Characterization by ENDOR Spectroscopy of the Iron-Alkyl Bond in a Synthetic Counterpart of Organometallic Intermediates in Radical SAM Enzymes

Members of the radical S-adenosyl-l-methionine (SAM) enzyme superfamily initiate a broad spectrum of radical transformations through reductive cleavage of SAM by a [4Fe-4S]¹⁺cluster it coordinates to generate the reactive 5′-deoxyadenosyl radical (5′-dAdo^•). However, 5′-dAdo^•is not directly liberated for reaction and instead binds to the unique Fe of the cluster to create the catalytically competent S = 1/2 organometallic intermediate ω. An alternative mode of reductive SAM cleavage, especially seen photochemically, instead liberates CH₃^•, which forms the analogous S = 1/2 organometallic intermediate with an Fe-CH₃bond, ω_M. The presence of a covalent Fe-C bond in both structures was established by the ENDOR observation of ¹³C and ¹H hyperfine couplings to the alkyl groups that show isotropic components indicative of Fe-C bond covalency. The synthetic [Fe₄S₄]³⁺-CH₃cluster, M-CH₃, is a crystallographically characterized analogue to ω_Mthat exhibits the same [Fe₄S₄]³⁺cluster state as ω and ω_M, and thus an analysis of its spectroscopic properties-and comparison with those of ω and ω_M-can be grounded in its crystal structure. We report cryogenic (2 K) EPR and ¹³C/^1/2H ENDOR measurements on isotopically labeled M-CH₃. At low temperatures, the complex exhibits EPR spectra from two distinct conformers/subpopulations. ENDOR shows that at 2 K, one contains a static methyl, but in the other, the methyl undergoes rapid tunneling/hopping rotation about the Fe-CH₃bond. This generates an averaged hyperfine coupling tensor whose analysis requires an extended treatment of rotational averaging.