Radical S-adenosyl-L-methionine (SAM) enzymes initiate a variety of diverse radical reactions through liberation of the highly reactive 5’d-Ado● radical. The unique iron of a [4Fe-4S] cluster is first coordinated to cofactor/cosubstrate SAM by its amino acid moiety, and the 5’-dAdo● radical was thought to be liberated directly following electron transfer from the [4Fe-4S]1+ cluster to reductively cleave the S-C5’ bond of SAM. However, it has recently been determined that this mechanism actually proceeds through an organometallic [4Fe-4S]3+-5’d-Ado intermediate, Ω, that contains a direct bond between the 5’C of 5’d-Ado● and the unique iron of the cluster, with an analogous [4Fe-4S]3+-CH3 organometallic intermediate, ΩM, also having been observed. To better understand the electronic structure of this intermediate, a synthetic alkyl cluster, and isotopologues, have been prepared. The alkylated cluster has g-values reflective of a [Fe4S4]3+ cluster (giso&gt;2). The Fe-CH3 bond has been characterized by 13C/1/2H ENDOR. The organometallic bond can be described as a methyl group bound to a ‘down’-spin valence-localized iron, and yields hyperfine couplings that are comparable in magnitude with enzymatic intermediates. Additionally, we find that in one conformer poor solvation of the methyl group results in hindered rotation about the Fe-C bond, while in a second conformer the methyl group is freely rotating about the Fe-C bond at 2K. In the rotating conformer, the three distinct anisotropic 1H hyperfine coupling tensors of the -C(1H)3 are averaged by rotation to a single, joint axial tensor. These studies on a crystallographically characterized analogue further validate EPR and ENDOR characterization of the enzymatic intermediate.

EPR and ENDOR characterization of the Fe-CH3 bond in a model complex for radical SAM organometallic intermediates Ω and ΩM

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