Probing the Role of Accessory FeS Clusters in Putative Sensory Group D [FeFe] Hydrogenase: Influence of the C-Terminal [4Fe-4S] Cluster on the Reactivity of Tam HydS

Hydrogenases are metalloenzymes that play key roles in H₂ metabolism. Beyond their catalytic function, [FeFe] hydrogenases from phylogenetic groups C and D have been proposed to act as H₂ sensors. These putative sensory enzymes contain, in addition to the canonical H₂-activating H-cluster, N-terminal [4Fe-4S] clusters as well as an atypical C-terminal [4Fe-4S] cluster, the latter being further associated with a Per-Arnt-Sim (PAS) domain in group C enzymes. The functional significance of these C-terminal clusters and their influence on enzymatic activity, however, remain poorly understood. Here we studied the accessory [4Fe-4S] clusters in the model group D enzyme from Thermoanaerobacter mathranii (TamHydS), by disrupting cluster formation in the N- and C-terminal domains, respectively. Spectroscopic and biochemical investigations indicated that one of the N-terminal [4Fe-4S] clusters is critical for the structural integrity of TamHydS. In contrast, disrupting the C-terminal [4Fe-4S] cluster through a cysteine to alanine mutation (C379A) resulted in a stable enzyme variant with a modified cluster. The C379A variant retained catalytic activity similar to the WT enzyme, although with a 2-fold enhancement of the H₂ oxidation rate observed under high-driving-force conditions. Based on electron paramagnetic resonance spectroscopy, we found the oxidation state of the C-terminal cluster to be unresponsive to the presence of H₂ gas. We inferred that the H₂-sensing or signaling function is unlikely to involve redox state changes in the C-terminal [4Fe-4S] cluster. Still, highly conserved charged residues around the [4Fe-4S] clusters of group D [FeFe] hydrogenases indicate a functional role of the C-terminal region.