An Anchoring Role for FeS Clusters:  Chelation of the Amino Acid Moiety of S-Adenosylmethionine to the Unique Iron Site of the 4Fe−4S Cluster of Pyruvate Formate-Lyase Activating Enzyme

Pyruvate formate-lyase activating enzyme (PFL-AE) generates the catalytically essential glycyl radical on pyruvate formate-lyase via the interaction of the catalytically active 4Fe-4S+ cluster with S-adenosylmethionine (AdoMet). Like other members of the Fe-S/AdoMet family of enzymes, PFL-AE is thought to function via generation of an AdoMet-derived 5'-deoxyadenosyl radical intermediate; however, the mechanistic steps by which this radical is generated remain to be elucidated. While all of the members of the Fe-S/AdoMet family of enzymes appear to have a unique iron site in the 4Fe-4S cluster, based on the presence of a conserved three-cysteine cluster binding motif, the role of this unique site has been elusive. Here we utilize 35-GHz pulsed electron nuclear double resonance (ENDOR) studies of the 4Fe-4S+ cluster of PFL-AE in complex with isotopically labeled AdoMet (denoted 1+/AdoMet) to show that the unique iron serves to anchor the AdoMet for catalysis. AdoMet labeled with 17O at the carboxylate shows a coupling of A = 12.2 MHz, consistent with direct coordination of the carboxylate to the unique iron of the cluster. This is supported by 13C-ENDOR with the carboxylato carbon labeled with 13C, which shows a hyperfine coupling of 0.71 MHz. AdoMet enriched with 15N at the amino position gives rise to a spectrum with A(15N) = 5.8 MHz, consistent with direct coordination of the amino group to a unique iron of the cluster. Together, the results demonstrate that the unique iron of the 4Fe-4S cluster anchors AdoMet by forming a classical N/O chelate with the amino and carboxylato groups of the methionine fragment.