title: Identification of FeS clusters in the glycyl-radical enzyme benzylsuccinate synthase via EPR and Mössbauer spectroscopy.
abstract: The anaerobic degradation pathway of toluene is initiated by the addition of the methyl group of toluene to the double bond of fumarate. This reaction is catalyzed by a novel glycyl-radical enzyme, (R)-benzylsuccinate synthase (BSS). The enzyme consists of three subunits, α, β, and γ, and differs from most other glycyl-radical enzymes in having additional cofactors. We have purified a Strep-tagged nonactivated BSS from recombinant Escherichia coli and identified the additional cofactors as FeS clusters by UV/vis, EPR, and Mössbauer spectroscopy. Analysis of the metal content as well as the EPR and Mössbauer spectra indicated that BSS contains magnetically coupled low-potential [4Fe-4S] clusters. Several enzyme preparations showed differing amounts of [3Fe-4S] clusters that could be reconstituted to [4Fe-4S] clusters, indicating that they arise from partial decay of the initial [4Fe-4S] clusters. The most likely location of these FeS clusters in the enzyme are the small β and γ subunits, which are unique for the BSS subfamily of glycyl-radical enzymes and contain conserved cysteines as potential ligands.
authors.affiliation: Laboratorium für Mikrobielle Biochemie, Philipps-Universität Marburg, Karl-von-Frisch-Strasse 8, 35043 Marburg, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Hilberg
authors.forename: Antonio J
authors.initials: AJ
authors.lastname: Pierik
authors.forename: Eckhard
authors.initials: E
authors.lastname: Bill
authors.forename: Thorsten
authors.initials: T
authors.lastname: Friedrich
authors.forename: Marie-Luise
authors.initials: ML
authors.lastname: Lippert
authors.forename: Johann
authors.initials: J
authors.lastname: Heider
mesh_terms: D055162:Biocatalysis; D019755:Carbon-Carbon Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000166:cytology / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000032:analysis* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015204:Spectroscopy, Mossbauer
chemical_list: D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; D019755:Carbon-Carbon Lyases; C114354:benzylsuccinate synthase