Control of electron transfer and catalysis in neuronal NOS by a hinge connecting its FMN and FAD-NADPH domains

Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 06/2012; 287(36):30105-16. DOI: 10.1074/jbc.M112.339697
Source: PubMed


In nitric-oxide synthases (NOSs), two flexible hinges connect the FMN domain to the rest of the enzyme and may guide its interactions with partner domains for electron transfer and catalysis. We investigated the role of the FMN-FAD/NADPH hinge in rat neuronal NOS (nNOS) by constructing mutants that either shortened or lengthened this hinge by 2, 4, and 6 residues. Shortening the hinge progressively inhibited electron flux through the calmodulin (CaM)-free and CaM-bound nNOS to cytochrome c, whereas hinge lengthening relieved repression of electron flux in CaM-free nNOS and had no impact or slowed electron flux through CaM-bound nNOS to cytochrome c. How hinge length influenced heme reduction depended on whether enzyme flavins were pre-reduced with NADPH prior to triggering heme reduction. Without pre-reduction, changing the hinge length was deleterious; with pre-reduction, the hinge shortening was deleterious, and hinge lengthening increased heme reduction rates beyond wild type. Flavin fluorescence and stopped-flow kinetic studies on CaM-bound enzymes suggested hinge lengthening slowed the domain-domain interaction needed for FMN reduction. All hinge length changes lowered NO synthesis activity and increased uncoupled NADPH consumption. We conclude that several aspects of catalysis are sensitive to FMN-FAD/NADPH hinge length and that the native hinge allows a best compromise among the FMN domain interactions and associated electron transfer events to maximize NO synthesis and minimize uncoupled NADPH consumption.

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    • "Moreover, the biphasic nature of the reduction of CaM-free nNOS by NADPH turned out to be also ionic strength dependent and could not therefore be caused by partial degradation or reoxidation of the sample. Using a series of amino acid deletions or insertions in the linker between the FMN and FAD domains, Haque et al. evidenced how the length of this small region controls ET between flavins and between FMN and heme [109,110]. Evidently, the control of ET by the linker length further strengthens the hypotheses of dynamical exchange between the shielded and deshielded forms in NOS. "
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