Extended data Figure 6 |. In vitro synthesis of 6-thiooctanoyl intermediate and lipoyl product by human lipoyl synthase LIAS requires ferredoxin FDX1 as an electron donor.

a Model of the multi-step reaction mechanism of lipoyl formation by human lipoyl synthase (LIAS) based on bacterial LipA¹. The LIAS enzyme contains two [4Fe-4S] clusters, the catalytic and auxiliary cluster, needed for reductive cleavage of S-adenosylmethionine (SAM) and as a source for sulfur insertion into the octanoyl precursor, respectively. (1) The reaction starts with the binding of SAM and the octanoyl substrate which is covalently attached to a lysinyl residue of a lipoyl carrier domain (LCD) of the H protein of the glycine cleavage system (GCS)^1,2. An electron donor (orange) transfers a single electron to the catalytic cluster which mediates reductive cleavage of SAM to methionine and a 5’-deoxyadenosyl radical (5’-dA·). This work identified human FDX1 as the physiological electron donor, efficiently replacing dithionite that typically is used as an artificial electron donor¹. (2) The radical abstracts a hydrogen atom from the octanoyl C6 carbon, forming 5’-deoxyadenosine (5’-dAH). (3) The octanoyl C6 carbon in turn forms a covalent bond with a sulfur atom of the auxiliary cluster, concomitant with partial degradation of the cluster. (4) A second SAM molecule binds to LIAS, and upon electron supply from FDX1 (or DT) again leads to 5’-dA· radical formation by the catalytic cluster and abstraction of a proton from the terminal C8 carbon of the octanoyl moiety. (5) A second sulfur atom is covalently attached to the thiooctanoyl molecule, (6) leading to formation of the mature lipoyl cofactor and further degradation of the Fe/S cluster. To enable multiple reaction cycles, the auxiliary cluster must be regenerated by a still unclear mechanism. In humans, the mature lipoyl cofactor is finally transferred to the target proteins, e.g., the E2 subunits of pyruvate (PDH) and α-ketoglutarate (KGDH) dehydrogenase complexes. BCKDH, branched-chain ketoacid dehydrogenase; OADH, 2-oxoadipate dehydrogenase³. b Time courses of 6-thiooctanoyl intermediate and lipoyl product formation in FDX1-catalyzed reactions (see Fig. 3b,c). Samples included 0.5 mM peptide substrate, 35 μM LIAS, 2 mM NADPH, 20 μM FDXR, FDX1 as indicated and 1 mM SAM. Formation of the 6-thiooctanoyl intermediate proceeded significantly faster than lipoyl formation, indicating the second sulfur insertion step to be rate-limiting under the experimental conditions. The result suggested to record the data of other experiments after 150 min incubation.