The HydG Enzyme Generates an Fe(CO)2(CN) Synthon in Assembly of the FeFe Hydrogenase H-Cluster

Three iron-sulfur proteins-HydE, HydF, and HydG-play a key role in the synthesis of the [2Fe](H) component of the catalytic H-cluster of FeFe hydrogenase. The radical S-adenosyl-L-methionine enzyme HydG lyses free tyrosine to produce p-cresol and the CO and CN- ligands of the [2Fe](H) cluster. Here, we applied stopped-flow Fourier transform infrared and electron-nuclear double resonance spectroscopies to probe the formation of HydG-bound Fe-containing species bearing CO and CN- ligands with spectroscopic signatures that evolve on the 1- to 1000-second time scale. Through study of the C-13, N-15, and Fe-57 isotopologs of these intermediates and products, we identify the final HydG-bound species as an organometallic Fe(CO)(2)(CN) synthon that is ultimately transferred to apohydrogenase to form the [2Fe](H) component of the H-cluster.