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

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Abstract 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.

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