Bradley J. Landgraf † and Squire J. Booker * † ‡ § † Department of Chemistry, ‡ Department of Biochemistry and Molecular Biology, and § The Howard Hughes Medical Institute, The Pennsylvania State University , University Park, Pennsylvania 16802, United States J. Am. Chem. Soc. , Article ASAP DOI: 10.1021/jacs.5b11035 http://pubs.acs.org/doi/abs/10.1021/jacs.5b11035 http://pubs.acs.org/doi/pdf/10.1021/jacs.5b11035 Abstract RimO is a member of the growing radical S-adenosylmethionine (SAM) superfamily of enzymes, which use a reduced [4Fe–4S] cluster to effect reductive cleavage of the 5′ C–S bond of SAM to form a 5′-deoxyadenosyl 5′-radical (5′-dA • ) intermediate. RimO uses this potent oxidant to catalyze the attachment of a methylthio group (−SCH 3 ) to C3 of aspartate 89 of protein S12, one of 21 proteins that compose the 30S subunit of the bacterial ribosome. However, the exact mechanism by which this transformation takes place has remained elusive. H...