Nitric oxide reductase (NOR) is a membrane bound enzyme involved in the metabolic denitrification pathway, reducing nitric oxide (NO) to nitrous oxide (N(2)O), subsequently promoting the formation of the NN bond. Three types of bacterial NOR are known, namely cNOR, qNOR and qCuNOR, that differ on the physiological electron donor. cNOR has been purified as a two subunit complex, the NorC, anchored to the cytoplasmic membrane, with a low-spin heme c, and the NorB subunit showing high structural homology with the HCuO subunit I, comprising a bis-histidine low-spin heme b and a binuclear iron centre. The binuclear iron centre is the catalytic site and it is formed by a heme b(3) coupled to a non-heme iron (Fe(B)) through a μ-oxo bridge. The catalytic mechanism is still under discussion and three hypotheses have been proposed: the trans-mechanism, the cis-Fe(B) and the cis-heme b(3) mechanisms. In the present work, the Pseudomonas nautica cNOR electrochemical behaviour was studied by cyclic voltammetry (CV), using a pyrolytic graphite electrode modified with the immobilised protein. The protein redox centres were observed and the formal redox potentials were determined. The binuclear iron centre presents the lowest redox potential value, and discrimination between the heme b(3) and Fe(B) redox processes was attained. Also, the number of electrons involved and correspondent surface electronic transfer rate constants were estimated. The pH dependence of the observed redox processes was determined and some new insights on the NOR catalytic mechanism are discussed.

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.

Superoxide reductases are a class of non-haem iron enzymes which catalyse the monovalent reduction of the superoxide anion O : 2 into hydrogen peroxide and water. Treponema pallidum (Tp), the syphilis spirochete, expresses the gene for a superoxide reductase called neelaredoxin, having the iron protein rubredoxin as the putative electron donor necessary to complete the catalytic cycle. In this work, we present the first cloning, overexpression in Escherichia coli and purification of the Tp rubredoxin. Spectroscopic characterization of this 6 kDa protein allowed us to calculate the molar absorption coefficient of the 490 nm feature of ferric iron, e=6.9±0.4 mM À1 cm À1 . Moreover, the midpoint potential of Tp rubredoxin, determined using a glassy carbon electrode, was À76±5 mV. Reduced rubredoxin can be efficiently reoxidized upon addition of Na 2 IrCl 6 -oxidized neelaredoxin, in agreement with a direct electron transfer between the two proteins, with a stoichiometry of the electron transfer reaction of one molecule of oxidized rubredoxin per one molecule of neelaredoxin. In addition, in presence of a steady-state concentration of superoxide anion, the physiological substrate of neelaredoxin, reoxidation of rubredoxin was also observed in presence of catalytic amounts of superoxide reductase, and the rate of rubredoxin reoxidation was shown to be proportional to the concentration of neelaredoxin, in agreement with a bimolecular reaction, with a calculated k app =180 min À1 . Interestingly, similar experiments performed with a rubredoxin from the sulfate-reducing bacteria Desulfovibrio vulgaris resulted in a much lower value of k app =4.5 min À1 . Altogether, these results demonstrated the existence for a superoxide-mediated electron transfer between rubredoxin and neelaredoxin and confirmed the physiological character of this electron transfer reaction.

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Knowledge gap reduced. Although this enzyme is responsible for the two‐electron reduction of nitric oxide to nitrous oxide, our knowledge of the sensitive, membrane‐bound nitric oxide reductase is less than that of the other enzymes of the denitrification pathway. In this communication, we report the first successful direct electrochemical experiments to reveal relevant catalytic aspects of this interesting and important enzyme.

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Cytochrome b is the main electron acceptor of cytochrome b reductase. The interacting domain between both human proteins has been unidentified up to date and very little is known about its redox properties modulation upon complex formation. In this article, we characterized the protein/protein interacting interface by solution NMR and molecular docking. In addition, upon complex formation, we measured an increase of cytochrome b reductase flavin autofluorescence that was dependent upon the presence of cytochrome b Data analysis of these results allowed us to calculate a dissociation constant value between proteins of 0.5±0.1μM and a 1:1 stoichiometry for the complex formation. In addition, a 30mV negative shift of cytochrome b reductase redox potential in presence of cytochrome b was also measured. These experiments suggest that the FAD group of cytochrome b reductase increase its solvent exposition upon complex formation promoting an efficient electron transfer between the proteins.

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