Biochemical and Kinetic Characterization of Radical S-Adenosyl-l-methionine Enzyme HydG

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• 作者：Rebecca C. Driesener ; Benjamin R. Duffus ; Eric M. Shepard ; Ian R. Bruzas ; Kaitlin S. Duschene ; Natalie J.-R. Coleman ; Alexander P. G. Marrison ; Enrico Salvadori ; Christopher W. M. Kay ; John W. Peters ; Joan B. Broderick ; Peter L. Roach
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：December 3, 2013
• 年：2013
• 卷：52
• 期：48
• 页码：8696-8707
• 全文大小：407K
• 年卷期：v.52,no.48(December 3, 2013)
• ISSN：1520-4995

文摘

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鈥搒ulfur 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鈥搗isible 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 (螖CTD) harboring only the N-terminal cluster demonstrates that both clusters have similar UV鈥搗isible 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鈥揗enten constants for AdoMet and l-tyrosine for reconstituted wild-type, C386S, and 螖CTD 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.