Biochemical and Kinetic Characterization of Radical <i>S</i>‑Adenosyl‑l‑methionine Enzyme HydG

The radical <i>S</i>-adenosyl-l-methionine (AdoMet) enzyme HydG is one of three maturase enzymes involved in [FeFe]-hydrogenase H-cluster assembly. It catalyzes l-tyrosine cleavage to yield the H-cluster cyanide and carbon monoxide ligands as well as <i>p</i>-cresol. <i>Clostridium acetobutylicum</i> HydG contains the conserved CX₃CX₂C motif coordinating the AdoMet binding [4Fe-4S] cluster and a C-terminal CX₂CX₂₂C motif proposed to coordinate a second [4Fe-4S] cluster. To improve our understanding of the roles of each of these iron–sulfur clusters in catalysis, we have generated HydG variants lacking either the N- or C-terminal cluster and examined these using spectroscopic and kinetic methods. We have used iron analyses, UV–visible spectroscopy, and electron paramagnetic resonance (EPR) spectroscopy of an N-terminal C96/100/103A triple HydG mutant that cannot coordinate the radical AdoMet cluster to unambiguously show that the C-terminal cysteine motif coordinates an auxiliary [4Fe-4S] cluster. Spectroscopic comparison with a C-terminally truncated HydG (ΔCTD) harboring only the N-terminal cluster demonstrates that both clusters have similar UV–visible and EPR spectral properties, but that AdoMet binding and cleavage occur only at the N-terminal radical AdoMet cluster. To elucidate which steps in the catalytic cycle of HydG require the auxiliary [4Fe-4S] cluster, we compared the Michaelis–Menten constants for AdoMet and l-tyrosine for reconstituted wild-type, C386S, and ΔCTD HydG and demonstrate that these C-terminal modifications do not affect the affinity for AdoMet but that the affinity for l-tyrosine is drastically reduced compared to that of wild-type HydG. Further detailed kinetic characterization of these HydG mutants demonstrates that the C-terminal cluster and residues are not essential for l-tyrosine cleavage to <i>p</i>-cresol but are necessary for conversion of a tyrosine-derived intermediate to cyanide and CO