Abstract
The neuronal nitric-oxide synthase (nNOS) flavoprotein domain (nNOSr) contains regulatory elements that repress its electron flux in the absence of bound calmodulin (CaM). The repression also requires bound NADP(H), but the mechanism is unclear. The crystal structure of a CaM-free nNOSr revealed an ionic interaction between Arg(1400) in the C-terminal tail regulatory element and the 2'-phosphate group of bound NADP(H). We tested the role of this interaction by substituting Ser and Glu for Arg(1400) in nNOSr and in the full-length nNOS enzyme. The CaM-free nNOSr mutants had cytochrome c reductase activities that were less repressed than in wild-type, and this effect could be mimicked in wild-type by using NADH instead of NADPH. The nNOSr mutants also had faster flavin reduction rates, greater apparent K(m) for NADPH, and greater rates of flavin auto-oxidation. Single-turnover cytochrome c reduction data linked these properties to an inability of NADP(H) to cause shielding of the FMN module in the CaM-free nNOSr mutants. The full-length nNOS mutants had no NO synthesis in the CaM-free state and had lower steady-state NO synthesis activities in the CaM-bound state compared with wild-type. However, the mutants had faster rates of ferric heme reduction and ferrous heme-NO complex formation. Slowing down heme reduction in R1400E nNOS with CaM analogues brought its NO synthesis activity back up to normal level. Our studies indicate that the Arg(1400)-2'-phosphate interaction is a means by which bound NADP(H) represses electron transfer into and out of CaM-free nNOSr. This interaction enables the C-terminal tail to regulate a conformational equilibrium of the FMN module that controls its electron transfer reactions in both the CaM-free and CaM-bound forms of nNOS.
Highlights
Nitric oxide (NO)2 has diverse biological functions and is generated in mammals by the NO synthase (NOS) enzymes (EC 1.14.13.39) [1, 2]
NO Synthesis Supported by CaM Substitutes—The above data establish that the lower NO synthesis activities of the Arg1400 mutants are associated with faster rates of ferric heme reduction and catalysis, as we have found for other neuronal nitric-oxide synthase (nNOS) mutants [56]
The electron transfer reactions of nNOS are regulated by bound NADP(H) and the C-terminal tail [32, 42], but the mechanisms are unclear
Summary
Materials and General Methods—All reagents and materials were obtained from Sigma or sources previously reported [47, 48]. In the case of NO synthesis and NADPH oxidation the oxyhemoglobin assay buffer solution contains 150 mM NaCl. Measurement of Apparent Km and Kcat for NADPH and NADH— Apparent Km and kcat values of wild-type and mutants nNOS enzymes were determined in the presence or absence of bound Ca2ϩ/CaM by analysis of cytochrome c reduction measured at 550 nm using quartz cuvettes or a 96-microwell Molecular Dynamics kinetic plate reader. For each protein sample used the maximum absorbance value at 457 nm was obtained by replacing the NADPH solution in one of the stopped-flow syringes with buffer only and recording two to three additional mixing events. To initiate NO synthesis an air-saturated solution that contained 100 mM EPPS, pH 7.6, 5 M nNOS or mutant, 150 mM NaCl, 10 M 6R-tetrahydrobiopterin, 0.4 mM dithiothreitol, 1 mM Arg, 0.5 mM EDTA, 1.2 mM Ca2ϩ, and 10 M CaM was rapidly mixed with a buffered solution containing 50 M NADPH. In the case of flavin reduction by excess NADPH, we used a four exponential equation to fit the absorbance change at 457 nm, as done in previous reports [34, 58]
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