FeFe hydrogenases are highly active, reversible enzymes for the interconversion of hydrogen with protons and electrons. Their active site H-cluster consists of a canonical 4Fe-4S cluster covalently linked to a unique 2FeH centre. Their catalytic mechanism has been studied extensively, but several details remain disputed, and two rival models exist in the literature. One crucial difference between these models is the structure and catalytic relevance of two states named HredH+ and HsredH+. In the first model, these states are catalytic intermediates containing a reduced Fe(I)Fe(I)H centre and a bridging CO ligand (µCO), while in the second model they are inactive states containing an oxidised Fe(II)Fe(II)H site and a bridging hydride ligand (µH-). The second proposal was initially based on the lack of a prominent absorption peak attributed to a µCO ligand in the infrared (IR) spectra of both states. Here, we provide evidence for the presence of a µCO ligand in the HredH+ and HsredH+ states using two-dimensional (2D) IR spectroscopy, firmly establishing the structure of these states as Fe(I)Fe(I)H with a µCO ligand. The results suggest that these states are catalytically relevant intermediates with crucial implications for understanding hydrogen conversion in nature and designing new synthetic catalysts.
Bernitzky et al. (Fri,) studied this question.