Spectroscopic and Structural Characterization of Reduced Desulfovibrio vulgaris Hildenborough W-FdhAB Reveals Stable Metal Coordination during Catalysis

Funding Information: This work was financially supported by Fundação para a Ciência e Tecnologia (FCT, Portugal) through fellowship SFRH/BD/116515/2016, COVID/BD/151766/2021, grant PTDC/BII-BBF/2050/2020, R&D units MOSTMICRO-ITQB (UIDB/04612/2020 and UIDP/04612/2020) and UCIBIO (UIDP/04378/2020 and UIDB/04378/2020), and associated laboratories LS4FUTURE (LA/P/0087/2020) and i4HB (LA/P/0140/2020). European Union’s Horizon 2020 research and innovation program (grant agreement no. 810856) is also acknowledged. This work was also funded by the French national research agency (ANR─MOLYERE project, grant number 16-CE-29-0010-01) and supported by the computing facilities of the CRCMM, “Centre Régional de Compétences en Modélisation Moléculaire de Marseille”. The authors are grateful to the EPR facilities at the French EPR network RENARD (IR CNRS 3443, now INFRANALYTICS, FR2054) and the Aix-Marseille University EPR center. Publisher Copyright: © 2022 American Chemical Society. All rights reserved. Metal-dependent formate dehydrogenases are important enzymes due to their activity of CO2reduction to formate. The tungsten-containing FdhAB formate dehydrogenase from Desulfovibrio vulgaris Hildenborough is a good example displaying high activity, simple composition, and a notable structural and catalytic robustness. Here, we report the first spectroscopic redox characterization of FdhAB metal centers by EPR. Titration with dithionite or formate leads to reduction of three [4Fe-4S]1+clusters, and full reduction requires Ti(III)-citrate. The redox potentials of the four [4Fe-4S]1+centers range between -250 and -530 mV. Two distinct WVsignals were detected, WDVand WFV, which differ in only the g2-value. This difference can be explained by small variations in the twist angle of the two pyranopterins, as determined through DFT calculations of model compounds. The redox potential of WVI/Vwas determined to be -370 mV when reduced by dithionite and -340 mV when reduced by formate. The crystal structure of dithionite-reduced FdhAB was determined at high resolution (1.5 Å), revealing the same structural alterations as reported for the formate-reduced structure. These results corroborate a stable six-ligand W coordination in the catalytic intermediate WVstate of FdhAB. publishersversion published