The radical S-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 p-cresol. Clostridium acetobutylicum 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 p-cresol but are necessary for conversion of a tyrosine-derived intermediate to cyanide and CO.

A system has been developed for subjecting protein crystals to hyperbaric pressures of oxygen gas in order to promote enzymatic reaction. Crystals of an oxygenase or oxidase enzyme are grown anaerobically by hanging drop vapor diffusion, under crystallization conditions modified to eliminate combustible materials such as plastic coverslips and grease. The crystalline enzyme:substrate complex can then be exposed to oxygen gas at pressures up to 60 bar using a custom-built device or “bomb.” In this way, reaction is initiated synchronously throughout the crystal and subsequent flash freezing allows the trapping of enzyme:product complexes in high occupancy. These complexes can then be structurally characterized by conventional monochromatic X-ray crystallography. The bomb is furnished from naval brass and lubricated with Fomblin RT15 perfluorinated polyether grease in order to ensure compatibility with the highly oxidizing environment.