The radical S-adenosylmethionine (SAM) enzyme HydG lyses free L-tyrosine to produce CO and CN- 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 H-2, C-13, and N-15 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-alpha-C-beta bond forms a transient 4-oxidobenzyl (4OB(center dot)) radical and a dehydroglycine bound to the C-terminal 4Fe-4S cluster. Electron and proton transfer to this 4OB(center dot) radical forms p-cresol, with the conversion of this dehydroglycine ligand to Fe-bound CO and CN-, a key intermediate in the assembly of the 2Fe subunit of the H cluster.