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9-Mercaptodethiobiotin Is Generated as a Ligand to the [2Fe–2S]+ Cluster during the Reaction Catalyzed by Biotin Synthase from Escherichia coli

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journal contribution
posted on 20.02.2016, 20:10 by Corey J. Fugate, Troy A. Stich, Esther G. Kim, William K. Myers, R. David Britt, Joseph T. Jarrett
Biotin synthase catalyzes formation of the thiophane ring through stepwise substitution of a sulfur atom for hydrogen atoms at the C9 and C6 positions of dethiobiotin. Biotin synthase is a radical S-adenosylmethionine (SAM) enzyme that reductively cleaves S-adenosylmethionine, generating 5′-deoxyadenosyl radicals that initially abstract a hydrogen atom from the C9 position of dethiobiotin. We have proposed that the resulting dethiobiotinyl radical is quenched by the μ-sulfide of the nearby [2Fe–2S]2+ cluster, resulting in coupled formation of 9-mercaptodethiobiotin and a reduced [2Fe–2S]+ cluster. This reduced FeS cluster is observed by electron paramagnetic resonance spectroscopy as a mixture of two orthorhombic spin systems. In the present work, we use isotopically labeled 9-mercaptodethiobiotin and enzyme to probe the ligand environment of the [2Fe–2S]+ cluster in this reaction intermediate. Hyperfine sublevel correlation spectroscopy (HYSCORE) spectra exhibit strong cross-peaks demonstrating strong isotropic coupling of the nuclear spin with the paramagnetic center. The hyperfine coupling constants are consistent with a structural model for the reaction intermediate in which 9-mercaptodethiobiotin is covalently coordinated to the remnant [2Fe–2S]+ cluster.