LAUSR

A quick freeze shows an enzyme's secrets Organic radicals are chemically useful in enzymatic reactions but are often hard to observe, owing to their short lifetimes. Dong et al. used rapid freeze-quench methods to trap two intermediates formed by a noncanonical radical S-adenosylmethionine (SAM) enzyme: a fragmented SAM molecule bound to the iron-sulfur cluster through an iron-carbon bond and a product-like radical. The structure of the SAM-bound enzyme reveals a noncolinear arrangement of carbon, sulfur, and iron atoms. The arrangement of bonds suggests that the organometallic intermediate may be created through a two-electron nucleophilic mechanism. A subsequent radical intermediate is formed on the protein substrate and resolves by oxidation to form the amino acid product diphthamide. Science, this issue p. 1247 An unusual radical enzyme forms an iron-carbon bond as the first step in the modification of a protein side chain. Diphthamide biosynthesis involves a carbon-carbon bond-forming reaction catalyzed by a radical S-adenosylmethionine (SAM) enzyme that cleaves a carbon-sulfur (C–S) bond in SAM to generate a 3-amino-3-carboxypropyl (ACP) radical. Using rapid freezing, we have captured an organometallic intermediate with an iron-carbon (Fe–C) bond between ACP and the enzyme’s [4Fe-4S] cluster. In the presence of the substrate protein, elongation factor 2, this intermediate converts to an organic radical, formed by addition of the ACP radical to a histidine side chain. Crystal structures of archaeal diphthamide biosynthetic radical SAM enzymes reveal that the carbon of the SAM C–S bond being cleaved is positioned near the unique cluster Fe, able to react with the cluster. Our results explain how selective C–S bond cleavage is achieved in this radical SAM enzyme.