flavin domains in all three isoforms are basically similar. The major flavin states are FMN, FMNH·, FMNH 2 , FAD, FADH·, and FADH 2 . The FMN/FMNH· couple is high potential (∼100 mV) in all three isoforms and is unlikely to be catalytically competent; the other three flavin couples form a nearly isopotential group clustered around -250 mV. Reduction of the flavins by the pyridine nucleotide couple at -325 mV is thus moderately thermodynamically favorable. The ferri/ferroheme couple in all three isoforms is ∼-270 mV in the presence of saturating arginine. Ca 2+ /calmodulin has no effect on the potentials of any of the couples in endothelial nitric-oxide synthase (eNOS) or neuronal nitric-oxide synthase (nNOS). The pH dependence of the flavin couples suggests the presence of ionizable groups coupled to the flavin redox/protonation states.