new considerations on substrate inhibition profile and catalytic mechanism

We would like to thank Fundacao para a Ciencia e Tecnologia for the financial support through grants SFRH/BD/39009/2007 (AGD), PDTC/QUI/64638/2006 (IM) and PDCT/QUI-BIOQ/1/6481/2010 (IM). REQUIMTE is funded by grant PEst-C/EQB/LA0006/2013 from FCT/MEC.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.publishersversionpublishe

Química Bioinorgânica e luz - fotossíntese, oxigénio e água

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Cytochrome b5 reductase is the component from neuronal synaptic plasma membrane vesicles that generates superoxide anion upon stimulation by cytochrome c

En este trabajo, medimos el efecto del citocromo c en la producción de aniones superóxidos dependientes de la NADH por las vesículas de la membrana plasmática sináptica del cerebro de las ratas. En estas membranas, el citocromo c estimuló la producción de aniones superóxido dependientes de NADH, que fue inhibida por anticuerpos contra el citocromo b5 reductasa que vinculan la producción a esta enzima. La medición del radical del anión superóxido generado por el citocromo b5 reductasa recombinante soluble purificado y de membrana corrobora la producción del radical por diferentes isoformas enzimáticas. En presencia del citocromo c, se midió una explosión de anión superóxido así como la reducción del citocromo c por la citocromo b5 reductasa. La formación de complejos entre ambas proteínas sugiere que el citocromo b5 reductasa es uno de los principales socios del citocromo c al ser liberado de las mitocondrias al citosol durante la apoptosis. La producción de aniones superóxido y la reducción del citocromo c son las consecuencias del consumo estimulado de NADH por el citocromo b5 reductasa al formarse complejos con el citocromo c y sugieren un papel importante de esta enzima como proteína antiapoptótica durante la muerte celular.In this work, we measured the effect of cytochrome c on the NADH-dependent superoxide anion production by synaptic plasma membrane vesicles from rat brain. In these membranes, the cytochrome c stimulated NADH-dependent superoxide anion production was inhibited by antibodies against cytochrome b5 reductase linking the production to this enzyme. Measurement of the superoxide anion radical generated by purified recombinant soluble and membrane cytochrome b5 reductase corroborates the production of the radical by different enzyme isoforms. In the presence of cytochrome c, a burst of superoxide anion as well as the reduction of cytochrome c by cytochrome b5 reductase was measured. Complex formation between both proteins suggests that cytochrome b5 reductase is one of the major partners of cytochrome c upon its release from mitochondria to the cytosol during apoptosis. Superoxide anion production and cytochrome c reduction are the consequences of the stimulated NADH consumption by cytochrome b5 reductase upon complex formation with cytochrome c and suggest a major role of this enzyme as an anti-apoptotic protein during cell death.• FCT/MEC (Portugal). Beca UID/Multi/04378/2013 • Acuerdo de Asociación PT2020(Portugal) y Fondos FEDER. Beca POCI-01-0145-FEDER-007728 • Ministerio de Economía y Competitividad (España). Subvención BFU2014-53641-P (I+D+i) • Junta de Extremadura y Fondos Europeos para el Desarrollo Estructural. Ayuda GR15139, para al Grupo BBB008 • Fondo Social Europeo y FCT/MCTES (Portugal). Beca postdoctoral SFRH/BPD/100069/2014, para Alejandro K. Samhan Arias • Fondo Social Europeo y FCT/MCTES (Portugal). Beca postdoctoral SFRH/BD/84543/2012, para Sofia FortalezaspeerReviewe