title: Coordination of adenosylmethionine to a unique iron site of the [4Fe-4S] of pyruvate formate-lyase activating enzyme: a Mössbauer spectroscopic study.
abstract: Pyruvate formate-lyase activating enzyme (PFL-AE) generates the catalytically essential glycyl radical of PFL. It is a member of the so-called "radical-SAM superfamily" of enzymes that use a [4Fe-4S] cluster and S-adenosylmethionine (AdoMet or SAM) to catalyze diverse radical-mediated reactions. Evidence suggests that this class of enzymes operate by common initial steps involving the generation of an AdoMet-derived adenosyl radical intermediate, of which the mechanism remains unresolved. The three-cysteine CX3CX2C cluster-binding motif common to all members of this superfamily suggests a unique Fe site in the [4Fe-4S] cluster, which presumably interacts with AdoMet to effect the reductive cleavage and radical generation. Here we employ a dual-iron-isotope (56Fe/57Fe) approach to demonstrate the existence of a unique Fe site in the [4Fe-4S] cluster of PFL-AE by Mössbauer spectroscopy. Coordination of AdoMet to this unique Fe site was made evident by the observation of a substantial increase in the isomer shift (delta) of the Mössbauer spectrum associated with the unique Fe site: delta = 0.42 mm/s in the absence of AdoMet increases to delta = 0.72 mm/s in the presence of AdoMet. Further, the Mössbauer data show that the binding of AdoMet to the unique Fe site occurs in the [4Fe-4S]2+ state, prior to the injection of the reducing equivalent required for catalysis. This observation indicates that AdoMet coordination is a necessary prerequisite to adenosyl radical generation.
authors.affiliation: Department of Physics, Emory University, Atlanta, Georgia 30322, USA.
authors.forename: Carsten
authors.initials: C
authors.lastname: Krebs
authors.forename: William E
authors.initials: WE
authors.lastname: Broderick
authors.forename: Timothy F
authors.initials: TF
authors.lastname: Henshaw
authors.forename: Joan B
authors.initials: JB
authors.lastname: Broderick
authors.forename: Boi Hanh
authors.initials: BH
authors.lastname: Huynh
mesh_terms: D000123:Acetyltransferases; D001665:Binding Sites; D002384:Catalysis; D004798:Enzymes / Q000737:chemistry* / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000737:chemistry* / Q000378:metabolism*; D015204:Spectroscopy, Mossbauer
chemical_list: D004798:Enzymes; D007506:Iron-Sulfur Proteins; D012436:S-Adenosylmethionine; D000123:Acetyltransferases; C040285:pyruvate formate-lyase activating enzyme