| Sumari: | The radical <em>S</em>-adenosylmethionine (SAM) enzyme HydG lyses free L-tyrosine to produce CO and CN<em><sup>−</sup></em> for the assembly of the catalytic H cluster of FeFe hydrogenase. We used electron paramagnetic resonance spectroscopy to detect and characterize HydG reaction intermediates generated with a set of <sup>2</sup>H, <sup>13</sup>C, and <sup>15</sup>N nuclear spin-labeled tyrosine substrates. We propose a detailed reaction mechanism in which the radical SAM reaction, initiated at an N-terminal 4Fe-4S cluster, generates a tyrosine radical bound to a C-terminal 4Fe-4S cluster. Heterolytic cleavage of this tyrosine radical at the C<sub>α</sub>-C<sub>β</sub> bond forms a transient 4-oxidobenzyl (4OB<em><sup>•</sup></em>) radical and a dehydroglycine bound to the C-terminal 4Fe-4S cluster. Electron and proton transfer to this 4OB<em><sup>•</sup></em> radical forms <em>p</em>-cresol, with the conversion of this dehydroglycine ligand to Fe-bound CO and CN<em><sup>−</sup></em>, a key intermediate in the assembly of the 2Fe subunit of the H cluster.
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