Biological radical sulfur insertion reactions.

ion at C-6 results in an allylic radical in which spin delocalization of the radical center makes it reactive with respect to the protein itself and thus results in covalent binding. b. Biotin Synthase is a Member of the RadicalSAM Family. Biotin synthase has been proposed to belong to the radical-SAM enzyme family for the following reasons.75 First it contains a (4Fe-4S)2+/1+ cluster required for catalysis3,73,75 and is coordinated by the three cysteines of the CXXXCXXC motif, which is a unique sequence common to members of that enzyme family. In addition, S-adenosylmethionine (AdoMet or SAM) and an electron-donating system consisting of NADPH, flavodoxin reductase, and flavodoxin were shown to be required for biotin synthase activity in vitro.84-87 Photoreduced deazaflavin could serve as the reducing agent as well.88,89 The requirement for these components is a signature for enzymes of the radical-SAM superfamily.3,73,75 The AdoMet/FeS/ecombination has been proposed, in the case of prototypic enzymes such as the anaerobic ribonucleotide reductase, pyruvate formate lyase, and lysine 2,3-aminomutase, to function as a source of methionine and a 5′-deoxyadenosyl radical, noted Ado° in Figure 15, resulting from reductive cleavage of AdoMet.3,73-75 The dissociation energy of the S-C(5′-deoxyadenosyl) bond of AdoMet is about 60 kcal/mol, and homolytic cleavage of that bond is possible only after a one-electron reduction. It has been demonstrated that the electron is provided by the reduced (4Fe-4S)+ and that the flavodoxin reductase/flavodoxin system serves to convert the cluster into this reduced active state (Figure 15). The highly energetic 5′-deoxyadenosyl radical, which has never been directly observed, is proposed to initiate the reaction by selectively abstracting a H atom from the substrate RH (Figure 15). With the first two enzymes mentioned above, the substrate is a glycine residue of the polypeptide chain and H atom abstraction leads to an oxygen-sensitive glycyl radical that is essential for enzyme activity.90,91 Experiments with proteins containing deuterated glycines have shown in both cases that the label is transferred to the 5′deoxyadenosyl group, strongly supporting the hypothesis of an intermediate 5′-deoxyadenosyl radical.90,92 Furthermore, a 5′-deoxyadenosyl group was trapped by a peptide analogue of the pyruvate formate lyase radical site.93 In the case of lysine 2,3aminomutase, the substrate is lysine, and the reaction involves the intermediate sequencial formation of substrateand product-derived radicals.74 Using a variety of substrate analogues, P. Frey et al. could observe several of these species by EPR spectroscopy, thus providing a strong support to the hypothesis of a radical mechanism in this radical-SAM enzyme family.74 Finally, using 3′,4′-anhydroadenosylmethionine as well as 2Hand 13C-labeled versions of that compound as an analogue of AdoMet, they could observe the stabilized allylic 5′-deoxy-3′,4′-anhydroadenosin-5′-yl radical, further supporting the mechanism proposed in Figure 15.94,95 The ability of reduced clusters to reduce sulfonium ions and cleave them in a reaction affording the most stable radical has been demonstrated with Fe4S4(SEt)4 and Fe4S4(SPh)4 model complexes and AdoMet analogues.96 In these experiments the selection of sulfonium salts was made on the basis of their irreversible reduction potentials so that the clusters were thermodynamically competent for reduction. However, with these model systems, several other processes occurred which have not been observed Biological Radical Sulfur Insertion Reactions Chemical Reviews, 2003, Vol. 103, No. 6 2159