The nitric oxide synthases (NOS-I, neuronal, NOS-II, inducible, and NOS-III, endothelial) are the most recent additions to the large number of heme proteins that contain cysteine thiolate-liganded protoporphyrin IX heme prosthetic groups. This group of oxygenating enzymes also includes one of the largest gene families, that of the cytochromes P450, which have been demonstrated to be involved in the hydroxylation of a variety of substrates, including endogenous compounds (steroids, fatty acids, and prostaglandins) and exogenous compounds (therapeutic drugs, environmental toxicants, and carcinogens). The substrates for cytochromes P450 are universally hydrophobic while the physiological substrate for the nitric oxide synthases is the amino acid L-arginine, a hydrophilic compound. This review will discuss the approaches being used to study the structure and mechanism of neuronal nitric oxide synthase in the context of its known prosthetic groups and regulation by Ca(2+)-calmodulin and/or tetrahydrobiopterin (BH4).

Neuronal nitric oxide synthase (nNOS) is a modular enzyme which consists of a flavin-containing reductase domain and a heme-containing oxygenase domain, linked by a stretch of amino acids which contains a calmodulin (CaM) binding site. CaM binding to nNOS facilitates the transfer of NADPH-derived electrons from the reductase domain to the oxygenase domain, resulting in the conversion of L-arginine to L-citrulline with the concomitant formation of a guanylate cyclase activating factor, putatively nitric oxide. Numerous studies have established that peroxynitrite-derived nitrogen oxides are present following nNOS turnover. Since peroxynitrite is formed by the diffusion-limited reaction between the two radical species, nitric oxide and O2.-, we employed the adrenochrome assay to examine whether nNOS was capable of producing O2.- during catalytic turnover in the presence of L-arginine. To differentiate between the role played by the reductase domain and that of the oxygenase domain in O2.- production, we compared its production by nNOS against that of a nNOS mutant (CYS-331), which was unable to transfer NADPH-derived electrons efficiently to the heme iron under special conditions, and against that of a flavoprotein module construct of nNOS. We report that O2.- production by nNOS and the CYS-331 mutant is CaM-dependent and that O2.- production can be modulated by substrates and inhibitors of nNOS. O2.- was also produced by the reductase domain of nNOS; however, it did not display the same CaM dependency. We conclude that both the reductase and oxygenase domains of nNOS produce O2.-, but that the reductase domain is both necessary and sufficient for O2.- production.

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A variety of monovalent anions and cations were effective in stimulating both calcium ion/calmodulin (Ca 2؉ /CaM)-independent NADPH-cytochrome c reductase activity of, and Ca 2؉ /CaM-dependent nitric oxide (NO ⅐ ) synthesis by, neuronal nitric oxide synthase (nNOS). The efficacy of the ions in stimulating both activities could be correlated, in general, with their efficacy in precipitating or stabilizing certain proteins, an order referred to as the Hofmeister ion series. In the hemoglobin capture assay, used for measurement of NO ⅐ production, apparent substrate inhibition by L-arginine was almost completely reversed by the addition of sodium perchlorate (NaClO 4 ), one of the more effective protein-destabilizing agents tested. Examination of this phenomenon by the assay of L-arginine conversion to L-citrulline revealed that the stimulatory effect of NaClO 4 on the reaction was observed only in the presence of oxyhemoglobin or superoxide anion (generated by xanthine and xanthine oxidase), both scavengers of NO ⅐ . Spectrophotometric examination of nNOS revealed that the addition of NaClO 4 and a superoxide-generating system, but neither alone, prevented the increase of heme absorption at 436 nm, which has been attributed to the nitrosyl complex. The data are consistent with the release of autoinhibitory NO ⅐ coordinated to the prosthetic group of nNOS, which, in conjunction with an NO ⅐ scavenger, causes stimulation of the reaction.The nitric-oxide synthases (NOSs) 1 comprise a family of calmodulin (CaM)-dependent flavoheme enzymes that catalyze the NADPH-dependent oxidation by molecular oxygen of Larginine to L-citrulline and nitric oxide (NO ⅐ ). The isoforms of NOS are grouped into three categories, neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). The first two constitutive isoforms are collectively referred to as cNOS. nNOS is a homodimer, which contains one molecule each of heme, FAD, FMN, and tetrahydrobiopterin (BH 4 ) per subunit (1-5). The binding sites for NADPH, FAD, and FMN are located in the carboxyl-terminal half of nNOS, which exhibits sequence homology to NADPH-cytochrome P-450 reductase and also contains FMN and FAD (6). The amino-terminal half contains the binding sites for heme, L-arginine, and BH 4 (7). Electron transfer from NADPH via the flavins is facilitated by the binding of Ca 2ϩ -calmodulin (Ca 2ϩ /CaM) and L-arginine (8, 9). Electron transfer to the artificial electron acceptor cytochrome c is stimulated by, but is not totally dependent on, Ca 2ϩ /CaM (10, 11) and represents the NADPH-cytochrome c reductase activity of nNOS.A primary area of interest regarding the NOSs is the control of their activities. cNOS requires Ca 2ϩ /CaM and is therefore sensitive to levels of Ca 2ϩ in the cell. On the other hand, iNOS contains tightly bound Ca 2ϩ /CaM and is not sensitive to cellular levels of Ca 2ϩ . cNOSs contain what appears to be an autoinhibitory element, whereas iNOS does not (12). There is also considerable evidence that cNOS and iNOS are feedback-inhibited by NO...

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