Lys842 in neuronal nitric-oxide synthase enables the autoinhibitory insert to antagonize calmodulin binding, increase FMN shielding, and suppress interflavin electron transfer

Abstract

Neuronal nitric-oxide synthase (nNOS) contains a unique autoinhibitory insert (AI) in its FMN subdomain that represses nNOSreductase activities and controls the calcium sensitivity of calmodulin (CaM) binding to nNOS. How the AI does this is unclear.Aconserved charged residue (Lys842) lies within a putative CaM binding helix in the middle of the AI. We investigated its role by substituting residues that neutralize (Ala) or reverse (Glu) the charge at Lys842. Compared with wild type nNOS, the mutant enzymes had greater cytochrome c reductase and NADPH oxidase activities in the CaM-free state, were able to bind CaM at lower calcium concentration, and had lower rates of heme reduction andNOsynthesis in one case (K842A). Moreover, stopped-flow spectrophotometric experiments with the nNOS reductase domain indicate that the CaM-free mutants had faster flavin reduction kinetics and had less shielding of their FMN subdomains compared with wild type and no longer increased their level of FMN shielding in response to NADPH binding. Thus, Lys842 is critical for the known functions of the AI and also enables two additional functions of the AI as newly identified here: suppression of electron transfer to FMN and control of the conformational equilibrium of the nNOS reductase domain. Its effect on the conformational equilibrium probably explains suppression of catalysis by the AI. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.