Biotin synthase catalyzes the oxidative addition of a sulfur atom to dethiobiotin (DTB) to generate the biotin thiophane ring. This reaction is initiated by the reductive cleavage of the sulfonium center of S-adenosyl-L-methionine (AdoMet), generating methionine and a transient 5'-deoxyadenosyl radical that functions as an oxidant by abstracting hydrogen atoms from DTB. Biotin synthase contains a highly conserved sequence motif, YNHNLD, in which Asn153 and Asp155 form hydrogen bonds with the ribose hydroxyl groups of AdoMet. In the present work, we constructed four individual site-directed mutations to change each of these two residues in order to probe their role in the active site. We used molecular weight filtration assays to show that for most of the mutant enzymes binding of the substrates was only slightly affected. In vitro assays demonstrate that several of the mutant enzymes were able to reductively cleave AdoMet, but none were able to produce a significant amount of biotin. Several of the mutants, especially Asn153Ser, were able to produce high levels of the stable intermediate 9-mercaptodethiobiotin. Some of the mutants, such as Asp155Asn and Asn153Ala, produced instead an alternate product tentatively identified by mass spectrometry as 5'-mercapto-5'-deoxyadenosine, generated by direct attack of the 5'-deoxyadenosyl radical on the [4Fe-4S](2+) cluster. Collectively, these results suggest that the protein residues that form hydrogen bonds to AdoMet and DTB are important for retaining intermediates during the catalytic cycle and for targeting the reactivity of the 5'-deoxyadenosyl radical.