Iron‐Sulfur‐Mediated C‐S Bond Formation: Mechanistic Insights from the state‐crossing tRNA Methylthiolation by the Radical SAM Enzyme MiaB

The radical S-adenosylmethionine (SAM) enzyme MiaB is a bifunctional catalyst that mediates the posttranscriptional methylthiolation of N⁶-isopentenyladenosine (i⁶A37) at position 37 in tRNA. Herein, density functional calculations were employed to elucidate the two stages of MiaB-catalyzed modification: methylation and sulfur insertion at the C² position of i⁶A37. MiaB contains two iron-sulfur clusters: a radical SAM cluster ([4Fe-4S]_RS) and an auxiliary cluster ([3Fe-4S]_Aux). Our calculations demonstrate that the [4Fe-4S]_RS cluster is essential for generating the potent oxidant 5'-deoxyadenosyl radical (5'-dAdo^•) via the reductive cleavage of SAM and that the [3Fe-4S]_Aux cluster serves both as the carrier of methyl and as a direct sulfur donor during catalysis. Furthermore, it is revealed that a state crossing occurs during the methylthio installation at the substrate, which follows C²-H abstraction by 5'-dAdo^•. Additionally, substituting Arg66 with glutamine highlights its critical role in stabilizing the substrate radical and modulating MiaB activity. Overall, our work advances the understanding of iron-sulfur cluster chemistry and inert C-H bond activation within the growing superfamily of radical SAM enzymes, offering insights for translating the catalytic advantages of natural enzymes into synthetic transition-metal complexes and functional materials.