Reducing CO 2 is a challenging chemical transformation that biology solves easily, with high efficiency and specificity. In particular, formate dehydrogenases are of great interest since they reduce CO 2 to formate, a valuable chemical fuel and hydrogen storage compound. The metal-dependent formate dehydrogenases of prokaryotes can show high activity for CO 2 reduction. Here, we report an expression system to produce recombinant W/Sec-FdhAB from Desulfovibrio vulgaris Hildenborough fully loaded with cofactors, its catalytic characterization and crystal structures in oxidized and reduced states. The enzyme has very high activity for CO 2 reduction and displays remarkable oxygen stability. The crystal structure of the formate-reduced enzyme shows Sec still coordinating the tungsten, supporting a mechanism of stable metal coordination during catalysis. Comparison of the oxidized and reduced structures shows significant changes close to the active site. The DvFdhAB is an excellent model for studying catalytic CO 2 reduction and probing the mechanism of this conversion.

Metal-dependent formate dehydrogenases (Fdh) from prokaryotic organisms are members of the dimethyl sulfoxide reductase family of mononuclear molybdenum-containing and tungsten-containing enzymes. Fdhs catalyze the oxidation of the formate anion to carbon dioxide in a redox reaction that involves the transfer of two electrons from the substrate to the active site. The active site in the oxidized state comprises a hexacoordinated molybdenum or tungsten ion in a distorted trigonal prismatic geometry. Using this structural model, we calculated the catalytic mechanism of Fdh through density functional theory tools. The simulated mechanism was correlated with the experimental kinetic properties of three different Fdhs isolated from three different Desulfovibrio species. Our studies indicate that the C-H bond break is an event involved in the rate-limiting step of the catalytic cycle. The role in catalysis of conserved amino acid residues involved in metal coordination and near the metal active site is discussed on the basis of experimental and theoretical results.

Spontaneous aggregation of folded and soluble native proteins in vivo is still a poorly understood process. A prototypic example is the D76N mutant of beta-2 microglobulin (β2m) that displays an aggressive aggregation propensity. Here we investigate the dynamics of β2m by X-ray crystallography, solid-state NMR, and molecular dynamics simulations to unveil the effects of the D76N mutation. Taken together, our data highlight the presence of minor disordered substates in crystalline β2m. The destabilization of the outer strands of D76N β2m accounts for the increased aggregation propensity. Furthermore, the computational modeling reveals a network of interactions with residue D76 as a keystone: this model allows predicting the stability of several point mutants. Overall, our study shows how the study of intrinsic dynamics in crystallo can provide crucial answers on protein stability and aggregation propensity. The comprehensive approach here presented may well be suited for the study of other folded amyloidogenic proteins.

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