Lys842 in neuronal nitric-oxide synthase enables the autoinhibitory insert to antagonize calmodulin binding, increase FMN shielding, and suppress interflavin electron transfer.
Neuronal nitric-oxide synthase (nNOS) contains a unique autoinhibitory insert (AI) in its FMN subdomain that represses nNOS reductase activities and controls the calcium sensitivity of calmodulin (CaM) binding to nNOS. How the AI does this is unclear. A conserved charged residue (Lys(842)) 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 Lys(842). 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 and NO synthesis 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, Lys(842) 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.
Guan
Zhi-Wen
ZW
Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA.
Haque
Mohammad Mahfuzul
MM
Wei
Chin-Chuan
CC
Garcin
Elsa D
ED
Getzoff
Elizabeth D
ED
Stuehr
Dennis J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000737:chemistry*; D002417:Cattle; D003579:Cytochrome Reductases / Q000737:chemistry; D005415:Flavins / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000737:chemistry*; D008969:Molecular Sequence Data; D009154:Mutation; D019255:NADPH Oxidases / Q000737:chemistry; D009474:Neurons / Q000201:enzymology*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D017386:Sequence Homology, Amino Acid
D002147:Calmodulin; D005415:Flavins; D019001:Nitric Oxide Synthase; D003579:Cytochrome Reductases; D019255:NADPH Oxidases; D008239:Lysine