The enzyme MiaB catalyzes the attachment of a methylthio (-SCH3 ) group at the C2 position of N-6- (isopentenyl)adenosine (i(6)A) in the final step of the biosynthesis of the hypermodified tRNA nucleotide 2-methythio-N-6 -(isopentenyl)adenosine (ms(2)i(6) A). MiaB belongs to the expanding subgroup of enzymes of the radical S-adenosylmethionine (SAM) superfamily that harbor one or more auxiliary [4Fe-4S] clusters in addition to the [4Fe-4S] cluster that all family members require for the reductive cleavage of SAM to afford the common 5'-deoxyadenosyl S'-radical (5'-dA(center dot)) intermediate. While the role of the radical SAM cluster in generating the 5'-dA(center dot) is well understood, the detailed role of the auxiliary cluster, which is essential for MiaB catalysis, remains unclear. It has been proposed that the auxiliary cluster may serve as a coordination site for exogenously derived sulfur destined for attachment to the substrate or that the cluster itself provides the sulfur atom and is sacrificed during turnover. In this work, we report spectroscopic and biochemical evidence that the auxiliary [4Fe-4S](2+) cluster in Bacteroides thetaiotaomicron (Bt) MiaB is converted to a [3Fe-4S](0) -like cluster during the methylation step of catalysis. Mossbauer characterization of the MiaB [3Fe-4S](0) -like cluster revealed unusual spectroscopic properties compared to those of other well-characterized cuboidal [3Fe-4S](0 )clusters. Specifically, the Fe sites of the mixed-valent moiety do not have identical Mossbauer parameters. Our results support a mechanism where the auxiliary [4Fe-4S] cluster is the direct sulfur source during catalysis.