title: Surface charge interactions of the FMN module govern catalysis by nitric-oxide synthase.
abstract: The FMN module of nitric-oxide synthase (NOS) plays a pivotal role by transferring NADPH-derived electrons to the enzyme heme for use in oxygen activation. The process may involve a swinging mechanism in which the same face of the FMN module accepts and provides electrons during catalysis. Crystal structure shows that this face of the FMN module is electronegative, whereas the complementary interacting surface is electropositive, implying that charge interactions enable function. We used site-directed mutagenesis to investigate the roles of six electronegative surface residues of the FMN module in electron transfer and catalysis in neuronal NOS. Results are interpreted in light of crystal structures of NOS and related flavoproteins. Neutralizing or reversing the negative charge of each residue altered the NO synthesis, NADPH oxidase, and cytochrome c reductase activities of neuronal NOS and also altered heme reduction. The largest effects occurred at the NOS-specific charged residue Glu(762). Together, the results suggest that electrostatic interactions of the FMN module help to regulate electron transfer and to minimize flavin autoxidation and the generation of reactive oxygen species during NOS catalysis.
authors.affiliation: Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA.
authors.forename: Koustubh
authors.initials: K
authors.lastname: Panda
authors.forename: Mohammad Mahfuzul
authors.initials: MM
authors.lastname: Haque
authors.forename: Elsa D
authors.initials: ED
authors.lastname: Garcin-Hosfield
authors.forename: Deborah
authors.initials: D
authors.lastname: Durra
authors.forename: Elizabeth D
authors.initials: ED
authors.lastname: Getzoff
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Stuehr
mesh_terms: D000818:Animals; D002384:Catalysis; D018360:Crystallography, X-Ray; D004583:Electrons; D005415:Flavins / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000737:chemistry*; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D051381:Rats; D017382:Reactive Oxygen Species / Q000737:chemistry
chemical_list: D005415:Flavins; D017382:Reactive Oxygen Species; D006418:Heme; D019001:Nitric Oxide Synthase; D009251:NADPH-Ferrihemoprotein Reductase; D010100:Oxygen