Ribosome methylation is important for life processes and is mainly catalyzed by radical S-Adenosylmethionine (SAM) enzymes. Two SAM molecules serve as the cofactor by providing the 5 ′-deoxyadenosyl radical for substrate activation and the methyl. Recently, Booker and coworkers (Science 2011, 332, 604) proposed an alternative mechanism for a pair of radical SAM enzymes, RlmN and Cfr, which respectively methylate the C2 and C8 of adenosine 2503. Their deuterium labeling experiments reveal that methyl group does not transfer directly from SAM to adenosine, instead it passes to Cys355 first, then onto adenosine. In this article, this new reaction mechanism is studied using density functional theory with B3LYP hybrid functional. The reaction system is simulated using small model compounds in the gas phase, and the protein environment is approximated using polarizable continuum model. The structures of the transition states and the intermediates are identified, and their free energies are calculated. The activation barriers indicate that the proposed reaction mechanism is plausible. The formation of a disulfide bond is found to be the rate-limiting step. © 2012 Wiley Periodicals, Inc.