title: 9-Mercaptodethiobiotin is generated as a ligand to the [2Fe-2S]+ cluster during the reaction catalyzed by biotin synthase from Escherichia coli.
abstract: 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.
authors.affiliation: Department of Chemistry, University of Hawai'i at Manoa, Honolulu, 96822, United States.
authors.forename: Corey J
authors.initials: CJ
authors.lastname: Fugate
authors.forename: Troy A
authors.initials: TA
authors.lastname: Stich
authors.forename: Esther G
authors.initials: EG
authors.lastname: Kim
authors.forename: William K
authors.initials: WK
authors.lastname: Myers
authors.forename: R David
authors.initials: RD
authors.lastname: Britt
authors.forename: Joseph T
authors.initials: JT
authors.lastname: Jarrett
mesh_terms: D055162:Biocatalysis; D001710:Biotin / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D015394:Molecular Structure; D013466:Sulfurtransferases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; D008024:Ligands; C083940:9-mercaptodethiobiotin; D001710:Biotin; D013466:Sulfurtransferases; C087072:biotin synthase, E coli