title: Biochemical and kinetic characterization of radical S-adenosyl-L-methionine enzyme HydG.
abstract: 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.
authors.affiliation: Chemistry, Faculty of Natural and Engineering Sciences, University of Southampton , Highfield SO17 1BJ, U.K.
authors.forename: Rebecca C
authors.initials: RC
authors.lastname: Driesener
authors.forename: Benjamin R
authors.initials: BR
authors.lastname: Duffus
authors.forename: Eric M
authors.initials: EM
authors.lastname: Shepard
authors.forename: Ian R
authors.initials: IR
authors.lastname: Bruzas
authors.forename: Kaitlin S
authors.initials: KS
authors.lastname: Duschene
authors.forename: Natalie J-R
authors.initials: NJ
authors.lastname: Coleman
authors.forename: Alexander P G
authors.initials: AP
authors.lastname: Marrison
authors.forename: Enrico
authors.initials: E
authors.lastname: Salvadori
authors.forename: Christopher W M
authors.initials: CW
authors.lastname: Kay
authors.forename: John W
authors.initials: JW
authors.lastname: Peters
authors.forename: Joan B
authors.initials: JB
authors.lastname: Broderick
authors.forename: Peter L
authors.initials: PL
authors.lastname: Roach
mesh_terms: D002384:Catalysis; D046969:Clostridium acetobutylicum / Q000201:enzymology* / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D006864:Hydrogenase / Q000737:chemistry* / Q000235:genetics; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D012436:S-Adenosylmethionine / Q000737:chemistry*
chemical_list: D007506:Iron-Sulfur Proteins; D012436:S-Adenosylmethionine; D006864:Hydrogenase