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 H2N–CH–CO2-. Fragmentation of this radical into H2N–CH2 and CO2 or H2CNH and CO2- provides plausible precursors for CN−/CO synthesis.