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

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