EPR Spectroscopic Studies of [FeFe]-Hydrogenase Maturation

Proton reduction and H₂ oxidation are key elementary reactions for solar fuel production. Hydrogenases interconvert H⁺ and H₂ with remarkable efficiency and have therefore received much attention in this context. For [FeFe]-hydrogenases, catalysis occurs at a unique cofactor called the H-cluster. In this article, we discuss ways in which EPR spectroscopy has elucidated aspects of the bioassembly of the H-cluster, with a focus on four case studies: EPR spectroscopic identification of a radical en route to the CO and CN^- ligands of the H-cluster, tracing ⁵⁷Fe from the maturase HydG into the H-cluster, characterization of the auxiliary Fe-S cluster in HydG, and isotopic labeling of the CN^- ligands of HydA for electronic structure studies of its H_ox state. Advances in cell-free maturation protocols have enabled several of these mechanistic studies, and understanding H-cluster maturation may in turn provide insights leading to improvements in hydrogenase production for biotechnological applications.