title: Steady-state kinetics with nitric oxide reductase (NOR): new considerations on substrate inhibition profile and catalytic mechanism.
abstract: Nitric oxide reductase (NOR) from denitrifying bacteria is an integral membrane protein that catalyses the two electron reduction of NO to N2O, as part of the denitrification process, being responsible for an exclusive reaction, the NN bond formation, the key step of this metabolic pathway. Additionally, this class of enzymes also presents residual oxidoreductase activity, reducing O2 to H2O in a four electron/proton reaction. In this work we report, for the first time, steady-state kinetics with the Pseudomonas nautica NOR, either in the presence of its physiological electron donor (cyt. c552) or immobilised on a graphite electrode surface, in the presence of its known substrates, namely NO or O2. The obtained results show that the enzyme has high affinity for its natural substrate, NO, and different kinetic profiles according to the electron donor used. The kinetic data, as shown by the pH dependence, is modelled by ionisable amino acid residues nearby the di-nuclear catalytic site. The catalytic mechanism is revised and a mononitrosyl-non-heme Fe complex (FeB(II)-NO) species is favoured as the first catalytic intermediate involved on the NO reduction.
authors.affiliation: Requimte, Centro de Química Fina e Biotecnologia, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, 2829-516 Monte de Caparica, Portugal. Electronic address: americogduarte@fct.unl.pt.
authors.forename: Américo G
authors.initials: AG
authors.lastname: Duarte
authors.affiliation: Requimte, Centro de Química Fina e Biotecnologia, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, 2829-516 Monte de Caparica, Portugal. Electronic address: c.cordas@fct.unl.pt.
authors.forename: Cristina M
authors.initials: CM
authors.lastname: Cordas
authors.affiliation: Requimte, Centro de Química Fina e Biotecnologia, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, 2829-516 Monte de Caparica, Portugal. Electronic address: jjgm@fct.unl.pt.
authors.forename: José J G
authors.initials: JJ
authors.lastname: Moura
authors.affiliation: Requimte, Centro de Química Fina e Biotecnologia, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, 2829-516 Monte de Caparica, Portugal. Electronic address: isabelmoura@fct.unl.pt.
authors.forename: Isabel
authors.initials: I
authors.lastname: Moura
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D001667:Binding, Competitive; D055162:Biocatalysis; D003574:Cytochrome c Group / Q000378:metabolism; D004579:Electron Transport; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D053520:Marinobacter / Q000201:enzymology*; D008954:Models, Biological; D009569:Nitric Oxide / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism; D013379:Substrate Specificity; D014867:Water / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003574:Cytochrome c Group; D021122:Protein Subunits; D014867:Water; D009569:Nitric Oxide; D010088:Oxidoreductases; C034747:nitric-oxide reductase; D010100:Oxygen
keywords: Electrochemistry; Enzyme kinetics; NO reduction; NOR