María Gabriela Rivas

NAD-dependent formate dehydrogenases (NAD-FdHs) are essential biocatalysts with distinct metabolic functions depending on their source. These enzymes have a great potential for industrial applications in NADH regeneration and carbon capture and conversion technologies. However, although they catalyze the reversible oxidation of formate to CO2, a primary bottleneck for their application in CO2 conversion is the pronounced reaction rate asymmetry, as the reverse reaction (CO2 reduction) rate is several orders of magnitude slower than the forward reaction (HCOO- oxidation). This asymmetry is fundamentally a kinetic manifestation of the thermodynamic equilibrium favoring formate oxidation, as defined by the Haldane relationship. While structural studies have identified conserved active sites, the role of specific molecular features, such as distinct amino acid residues and loops, in determining substrate binding affinities and why CO2 reduction kinetics is so slow even in absence of formate and NAD+ remains unexplored in the current literature. This work addresses some of these issues through fluorescence spectroscopy and Molecular Dynamics simulations. Results showed that the yeast NAD-FdH has independent binding sites for the NAD(H) and that the affinity for NADH is ~200-fold higher than for NAD+. On the other hand, the bacterial enzyme exhibits negative cooperativity for NAD+ binding and positive cooperativity for NADH binding, and their affinities are roughly of the same order. Based on these findings, we discuss the factors that could explain why some NAD-FdHs are more proficient in CO2 reduction than others.

We report the crystal structure of the copper‐containing nitrite reductase (NirK) from the Gram‐negative bacterium Sinorhizobium meliloti 2011 (Sm), together with complex structural alignment and docking studies with both non‐cognate and the physiologically related pseudoazurins, SmPaz1 and SmPaz2, respectively. S. meliloti is a rhizobacterium used for the formulation of Medicago sativa bionoculants, and SmNirK plays a key role in this symbiosis through the denitrification pathway. The structure of SmNirK, solved at a resolution of 2.5 Å, showed a striking resemblance with the overall structure of the well‐known Class I NirKs composed of two Greek key β‐barrel domains. The activity of SmNirK is ~12% of the activity reported for classical NirKs, which could be attributed to several factors such as subtle structural differences in the secondary proton channel, solvent accessibility of the substrate channel, and that the denitrifying activity has to be finely regulated within the endosymbiont. In vitro kinetics performed in homogenous and heterogeneous media showed that both SmPaz1 and SmPaz2, which are coded in different regions of the genome, donate electrons to SmNirK with similar performance. Even though the energetics of the interprotein electron transfer (ET) process is not favorable with either electron donors, adduct formation mediated by conserved residues allows minimizing the distance between the copper centers involved in the interprotein ET process.

Molybdenum and tungsten are taken up by bacteria and archaea as their soluble oxyanions through high affinity transport systems belonging to the ATP-binding cassette (ABC) transporters. The component A (ModA/TupA) of these transporters is the first selection gate from which the cell differentiates between MoO₄²⁻, WO₄²⁻ and other similar oxyanions. We report the biochemical characterization and the crystal structure of the apo-TupA from Desulfovibrio desulfuricans G20, at 1.4 Å resolution. Small Angle X-ray Scattering data suggests that the protein adopts a closed and more stable conformation upon ion binding. The role of the arginine 118 in the selectivity of the oxyanion was also investigated and three mutants were constructed: R118K, R118E and R118Q. Isothermal titration calorimetry clearly shows the relevance of this residue for metal discrimination and oxyanion binding. In this sense, the three variants lost the ability to coordinate molybdate and the R118K mutant keeps an extremely high affinity for tungstate. These results contribute to an understanding of the metal-protein interaction, making it a suitable candidate for a recognition element of a biosensor for tungsten detection.

Respiratory nitrate reductases (Nars) are oxidoreductases that depend on complex cofactors that contain different transition metals. Nars are produced by microorganisms that can grow under denitrifying conditions and catalyse the first step of the denitrification, a geochemical process that involves the conversion of nitrogen oxoanions and oxides into dinitrogen (N2[g]). Nars are complex enzymes with an (αβγ)2 biological assembly. The α- and β-subunits harbour the Mo-bisPGD cofactor and iron–sulphur clusters, while the integral membrane γ-subunit contains two b-type haems. The latter are involved in the oxidation of the membrane quinol pool and the generation of a proton gradient that energises the bacterium cell. In the present chapter, we update some general aspects of molybdo-enzymes and the present knowledge on Nars. Firstly, the special chemical properties that make Mo an efficient O-atom transfer catalyst are briefly presented. Next, the classification of molybdo-enzymes into different families based on the structural properties of the active site is discussed. Then, we review in detail the structural and spectroscopic properties of each subunit comprising the Nar heterotrimer, as well as the complex regulation of nar gene expression, the metabolic role of Nars within the bacterium cell and some key aspects of the catalytic mechanism.

Chlorite dismutase (Cld) catalyzes the reduction of chlorite to chloride and dioxygen. Here, the ligand binding to Cld of Magnetospirillum sp. (MaCld) is investigated with X-ray crystallography and electron paramagnetic resonance (EPR). EPR reveals a large heterogeneity in the structure of wild-type MaCld, showing a variety of low- and high-spin ferric heme forms. Addition of an axial ligand, such as azide or imidazole, removes this heterogeneity almost entirely. This is in line with the two high resolution crystal structures of MaCld obtained in the presence of azide and thiocyanate that show the coordination of the ligands to the heme iron. The crystal structure of the MaCld–azide complex reveals a single well-defined orientation of the azide molecule in the heme pocket. EPR shows, however, a pH-dependent heme structure, probably due to acid–base transitions of the surrounding amino-acid residues stabilizing azide. For the azide and imidazole complex of MaCld, the hyperfine and nuclear quadrupole interactions with the close-by ¹⁴N and ¹H nuclei are determined using pulsed EPR. These values are compared to the corresponding data for the low-spin forms observed in the ferric wild-type MaCld and to existing EPR data on azide and imidazole complexes of other heme proteins.

The activity of sulfate-reducing bacteria (SRB) intensifies the problems associated to corrosion of metals and the solution entails significant economic costs. Although molybdate can be used to control the negative effects of these organisms, the mechanisms triggered in the cells exposed to Mo-excess are poorly understood. In this work, the effects of molybdate ions on the growth and morphology of the SRB Desulfovibrio alaskensis G20 (DaG20) were investigated. In addition, the cellular localization, ion uptake and regulation of protein expression were studied. We found that molybdate concentrations ranging between 50 and 150 µM produce a twofold increase in the doubling time with this effect being more significant at 200 µM molybdate (five times increase in the doubling time). It was also observed that 500 µM molybdate completely inhibits the cellular growth. On the context of protein regulation, we found that several enzymes involved in energy metabolism, cellular division and metal uptake processes were particularly influenced under the conditions tested. An overall description of some of the mechanisms involved in the DaG20 adaptation to molybdate-stress conditions is discussed.