ENDOR Spectroscopy Reveals the “Free” 5′-Deoxyadenosyl Radical in a Radical SAM Enzyme Active Site Actually is Chaperoned by Close Interaction with the Methionine-Bound [4Fe–4S]2+ Cluster

• DOI: 10.1021/jacs.3c09428
• Publication Date: 2024-02-03T16:05:17Z
• Authors: Hao Yang; Madeline B. Ho; Maike N. Lundahl; Martín A. Mosquera; William E. Broderick; Joan B. Broderick; Brian M. Hoffman
• Keywords: Unpaired electron

1/2H and 13C hyperfine coupling constants to 5′-deoxyadenosyl (5′-dAdo•) radical trapped within the active site of S-adenosyl-l-methionine (SAM) enzyme, pyruvate formate lyase-activating enzyme (PFL-AE), both in absence substrate presence a reactive peptide-model PFL substrate, are completely characteristic classical organic free whose unpaired electron is localized 2pπ orbital sp2 C5′-carbon (J. Am. Chem. Soc. 2019, 141, 12139–12146). However, prior electron-nuclear double resonance (ENDOR) measurements had indicated that this 5′-dAdo• never truly "free": tight van der Waals contact with its target partners active-site residues guide it carrying out exquisitely precise, regioselective reactions hallmarks RS enzymes. Here, our understanding how chaperones extended through finding apparently unexceptional has an anomalous g-tensor exhibits significant 57Fe, 13C, 15N, 2H couplings adjacent, isotopically labeled, methionine-bound [4Fe–4S]2+ cluster cogenerated during homolytic cleavage cluster-bound SAM. The origin 57Fe nonbonded radical-cluster illuminated by formal exchange-coupling model broken symmetry–density functional theory computations. Incorporation ENDOR-derived distances from C5′(dAdo•) labeled-methionine as structural constraints yields for positioning short, C5′-Fe distance (∼3 Å). This involves substantial motion toward unique Fe upon S–C(5′) bond-cleavage, plausibly initial step formation Fe–C5′ bond organometallic complex, Ω, central intermediate catalysis radical-SAM