Unique Spectroscopic Properties of the H-Cluster in a Putative Sensory [FeFe] Hydrogenase

Sensory type [FeFe] hydrogenases are predicted to play a role in transcriptional regulation by detecting the H2 level of the cellular environment. These hydrogenases contain the hydrogenase domain with distinct modifications in the active site pocket, followed by a Per-Arnt-Sim (PAS) domain. As yet, neither the physiological function nor the biochemical or spectroscopic properties of these enzymes have been explored. Here, we present the characterization of an artificially maturated, putative sensory [FeFe] hydrogenase from Thermotoga maritima (HydS). This enzyme shows lower hydrogen conversion activity than prototypical [FeFe] hydrogenases and a reduced inhibition by CO. Using FTIR spectroelectrochemistry and EPR spectroscopy, three redox states of the active site were identified. The spectroscopic signatures of the most oxidized state closely resemble those of the Hox state from the prototypical [FeFe] hydrogenases, while the FTIR spectra of both singly and doubly reduced states show large differences. ...