Characterization of auxiliary iron–sulfur clusters in a radical S ‐adenosylmethionine enzyme PqqE from Methylobacterium extorquens AM 1

PqqE is a radical S ‐adenosyl‐ l ‐methionine ( SAM ) enzyme that catalyzes the initial reaction of pyrroloquinoline quinone ( PQQ ) biosynthesis. PqqE belongs to the SPASM (subtilosin/ PQQ /anaerobic sulfatase/mycofactocin maturating enzymes) subfamily of the radical SAM superfamily and contains multiple Fe – S clusters. To characterize the Fe – S clusters in PqqE from Methylobacterium extorquens AM 1, Cys residues conserved in the N‐terminal signature motif ( CX 3 CX 2 C) and the C‐terminal seven‐cysteine motif ( CX 9–15 GX 4 CX n CX 2 CX 5 CX 3 CX n C; n  = an unspecified number) were individually or simultaneously mutated into Ser. Biochemical and Mössbauer spectral analyses of as‐purified and reconstituted mutant enzymes confirmed the presence of three Fe – S clusters in PqqE: one [4Fe – 4S] 2+ cluster at the N‐terminal region that is essential for the reductive homolytic cleavage of SAM into methionine and 5′‐deoxyadenosyl radical, and one each [4Fe – 4S] 2+ and [2Fe – 2S] 2+ auxiliary clusters in the C‐terminal SPASM domain, which are assumed to serve for electron transfer between the buried active site and the protein surface. The presence of [2Fe – 2S] 2+ cluster is a novel finding for radical SAM enzyme belonging to the SPASM subfamily. Moreover, we found uncommon ligation of the auxiliary [4Fe – 4S] 2+ cluster with sulfur atoms of three Cys residues and a carboxyl oxygen atom of a conserved Asp residue.