Reductive cleavage of S-adenosylmethionine by biotin synthase from Escherichia coli.

Biotin synthase (BioB) catalyzes the insertion of a sulfur atom between the C6 and C9 carbons of dethiobiotin. Reconstituted BioB from Escherichia coli contains a 4Fe-4S cluster thought to be involved in the reduction and cleavage of S-adenosylmethionine (AdoMet), generating methionine and the reactive 5'-deoxyadenosyl radical responsible for dethiobiotin H-abstraction. Using EPR and Mössbauer spectroscopy as well as methionine quantitation we demonstrate that the reduced S = 1/2 4Fe-4S cluster is indeed capable of injecting one electron into AdoMet, generating one equivalent of both methionine and S = 0 4Fe-4S cluster. Dethiobiotin is not required for the reaction. Using site-directed mutagenesis we show also that, among the eight cysteines of BioB, only three (Cys-53, Cys-57, Cys-60) are essential for AdoMet reductive cleavage, suggesting that these cysteines are involved in chelation of the 4Fe-4S cluster.