Electron-nuclear double resonance spectroscopic evidence that S-adenosylmethionine binds in contact with the catalytically active [4Fe-4S]⁺ cluster of pyruvate formate-lyase activating enzyme

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). ²H and ¹³C 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 ∼1 MHz (∼6-7 MHz for ¹H), 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. 2H and ¹³C 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 ∼3.0-3.8 Å, while the distance from the methyl carbon to the nearest iron is ∼4-5 Å. 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.