[FeFe] hydrogenases provide a blueprint for developing sustainable hydrogen-based energy technologies due to their efficiency even when utilizing earth-abundant metals. They are phylogenetically divided into Groups A–G.1-4 However, their structural and functional diversity remains underexplored. Here, we report the characterization of a single-domain Group A [FeFe] hydrogenase from a metabolically constrained nanosized microorganism. The enzyme displays activity and H-cluster fingerprint spectroscopic signatures despite an apparent lack of canonical H-cluster maturation machinery. Furthermore, the enzyme displays an unexpected extreme catalytic bias.
Analysis of structural models reveals an overall high similarity to previously characterized prototypical Group A [FeFe] hydrogenases and conserved key second coordination sphere residues surrounding the active site cofactor (H-cluster).5-6 The one identified exception being a methionine substituted by leucine. We report the enzyme's catalytic and spectroscopic properties, displaying a pronounced bias towards H₂ oxidation—a rare feature among characterized [FeFe] hydrogenases. Following artificial maturation7-8, the enzyme is isolated in an inactive state, requiring reductive activation to respond to H2 and interconvert redox intermediates, consistent with negligible oxidation activity in electrochemistry and in vitro assays unless exposed to negative potentials or reductants.
Its strong catalytic preference towards H2 oxidation suggests a unique physiological role within the nanosized microorganism, potentially linked to unidentified metabolic pathways beyond the predicted role involving fermentative H2 production.
Overall, these findings expand our understanding of the functional diversity and evolutionary adaptations of [FeFe] hydrogenases with implications for bio-inspired catalysts in sustainable energy applications, particularly fuel cells.9