A glycyl free radical as the precursor in the synthesis of carbon monoxide and cyanide by the [FeFe]‐hydrogenase maturase HydG

HydG uses tyrosine to synthesize the CN(-)/CO ligands of [FeFe]-hydrogenase active site. We have mutated two of the [4Fe-4S]-cluster cysteine ligands of the HydG C-terminal domain (CTD) to serine. The double mutant can still synthesize CN(-) but not CO. In a mutant lacking the CTD both CN(-) and CO synthesis are abolished. Like in ThiH, the initial steps of CN(-) synthesis are carried out in the TIM-barrel domain of HydG but some component(s) of the CTD are later needed. The mutants indicate that CO synthesis is metal-based and occurs in the CTD. We postulate that CN(-)/CO synthesis is initiated by H(2)N-*CH-CO(2)(-). Fragmentation of this radical into H(2)N-*CH(2) and CO(2) or H(2)C=NH and *CO(2)(-) provides plausible precursors for CN(-)/CO synthesis.