Fig. 1. Schematic representation of the whole-cell assembly and spectroscopic investigation of the resulting semi-synthetic H-cluster. Genetic modification of E. coli for expression of [FeFe]-hydrogenase apo-protein, followed by synthetic maturation generates functional hydrogenase (the H-cluster is shown in the Hox state). The reactivity of this semi-synthetic enzyme is probed in whole cells by EPR and ATR FTIR spectroscopy. Proposed key intermediates were observed in cells for the first time, and conditions for accumulating a protonated hydride state (denoted HhydH+) are reported. In parallel, the integrity of the cells is verified by AFM imaging and near-field IR spectroscopy.
