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Biochemical and Kinetic Characterization of Radical S-Adenosyl-L-methionine Enzyme HydG.
- Source :
-
Biochemistry . 12/3/2013, Vol. 52 Issue 48, p8696-8707. 12p. - Publication Year :
- 2013
-
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. [ABSTRACT FROM AUTHOR]
- Subjects :
- *ADENOSYLMETHIONINE
*TYROSINE
*SPECTRUM analysis
*METHIONINE
*ENZYMES
*CATALYSTS
Subjects
Details
- Language :
- English
- ISSN :
- 00062960
- Volume :
- 52
- Issue :
- 48
- Database :
- Academic Search Index
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 93248278
- Full Text :
- https://doi.org/10.1021/bi401143s