Amino Acids in the RSSY Motif of Lipoyl Synthase Control Substrate Binding and Reactivity^†

The last step in the biosynthesis of the lipoyl cofactor (LipCo) is the addition of two sulfur atoms at C6 and C8 of an n-octanoyl chain attached in an amide linkage to a target lysyl residue of a lipoyl carrier protein. This reaction is catalyzed by lipoyl synthase, a member of the radical S-adenosylmethionine (SAM) superfamily. Lipoyl synthase requires two [4Fe-4S] clusters. One cluster is used to cleave SAM reductively to generate two 5'-deoxyadenosyl 5'-radicals (5'-dA•), which abstract the C6 and C8 hydrogen atoms (H•) of the substrate in two sequential steps. The second cluster, termed the auxiliary cluster, is consumed during turnover to provide the attached sulfur atoms. The auxiliary cluster is ligated by three cysteines in a CX₄CX₅C motif and one serine residue (Ser308 in Escherichia coli) in a highly conserved R³⁰⁶SS³⁰⁸Y motif in the C-terminal region of the protein. Here, we show that Arg306 and Ser308 are absolutely required for LipCo formation. Substitution of Arg306 with Lys results in an essentially inactive protein due to poor substrate binding and positioning in the active site. Multiple substitutions of Ser308 were engineered. Most notable were the S308C and S308A variants, which greatly diminished LipCo formation. However, the S308C variant resulted in greater production of the 6-mercaptooctanoyl intermediate and the formation of a desaturated product, identified as a 6-octenoyl group. Furthermore, the 3Fe cluster formed during degradation of the auxiliary cluster during C6 sulfur substitution in the wild-type reaction is not observed with the S308C variant. Instead, the auxiliary cluster remains tetranuclear and forms a monothiolated cross-linked species with a high-spin, S = 7/2, configuration that decays to the 6-octenoyl-containing product. Other amino acids in the RSSY motif were not essential for catalysis.