Kinetics of CO and NO Ligation with the Cys331 → Ala Mutant of Neuronal Nitric-oxide Synthase

Abstract

Nitric-oxide synthases (NOS) catalyze the conversion of L-arginine to NO, which then stimulates many physiological processes. In the active form, each NOS is a dimer; each strand has both a heme-binding oxygenase domain and a reductase domain. In neuronal NOS (nNOS), there is a conserved cysteine motif (CX 4 C) that participates in a ZnS 4 center, which stabilizes the dimer interface and/or the flavoprotein-heme domain interface. Previously, the Cys 331 3 Ala mutant was produced, and it proved to be inactive in catalysis and to have structural defects that disrupt the binding of L-Arg and tetrahydrobiopterin (BH 4 ). Because binding L-Arg and BH 4 to wild type nNOS profoundly affects CO binding with little effect on NO binding, ligand binding to the mutant was characterized as follows. 1) The mutant initially has behavior different from native protein but reminiscent of isolated heme domain subchains. 2) Adding L-Arg and BH 4 has little effect immediately but substantial effect after extended incubation. 3) Incubation for 12 h restores behavior similar but not quite identical to that of wild type nNOS. Such incubation was shown previously to restore most but not all catalytic activity. These kinetic studies substantiate the hypothesis that zinc content is related to a structural rather than a catalytic role in maintaining active nNOS. Nitric-oxide synthases (NOS)1 constitute a family of hemethiolate-liganded proteins that catalyze the conversion of L-Arg to NO and L-citrulline, requiring NADPH and molecular O 2 (1). Three isoforms of mammalian NOS are known. Neuronal nitric-oxide synthase (nNOS) and endothelial nitric-oxide synthase (eNOS) are constitutively expressed and are Ca 2ϩ / calmodulin-dependent; inducible nitric-oxide synthase (iNOS) is immunostimulated and Ca 2ϩ -independent. All three are homodimers in their physiologic states; each monomer has a molar mass between about 130 and 160 kDa. In each monomer, an oxygenase domain is recognized that contains the heme (2-5) and requires tetrahydrobiopterin (BH 4 ) (6 -8). All isoforms also have a reductase domain, which is not of direct interest here.A recent study (9) prepared nNOS (rat brain) protein with the point mutation C331A expressed in Escherichia coli. The mutant was characterized with respect to catalytic activity, the binding of BH 4 and substrates, the state of the iron as deduced from EPR, and UV-visible spectral features. Because E. coli produces neither BH 4 nor calmodulin, the preparation is valuable for investigating reconstitution with those factors. That report should be consulted for the several hypotheses that motivated the investigation and for an extensive list of citations. In this study, we added a brief account of the kinetics of ligand binding. The major effect of the mutation was that C331A failed to bind either BH 4 or L-Arg. Arginine affinity was suppressed at least 100-fold, and the binding of BH 4 was undetectable at least for some time after mixing. However, extended incubation (ϳ12 h) with L-Arg restored essentially normal ...

Control of Nitric Oxide Synthase Dimer Assembly by a Heme−NO-Dependent Mechanism

“…Thus, we suspect that imidazole protects against irreversible damage by antagonizing heme−NO complex formation in iNOS monomer. This differs mechanistically from the protection obtained when H 4 B and Arg bind to iNOS dimer, because H 4 B and Arg do not bind directly to the ferric heme or antagonize its NO binding ( − ).…”

“…An extensive dimer interface is created between two oxygenase domains, and this helps activate NOS by sequestering the heme from bulk solvent, creating functional binding sites for Arg and H 4 B, and allowing electron transfer between reductase and oxygenase domains located on adjacent subunits (16)(17)(18)(19)(20). In iNOS and nNOS, binding H 4 B with or without Arg to the newly formed dimer favors additional structural changes, which although not apparent in existing crystal structures (11), are indicated by changes in proteolytic susceptibility (21), heme ligand binding (22)(23)(24)(25), and increased resistance toward dimer dissociation by detergent (26)(27)(28). In this way, H 4 B and Arg binding cause a transition from "loose" to "tight" NOS dimer.…”

Analysis of the kinetics of CO binding to neuronal nitric oxide synthase by flash photolysis: dual effects of substrates, inhibitors, and tetrahydrobiopterin

“…Accordingly, in this study, we employ CO to explore the structure and character of the ligand access channel and heme distal side. Previous studies on CO binding kinetics [6][7][8][9][10][11][12], CO binding equilibrium [13] and vibrational spectra [14,15] of the CO-bound heme complex of full-length NOS provide useful information on the structure and character of the CO access channel and heme distal side environment. CO binding studies on the isolated heme-bound oxygenase domain, instead of the full-length enzyme, should provide more precise information, since the effect of the reductase domain on CO binding is excluded by removal.…”

Oxidation of the zinc-thiolate complex and uncoupling of endothelial nitric oxide synthase by peroxynitrite

“…The catalytic mechanisms of NOS involve flavin-mediated electron transport from C-terminal-bound NADPH to the N-terminal heme center, where oxygen is reduced and incorporated into the guanidine group of L-arginine, giving rise to NO and L-citrulline. All three NOS's are dimeric enzymes comprised of two identical subunits, and NOS is catalytically active only in dimeric form (4)(5)(6)(7)(8)(9)(10). X-ray crystallography for all three isoforms of NOS shows a zinc thiolate (ZnS 4 ) cluster formed by a zinc ion coordinated in a tetrahedral conformation with pairs of symmetrically oriented and phylogenetically conserved cysteine residues at the dimer interface (4)(5)(6)(7)(8)(9)(10)(11)(12). Mutation within a C(× 4 )C motif prevents the binding of zinc, BH 4 , or L-arginine and eliminates enzyme activity (8)(9)(10)(11)(12), suggesting that stabilization of the dimer interface by the zinc-thiolate center is key for catalytic activity.…”

“…X-ray crystallography for all three isoforms of NOS shows a zinc thiolate (ZnS 4 ) cluster formed by a zinc ion coordinated in a tetrahedral conformation with pairs of symmetrically oriented and phylogenetically conserved cysteine residues at the dimer interface (4)(5)(6)(7)(8)(9)(10)(11)(12). Mutation within a C(× 4 )C motif prevents the binding of zinc, BH 4 , or L-arginine and eliminates enzyme activity (8)(9)(10)(11)(12), suggesting that stabilization of the dimer interface by the zinc-thiolate center is key for catalytic activity. Regulation of NOS subunit interactions could, therefore, provide a mechanism for modulation of enzyme activity in vivo.…”