Benzylsuccinate synthase is a member of the glycyl radical family of enzymes. It catalyzes the addition of toluene to fumarate to form benzylsuccinate as the first step in the anaerobic pathway of toluene fermentation. The enzyme comprises three subunits, alpha, beta, and gamma, that in Thauera aromatica strain T1 are encoded by the tutD, tutG, and tutF genes, respectively. The large alpha-subunit contains the essential glycine and cysteine residues that are conserved in all glycyl radical enzymes. However, the function of the small beta- and gamma-subunits has remained unclear. We have overexpressed all three subunits of benzylsuccinate synthase in Escherichia coli, both individually and in combination. Coexpression of the gamma-subunit (but not the beta-subunit) is essential for efficient expression of the alpha-subunit. The benzylsuccinate synthase complex lacking the glycyl radical could be purified as an alpha(2)beta(2)gamma(2) hexamer by nickel affinity chromatography through a "His(6)" affinity tag engineered onto the C-terminus of the alpha-subunit. Unexpectedly, BSS was found to contain two iron-sulfur clusters, one associated with the beta-subunit and the other with the gamma-subunit that appear to be necessary for the structural integrity of the complex. The spectroscopic properties of these clusters suggest that they are most likely [4Fe-4S] clusters. Removal of iron with chelating agents results in dissociation of the complex; similarly, a mutant gamma-subunit lacking the [4Fe-4S] cluster is unable to stabilize the alpha-subunit when the proteins are coexpressed.