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J Am Chem Soc. 2002 Sep 25;124(38):11270-1.

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.

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Department of Chemistry, Northwestern University, Evanston, Illinois 60208-3113, USA.


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.

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