Isolation and characterization of an intermediate in lipoyl cofactor biosynthesis

Lipoyl synthase (LS) is a member of the radical SAM superfamily, which use an iron–sulfur cluster to transfer an electron to S‐adenosyl‐L‐methionine (SAM), resulting in the cleavage of SAM into methionine and a very powerful 5′‐deoxyadenosyl 5′‐radical (5′‐dA•). LS uses the 5′‐dA• to abstract hydrogen atoms from C6 and C8 of protein‐bound octanoic acid, activating the cofactor for sulfur insertion. The second iron‐sulfur cluster of LS is proposed to be the sulfur donor. We show in this study that the second [4Fe‐4S] cluster is directly modified during the catalytic cycle and that the cluster is destroyed during catalysis. Recently, a monothiolated intermediate has been detected by LC‐MS. This intermediate can be trapped by adding only one equivalent of SAM to the reaction and isolated by gel filtration since it is covalently linked to the enzyme. We have characterized the trapped intermediate using EPR and Mössbauer spectroscopies and have shown that the intermediate is competent to form the lipoyl product. This work is supported by National Institutes of Health Grant GM‐63847.