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-CH3 bond, ΩM. 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+-CH3 cluster, M-CH3, is a crystallographically characterized analogue to ΩM that exhibits the same [Fe4S4]3+ cluster state as Ω and ΩM, and thus an analysis of its spectroscopic properties─and comparison with those of Ω and ΩM─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-CH3 bond. This generates an averaged hyperfine coupling tensor whose analysis requires an extended treatment of rotational averaging. The methyl group 13C/1/2H hyperfine couplings are compared with the corresponding values for Ω and ΩM.