Identification of FeS clusters in the glycyl-radical enzyme benzylsuccinate synthase via EPR and Mössbauer spectroscopy.

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.