Abstract
[FeFe] hydrogenases are highly active enzymes for interconverting protons and electrons with hydrogen (H(2)). Their active site H-cluster is formed of a canonical [4Fe-4S] cluster ([4Fe-4S](H)) covalently attached to a unique [2Fe] subcluster ([2Fe](H)), where both sites are redox active. Heterolytic splitting and formation of H(2) takes place at [2Fe](H), while [4Fe-4S](H) stores electrons. The detailed catalytic mechanism of these enzymes is under intense investigation, with two dominant models existing in the literature. In one model, an alternative form of the active oxidized state H(ox), named H(ox)H, which forms at low pH in the presence of the nonphysiological reductant sodium dithionite (NaDT), is believed to play a crucial role. H(ox)H was previously suggested to have a protonated [4Fe-4S](H). Here, we show that H(ox)H forms by simple addition of sodium sulfite (Na(2)SO(3), the dominant oxidation product of NaDT) at low pH. The low pH requirement indicates that sulfur dioxide (SO(2)) is the species involved. Spectroscopy supports binding at or near [4Fe-4S](H), causing its redox potential to increase by ∼60 mV. This potential shift detunes the redox potentials of the subclusters of the H-cluster, lowering activity, as shown in protein film electrochemistry (PFE). Together, these results indicate that H(ox)H and its one-electron reduced counterpart H(red)'H are artifacts of using a nonphysiological reductant, and not crucial catalytic intermediates. We propose renaming these states as the "dithionite (DT) inhibited" states H(ox)-DT(i) and H(red)-DT(i). The broader potential implications of using a nonphysiological reductant in spectroscopic and mechanistic studies of enzymes are highlighted.