ENDOR Spectroscopy Reveals the "Free"5-Deoxyadenosyl Radicalin a Radical SAM Enzyme Active Site Actually is Chaperoned by Close Interaction with the Methionine-Bound [4Fe-4S]²⁺ Cluste

H-1/2 and C-13 hyperfine coupling constants to 5 ' -deoxyadenosyl (5 '-dAdo center dot) 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 2p pi orbital of the sp(2) C5 '-carbon (J. Am. Chem. Soc. 2019, 141, 12139-12146). However, prior electron-nuclear double resonance (ENDOR) measurements had indicated that this 5 '-dAdo center dot 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 center dot is extended through the finding that this apparently unexceptional organic free radical has an anomalous g-tensor and exhibits significant Fe-57, C-13, N-15, and H-2 hyperfine couplings to the adjacent, isotopically labeled, methionine-bound [4Fe-4S](2+ )cluster cogenerated with 5 '-dAdo center dot during homolytic cleavage of cluster-bound SAM. The origin of the Fe-57 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 center dot) to labeled-methionine as structural constraints yields a model for active-site positioning of 5 '-dAdo center dot with a short, nonbonded C5 '-Fe distance (similar to 3 angstrom). This distance involves substantial motion of 5 '-dAdo center dot 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, Omega, the central intermediate in catalysis by radical-SAM enzymes.