The Mo/Cu-dependent CO dehydrogenase from O. carboxydovorans is an enzyme that is able to catalyse CO oxidation to CO 2 ; moreover, it also expresses hydrogenase activity, as it is able to oxidize H 2 . Here, we have studied the dihydrogen oxidation catalysis by this enzyme using QM/MM calculations. Our results indicate that the equatorial oxo ligand of Mo is the best suited base for catalysis. Moreover, extraction of the first proton from H 2 by means of this basic centre leads to the formation of a Mo–OH–Cu I H hydride that allows for the stabilization of the copper hydride, otherwise known to be very unstable. In light of our results, two mechanisms for the hydrogenase activity of the enzyme are proposed. The first reactive channel depends on protonation of the sulphur atom of a Cu-bound cysteine residues, which appears to favour the binding and activation of the substrate. The second reactive channel involves a frustrated Lewis pair, formed by the equatorial oxo group bound to Mo and by the copper centre. In this case, no binding of the hydrogen molecule to the Cu center is observed but once H 2 enters into the active site, it can be split following a low-energy path.

Some microorganisms, like the aerobic soil bacteria, Oligotropha carboxidovorans, have the capability to oxidize the highly toxic atmospheric gas carbon monoxide (CO) into CO 2 through CO dehydrogenase enzymes, whose active site contains a bimetallic MoCu center. Over the last decades, a number of experimental and theoretical investigations were devoted to understanding the mechanism of CO oxidation and, in particular, the role of a very stable thiocarbonate intermediate that may be formed during the catalytic cycle. The occurrence of such an intermediate was reported to make the CO 2 release step kinetically difficult. In this work, by using an accurate QM/MM approach and energy refinement by means of the BigQM method, we were able to determine the role of such an intermediate and propose a novel mechanism for the oxidation of CO into CO 2 by Mo/Cu CO dehydrogenase. Surprisingly, we found that the detachment of CO 2 occurs directly from the product of the MoO nucleophilic attack reaction on the carbon of CO aided by the transient coordination of the active site glutamate to the Mo ion. The estimated activation barrier is in good agreement with the experimental one, while the thiocarbonate turned out to not interfere with the CO-oxidation catalytic cycle. The results highlight the importance of the environmental effects in the assembly of the molecular model and in the choice of the computational protocol. Our accurate modeling of the enzyme also allowed us to exclude the involvement of a frustrated Lewis pair in the CO-oxidation mechanism, which has recently been suggested based on an analysis of structural and electronic features of synthetic mimics of the Mo/Cu CO dehydrogenase active site.

We present a theoretical investigation providing key insights on a long-standing controversial issue that dominated the debate on carbon monoxide oxidation by Mo-Cu CO-dehydrogenases. Previous investigations gravitate around the possible occurrence of a thiocarbonate intermediate, that was repeatedly reported to behave as a thermodynamic sink on the catalytic energy landscape. By using a hierarchy of quantum mechanical and hybrid quantum/classical models of the enzyme, we show that no such energy sink is present on the catalytic energy profile. Consequent perspectives for the definition of a novel mechanistic proposal for the enzyme-catalyzed CO-oxidation are discussed in light of the recent literature.

The aerobic CO dehydrogenase from Oligotropha carboxidovorans is an environmentally crucial bacterial enzyme for maintenance of subtoxic concentration of CO in the lower atmosphere, as it allows for the oxidation of CO to CO2 which takes place at its Mo−Cu heterobimetallic active site. Despite extensive experimental and theoretical efforts, significant uncertainties still concern the reaction mechanism for the CO oxidation. In this work, we used the hybrid quantum mechanical/molecular mechanical approach to evaluate whether a water molecule present in the active site might act as a nucleophile upon formation of the new C−O bond, a hypothesis recently suggested in the literature. Our study shows that activation of H2O can be favoured by the presence of the Mo=Oeq group. However, overall our results suggest that mechanisms other than the nucleophilic attack by Mo=Oeq to the activated carbon of the CO substrate are not likely to constitute reactive channels for the oxidation of CO by the enzyme.

The present paper reports a study on the energetics of protonation of a hydrogenase biomimetic complex, [Fe 2 (μ-adt)(CO) 4 (PMe 3 ) 2 ] (adt = Nbenzylazadithiolate), and of its homologue featuring triphenylphosphine ligands in place of trimethylphosphines. Formation of a terminal hydride on one of the Fe centres was considered first, given the key relevance of terminal hydride species in the enzymatic mechanism. Theoretical calculations highlight that, in a vacuum, terminal protonation of the selected Fe ion in the PPh 3 -bearing organometallic complex is highly favoured when compared to the analogous reaction involving the PMe 3 -containing species, but the trend is inverted