The Role of Selenocysteine in Catalysis and Oxygen Tolerance of a W-Dependent Formate Dehydrogenase
Ana Rita Oliveira, Guilherme Vilela‐Alves, Cristiano Mota, Christophe Léger, Vincent Fourmond, Frédéric Biaso, Bruno Guigliarelli, Maria João Romão, Inês A. C. Pereira
Abstract
Metal-dependent formate dehydrogenases (FDHs) catalyze, under mild conditions, the reversible reduction of CO 2 to formate, a versatile C1 feedstock that can contribute to a carbon-neutral economy. Metal-dependent FDHs are the most widespread selenoproteins found in bacteria, and around 44% of them include selenocysteine (Sec) as a ligand to the Mo/W active site. In the sulfate-reducer Nitratidesulfovibrio vulgaris Hildenborough, the main FDH responsible for CO 2 reduction is the W/Sec-dependent FdhAB, which is among the most active CO 2 reductases reported so far. In contrast to most metal-dependent FDHs, this enzyme is relatively O 2 -tolerant and can be purified aerobically. In this work, we evaluated the role of Sec in the catalytic and stability properties of the W/Sec-FdhAB. For that, a Sec-to-Cys variant (U192C) was created, its catalytic and spectroscopic properties were characterized, and its crystal structure was determined. Sec substitution by Cys strongly affects activity, decreases the K M for formate, and increases susceptibility to O 2 . While Sec-to-Cys replacement induces only weak changes of the W V EPR signals, using 77 Se-labeled enzyme, we could show that Sec undoubtedly coordinates the W metal in the W V redox state. The crystal structure of U192C confirmed previous findings on the redox switch mechanism of activation and protection of FdhAB, while revealing a putative catalytic intermediate of FdhAB with Arg441 orienting a CO 2 substrate analog (probably SO 2 ) in the active site. Overall, the results indicate that Sec plays a critical role in the high activity displayed by W/Sec-FdhAB, and that it may also be involved in or modulate the proton transfer to and from the active site.