Sofia Pauleta

Anaerobic organisms have molecular systems to detoxify reactive oxygen species when transiently exposed to oxygen. One of these systems is superoxide reductase, which reduces O2 (.-) to H2 O2 without production of molecular oxygen. In order to complete the reduction of superoxide anion, superoxide reductase requires an electron, delivered by its redox partners, which in Desulfovibrio gigas are rubredoxin and/or desulforedoxin. In this work, we characterized the interaction of Desulfovibrio gigas superoxide reductase with both electron donors by using steady-state kinetics, 2D NMR titrations, and backbone relaxation measurements. The rubredoxin surface involved in the electron transfer complex with superoxide reductase comprises the solvent-exposed hydrophobic residues in the vicinity of its metal center (Cys9, Gly10, Cys42, Gly43, and Ala44), and a Kd of 3 muM at 59 mM ionic strength was estimated by NMR. The ionic strength dependence of superoxide-mediated rubredoxin oxidation by superoxide reductase has a maximum kapp of (37 +/- 12) min(-1) at 157 mM. Relative to the electron donor desulforedoxin, its complex with superoxide reductase was not detected by chemical shift perturbation, though this protein is able to transfer electrons to superoxide reductase with a maximum kapp of (31 +/- 7) min(-1) at an ionic strength of 57 mM. Competition experiments using steady-state kinetics and NMR spectroscopy (backbone relaxation measurements and use of a paramagnetic relaxation enhancement probe) with Fe-desulforedoxin in the presence of (15) N-Zn-rubredoxin showed that these two electron donors compete for the same site on the enzyme surface, as shown in the model structure of the complex generated by using restrained molecular docking calculations. These combined strategies indicate that the two small electron donors bind in different manners, with the desulforedoxin complex being a short lived electron transfer complex or more dynamic, with many equivalent kinetically competent orientations.

The electron transfer pathways for the enzymes involved in the four sequential steps of the denitrification pathway are reviewed. In addition, brief information on the electron transfer events is also provided on two enzymes that participate in the dissimilatory nitrate reduction to ammonia. The two main aspects discussed are the intra- and inter-molecular electron transfer pathways and the molecular recognition processes involving the redox partners. When available, information on the residues that are involved in these pathways is given, and their role in electron transfer and/or the formation of the transient electron transfer complexes is discussed. © The Royal Society of Chemistry 2017.

Two cyclen-derived Gd probes, [Gd-DOTAM](3+) and [Gd-DOTP](5-) (DOTAM = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetamide; DOTP = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylenephosphonate)), were assessed as paramagnetic relaxation enhancement (PRE)-inducing probes for characterization of protein-protein interactions. Two proteins, Desulfovibrio gigas rubredoxin and Desulfovibrio gigas cytochrome c(3), were used as model partners. In a (1)H NMR titration it was shown that [Gd-DOTP](5-) binds to cytochrome c(3) near heme IV, causing pronounced PREs, characterized by line width broadenings of the heme methyl resonances at ratios as low as 0.08. A K(d) of 23 +/- 1 muM was calculated based on chemical shift perturbation of selected heme methyl resonances belonging to three different heme groups, caused by allosteric effects upon [Gd-DOTP](5-) binding to cytochrome c(3) at a molar ratio of 2. The other probe, [Gd-DOTAM](3+), caused PREs on a well-defined patch near the metal center of rubredoxin (especially the patch constituted by residues D19-G23 and W37-S45, which broaden beyond detection). This effect was partially reversed for some resonances (C6-Y11, in particular) when cytochrome c(3) was added to this system. Both probes were successful in causing reversible PREs at the partner binding site, thus showing to be good probes to identify partners' binding sites and since the interaction is reversible to structurally characterize protein complexes by better defining the complex interface.

The importance of understanding interactomes makes preeminent the study of protein interactions and protein complexes. Traditionally, protein interactions have been elucidated by experimental methods or, with lower impact, by simulation with protein docking algorithms. This article describes features and applications of the BiGGER docking algorithm, which stands at the interface of these two approaches. BiGGER is a user-friendly docking algorithm that was specifically designed to incorporate experimental data at different stages of the simulation, to either guide the search for correct structures or help evaluate the results, in order to combine the reliability of hard data with the convenience of simulations. Herein, the applications of BiGGER are described by illustrative applications divided in three Case Studies: (Case Study A) in which no specific contact data is available; (Case Study B) when different experimental data (e.g., site-directed mutagenesis, properties of the complex, NMR chemical shift perturbation mapping, electron tunneling) on one of the partners is available; and (Case Study C) when experimental data are available for both interacting surfaces, which are used during the search and/or evaluation stage of the docking. This algorithm has been extensively used, evidencing its usefulness in a wide range of different biological research fields.

The paramagnetic effect due to the presence of a metal center with unpaired electrons is no longer considered a hindrance in protein NMR spectroscopy. In the present work, the paramagnetic effect due to the presence of a metal center with impaired electrons was used to map the interface of an electron transfer complex. Desulfovibrio gigas cytochrome c(3) was chosen as target to study the effect of the paramagnetic probe, Fe-rubredoxin, which produced specific line broadening in the heme IV methyl resonances M2(1) and M18(1). The rubredoxin binding surface in the complex with cytochrome c(3) was identified in a heteronuclear 2D NMR titration. The identified heme methyls on cytochrome c(3) are involved in the binding interface of the complex, a result that is in agreement with the predicted complexes obtained by restrained molecular docking, which shows a cluster of possible solutions near heme IV. The use of a paramagnetic probe in (1)HNMR titration and the mapping of the complex interface, in combination with a molecular simulation algorithm proved to be a valuable strategy to study electron transfer complexes involving non-heme iron proteins and cytochromes. (C) 2009 Elsevier Inc. All rights reserved.

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.