Pereira, I. C. | Search bioRxiv

bioRxiv2024

Substrate-dependent oxidative inactivation of a W-dependent formate dehydrogenase involving selenocysteine displacement.

Metal-dependent formate dehydrogenases are very promising targets for enzyme optimization and design of bio-inspired catalysts for CO2 reduction, towards novel strategies for climate change mitigation. For effective application of these enzymes, the catalytic mechanism must be fully understood, and the molecular determinants clarified. Despite numerous studies, several doubts persist, namely regarding the role played by the possible dissociation of the SeCys ligand from the Mo/W active site. Additionally, the O2 sensitivity of these enzymes must also be understood as it poses an important obstacle for biotechnological applications. Here we present a combined biochemical, spectroscopic, and structural characterization of Desulfovibrio vulgaris FdhAB (DvFdhAB) when exposed to oxygen in the presence of a substrate (formate or CO2). This study reveals that O2 inactivation is promoted by the presence of either substrate and involves forming a new species in the active site, captured in the crystal structures, where the SeCys ligand is displaced from tungsten coordination and replaced by a dioxygen or peroxide molecule. This new form was reproducibly obtained and supports the conclusion that, although W-DvFdhAB can catalyze the oxidation of formate in the presence of oxygen for some minutes, it gets irreversibly inactivated after prolonged O2 exposure in the presence of either substrate. These results reveal that oxidative inactivation does not require reduction of the metal, as widely assumed, as it can also occur in the oxidized state in the presence of CO2.

biochemistry↗

bioRxiv2023

DsrMKJOP is the terminal reductase complex in anaerobic sulfate respiration

Microbial dissimilatory sulfate reduction is a key process in the Earth biogeochemical sulfur cycle. In spite of its importance to the sulfur and carbon cycles, industrial processes and human health, it is still not clear how reduction of sulfate to sulfide is coupled to energy conservation. A central step in the pathway is the reduction of sulfite by the DsrAB dissimilatory sulfite reductase, which leads to the production of a DsrC-trisulfide. A membrane-bound complex, DsrMKJOP, is present in most organisms that have DsrAB and DsrC, and its involvement in energy conservation has been inferred from sequence analysis, but its precise function was so far not determined. Here, we present studies revealing that the DsrMKJOP complex of the sulfate reducer Archaeoglobus fulgidus works as a menadiol:DsrC-trisulfide oxidoreductase. Our results reveal a close interaction between the DsrC-trisulfide and the DsrMKJOP complex and show that electrons from the quinone pool reduce consecutively the DsrM hemes b, the DsrK noncubane [4Fe-4S]3+/2+ catalytic center, and finally the DsrC-trisulfide with concomitant release of sulfide. These results clarify the role of this widespread respiratory membrane complex and indicate that DsrMKJOP will provide the missing link to energy conservation by generating a proton motive force across the membrane in the last step of dissimilatory sulfate reduction. Significance StatementDissimilatory sulfate reduction (DSR) is a vital microbial process in anoxic environments, namely in sulfate-rich marine sediments that harbor a vast microbial ecosystem. DSR drives the global biogeochemical sulfur cycle and is crucial in remineralization of organic matter on the seafloor. It also has huge environmental impact by preventing release of the greenhouse gas methane from these sediments, through its oxidation coupled to sulfate reduction. Despite its high ecological importance, it is still not clear how microorganisms derive energy to grow through DSR. Here, we disclose the physiological function of a widespread membrane complex in DSR, showing it acts as the terminal reductase in the respiratory chain and providing important insights into how sulfate/sulfite reduction is linked to energy conservation.

microbiology↗