| Summary: | Three iron-sulfur proteins–HydE, HydF, and HydG–play a key role in the synthesis of the [2Fe]<sub>H</sub> component of the catalytic H-cluster of FeFe hydrogenase. The radical <em>S</em>-adenosyl-<span style="font-variant: small-caps;">l</span>-methionine enzyme HydG lyses free tyrosine to produce <em>p</em>-cresol and the CO and CN<sup>−</sup> ligands of the [2Fe]<sub>H</sub> 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<sup>−</sup> ligands with spectroscopic signatures that evolve on the 1- to 1000-second time scale. Through study of the <sup>13</sup>C, <sup>15</sup>N, and <sup>57</sup>Fe isotopologs of these intermediates and products, we identify the final HydG-bound species as an organometallic Fe(CO)<sub>2</sub>(CN) synthon that is ultimately transferred to apohydrogenase to form the [2Fe]<sub>H</sub> component of the H-cluster.
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