Abstract
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.