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Identifying redox partners and the interaction surfaces is crucial for fully understanding electron flow in a respiratory chain. In this study, we focused on the interaction of nitrous oxide reductase (NOR), which catalyzes the final step in bacterial denitrification, with its physiological electron donor, either a c-type cytochrome or a type 1 copper protein. The comparison between the interaction of NOR from three different microorganisms, Pseudomonas nautica, Paracoccus denitrificans, and Achromobacter cycloclastes, with their physiological electron donors was performed through the analysis of the primary sequence alignment, electrostatic surface, and molecular docking simulations, using the bimolecular complex generation with global evaluation and ranking algorithm. The docking results were analyzed taking into account the experimental data, since the interaction is suggested to have either a hydrophobic nature, in the case of P. nautica NOR, or an electrostatic nature, in the case of P. denitrificans NOR and A. cycloclastes NOR . A set of well-conserved residues on the NOR surface were identified as being part of the electron transfer pathway from the redox partner to NOR (Ala495, Asp519, Val524, His566 and Leu568 numbered according to the P. nautica NOR sequence). Moreover, we built a model for Wolinella succinogenes NOR, an enzyme that has an additional c-type-heme-containing domain. The structures of the NOR domain and the c-type-heme-containing domain were modeled and the full-length structure was obtained by molecular docking simulation of these two domains. The orientation of the c-type-heme-containing domain relative to the NOR domain is similar to that found in the other electron transfer complexes. [ABSTRACT FROM AUTHOR]

Copper-containing nitrous oxide reductase catalyzes a 2-electron reduction of the green-house gas N2O to yield N2. It contains two metal centers, the binuclear electron transfer site CuA, and the unique, tetranuclear CuZ center that is the site of substrate binding. Different forms of the enzyme were described previously, representing variations in oxidation state and composition of the metal sites. Hypothesizing that many reported discrepancies in the structural data may be due to radiation damage during data collection, we determined the structure of anoxically isolated Marinobacter nauticus N2OR from diffraction data obtained with low-intensity X-rays from an in-house rotating anode generator and an image plate detector. The data set was of exceptional quality and yielded a structure at 1.5 Å resolution in a new crystal form. The CuA site of the enzyme shows two distinct conformations with potential relevance for intramolecular electron transfer, and the CuZ cluster is present in a [4Cu:2S] configuration. In addition, the structure contains three additional types of ions, and an analysis of anomalous scattering contributions confirms them to be Ca2+, K+, and Cl–. The uniformity of the present structure supports the hypothesis that many earlier analyses showed inhomogeneities due to radiation effects. Adding to the earlier description of the same enzyme with a [4Cu:S] CuZ site, a mechanistic model is presented, with a structurally flexible CuZ center that does not require the complete dissociation of a sulfide prior to N2O binding. The [4Cu:2S] CuZ site in M. nauticus N 2O reductase. The electron density map shown is contoured at the 5 σ level, highlighting the presence of two sulfide ligands. 705x677mm (72 x 72 DPI) [ABSTRACT FROM AUTHOR]

In this work we present a kinetic study of the superoxide-mediated electron transfer reactions between rubredoxin-type proteins and members of the three different classes of superoxide reductases (SORs). SORs from the sulfate-reducing bacteria Desulfovibrio vulgaris ( Dv) and D. gigas ( Dg) were chosen as prototypes of classes I and II, respectively, while SOR from the syphilis spyrochete Treponema pallidum ( Tp) was representative of class III. Our results show evidence for different behaviors of SORs toward electron acceptance, with a trend to specificity for the electron donor and acceptor from the same organism. Comparison of the different k app values, 176.9±25.0 min−1 in the case of the Tp/ Tp electron transfer, 31.8±3.6 min−1 for the Dg/ Dg electron transfer, and 6.9±1.3 min−1 for Dv/ Dv, could suggest an adaptation of the superoxide-mediated electron transfer efficiency to various environmental conditions. We also demonstrate that, in Dg, another iron–sulfur protein, a desulforedoxin, is able to transfer electrons to SOR more efficiently than rubredoxin, with a k app value of 108.8±12.0 min−1, and was then assigned as the potential physiological electron donor in this organism. [ABSTRACT FROM AUTHOR]

Superoxide reductases are a class of non-haem iron enzymes which catalyse the monovalent reduction of the superoxide anioninto 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 inEscherichia coliand 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-1 cm-1. Moreover, the midpoint potential of Tp rubredoxin, determined using a glassy carbon electrode, was -76±5 mV. Reduced rubredoxin can be efficiently reoxidized upon addition of Na2IrCl6-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 calculatedkapp=180 min-1. Interestingly, similar experiments performed with a rubredoxin from the sulfate-reducing bacteriaDesulfovibrio vulgarisresulted in a much lower value ofkapp=4.5 min-1. 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. [ABSTRACT FROM AUTHOR]