Indications for a universal hydrogen catalysis mechanism in [FeFe]-hydrogenases of different phylogenetic groups

[FeFe]-hydrogenases are metalloenzymes catalysing bidirectional hydrogen (H₂) turnover. These enzymes are generally considered to be extremely efficient and fast catalysts. However, [FeFe]-hydrogenases constitute a very diverse enzyme family that can be divided into several distinct phylogenetic groups, denoted as groups A-G. Very little is known about the properties of [FeFe]-hydrogenases outside of the intensively studied group A, but recent studies on putatively sensory group C and D enzymes have revealed distinct differences in reactivity. The variation in structure, reactivity and physiological function observed between phylogenetic groups raises the question if all [FeFe]-hydrogenases follow the same mechanism for H₂ turnover. Here, we provide the first detailed spectroscopic investigation of a slow-acting putatively sensory group D [FeFe]-hydrogenase from <i>Thermoanaerobacter mathranii</i> (<i>Tam</i>HydS). Photo-reduction enabled us to characterize redox states in group D [FeFe]-hydrogenase <i>via</i> infrared spectroscopy under catalytic conditions. The sequential population of redox states similar to group A [FeFe]-hydrogenases supports the notion that group A and D [FeFe]-hydrogenases follow a universal catalytic mechanism. However, clear differences between enzymes from different phylogenetic groups become evident when comparing the relative stability and protonation state of suggested key catalytic intermediates. Moreover, the spectroscopic data collected on <i>Tam</i>HydS provides new insight into the structure of the reduced active site, lending further support for the notion of a retained bridging CO ligand throughout the entire catalytic cycle.