Pyruvate formate-lyase activating enzyme (PFL-AE) is a representative member of an emerging family of enzymes that utilize iron-sulfur clusters and S-adenosylmethionine (AdoMet) to initiate radical catalysis. Although these enzymes have diverse functions, evidence is emerging that they operate by a common mechanism in which a [4Fe-4S](+) interacts with AdoMet to generate a 5'-deoxyadenosyl radical intermediate. To date, however, it has been unclear whether the iron-sulfur cluster is a simple electron-transfer center or whether it participates directly in the radical generation chemistry. Here we utilize electron paramagnetic resonance (EPR) and pulsed 35 GHz electron-nuclear double resonance (ENDOR) spectroscopy to address this question. EPR spectroscopy reveals a dramatic effect of AdoMet on the EPR spectrum of the [4Fe-4S]+ of PFL-AE, changing it from rhombic (g = 2.02, 1.94, 1.88) to nearly axial (g = 2.01, 1,88, 1.87). H-2 and C-13 ENDOR spectroscopy was performed on [4Fe-4S]+-PFL-AE (S = 1/2) in the presence of AdoMet labeled at the methyl position with either 2H or 13C (denoted [1+/AdoMet]). The observation of a substantial 2H coupling of similar to1 MHz (similar to6-7 MHz for H-1), as well as hyperfine-split signals from the 13C, manifestly require that AdoMet lie close to the cluster. 2H and 13C ENDOR data were also obtained for the interaction of AdoMet with the diamagnetic [4Fe-4S](2+) state of PFL-AE, which is visualized through cryoreduction of the frozen [4Fe-4S](2+)/AdoMet complex to form the reduced state (denoted [2+/AdoMet](red)) trapped in the structure of the oxidized state. H-2 and C-13 ENDOR spectra for [2+/AdoMet](red) are essentially identical to those obtained for the [1+/AdoMet] samples, showing that the cofactor binds in the same geometry to both the 1+ and 2+ states of PFL-AE. Analysis of 2D field-frequency 13C ENDOR data reveals an isotropic hyperfine contribution, which requires that AdoMet lie in contact with the cluster, weakly interacting with it through an incipient bond/antibond. From the anisotropic hyperfine contributions for the 2H and 13C ENDOR, we have estimated the distance from the closest methyl proton of AdoMet to the closest iron of the cluster to be similar to3.0-3.8 Angstrom, while the distance from the methyl carbon to the nearest iron is similar to4-5 Angstrom. We have used this information to construct a model for the interaction of AdoMet with the [4Fe-4S](2+/+) cluster of PFL-AE and have proposed a mechanism for radical generation that is consistent with these results.