title: Energy landscapes and catalysis in nitric-oxide synthase.
abstract: Nitric oxide (NO) plays diverse roles in mammalian physiology. It is involved in blood pressure regulation, neurotransmission, and immune response, and is generated through complex electron transfer reactions catalyzed by NO synthases (NOS). In neuronal NOS (nNOS), protein domain dynamics and calmodulin binding are implicated in regulating electron flow from NADPH, through the FAD and FMN cofactors, to the heme oxygenase domain, the site of NO generation. Simple models based on crystal structures of nNOS reductase have invoked a role for large scale motions of the FMN-binding domain in shuttling electrons from the FAD-binding domain to the heme oxygenase domain. However, molecular level insight of the dynamic structural transitions in NOS enzymes during enzyme catalysis is lacking. We use pulsed electron-electron double resonance spectroscopy to derive inter-domain distance relationships in multiple conformational states of nNOS. These distance relationships are correlated with enzymatic activity through variable pressure kinetic studies of electron transfer and turnover. The binding of NADPH and calmodulin are shown to influence interdomain distance relationships as well as reaction chemistry. An important effect of calmodulin binding is to suppress adventitious electron transfer from nNOS to molecular oxygen and thereby preventing accumulation of reactive oxygen species. A complex landscape of conformations is required for nNOS catalysis beyond the simple models derived from static crystal structures of nNOS reductase. Detailed understanding of this landscape advances our understanding of nNOS catalysis/electron transfer, and could provide new opportunities for the discovery of small molecule inhibitors that bind at dynamic protein interfaces of this multidimensional energy landscape.
authors.affiliation: From the Manchester Institute of Biotechnology and Faculty of Life Sciences, University of Manchester, Manchester M1 7DN, United Kingdom.
authors.forename: Anna
authors.initials: A
authors.lastname: Sobolewska-Stawiarz
authors.affiliation: From the Manchester Institute of Biotechnology and Faculty of Life Sciences, University of Manchester, Manchester M1 7DN, United Kingdom.
authors.forename: Nicole G H
authors.initials: NGH
authors.lastname: Leferink
authors.affiliation: From the Manchester Institute of Biotechnology and Faculty of Life Sciences, University of Manchester, Manchester M1 7DN, United Kingdom.
authors.forename: Karl
authors.initials: K
authors.lastname: Fisher
authors.affiliation: From the Manchester Institute of Biotechnology and Faculty of Life Sciences, University of Manchester, Manchester M1 7DN, United Kingdom.
authors.forename: Derren J
authors.initials: DJ
authors.lastname: Heyes
authors.affiliation: From the Manchester Institute of Biotechnology and Faculty of Life Sciences, University of Manchester, Manchester M1 7DN, United Kingdom.
authors.forename: Sam
authors.initials: S
authors.lastname: Hay
authors.affiliation: From the Manchester Institute of Biotechnology and Faculty of Life Sciences, University of Manchester, Manchester M1 7DN, United Kingdom.
authors.forename: Stephen E J
authors.initials: SEJ
authors.lastname: Rigby
authors.affiliation: From the Manchester Institute of Biotechnology and Faculty of Life Sciences, University of Manchester, Manchester M1 7DN, United Kingdom. Electronic address: nigel.scrutton@manchester.as.uk.
authors.forename: Nigel S
authors.initials: NS
authors.lastname: Scrutton
mesh_terms: D000818:Animals; D002147:Calmodulin / Q000378:metabolism; D002384:Catalysis; D004578:Electron Spin Resonance Spectroscopy; D008958:Models, Molecular; D009249:NADP / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D002147:Calmodulin; D011994:Recombinant Proteins; D009249:NADP; D052248:Nitric Oxide Synthase Type I
keywords: Calmodulin; Enzyme Kinetics; Flavoproteins; Free Energy Landscapes; High Pressure Stopped Flow; Nitric-oxide Synthase; PELDOR Spectroscopy; Protein Dynamics