Structure of the α ₂ ε ₂ Ni-dependent CO dehydrogenase component of the <i>Methanosarcina barkeri</i> acetyl-CoA decarbonylase/synthase complex

<jats:p> Ni-dependent carbon monoxide dehydrogenases (Ni-CODHs) are a diverse family of enzymes that catalyze reversible CO:CO <jats:sub>2</jats:sub> oxidoreductase activity in acetogens, methanogens, and some CO-using bacteria. Crystallography of Ni-CODHs from CO-using bacteria and acetogens has revealed the overall fold of the Ni-CODH core and has suggested structures for the C cluster that mediates CO:CO <jats:sub>2</jats:sub> interconversion. Despite these advances, the mechanism of CO oxidation has remained elusive. Herein, we report the structure of a distinct class of Ni-CODH from methanogenic archaea: the α <jats:sub>2</jats:sub> ε <jats:sub>2</jats:sub> component from the α <jats:sub>8</jats:sub> β <jats:sub>8</jats:sub> γ <jats:sub>8</jats:sub> δ <jats:sub>8</jats:sub> ε <jats:sub>8</jats:sub> CODH/acetyl-CoA decarbonylase/synthase complex, an enzyme responsible for the majority of biogenic methane production on Earth. The structure of this Ni-CODH component provides support for a hitherto unobserved state in which both CO and H <jats:sub>2</jats:sub> O/OH <jats:sup>-</jats:sup> bind to the Ni and the exogenous FCII iron of the C cluster, respectively, and offers insight into the structures and functional roles of the ε-subunit and FeS domain not present in nonmethanogenic Ni-CODHs. </jats:p>