Biotin synthase (BioB) catalyses the final step in the biosynthesis of biotin. Aerobically purified biotin synthase contains one [2Fe-2S](2+) cluster per monomer. However, active BioB contains in addition a [4Fe-4S](2+) cluster which can be formed either by reconstitution with iron and sulfide, or on reduction with sodium dithionite. Here, we use EPR spectroscopy to show that mutations in the conserved YNHNLD sequence of Escherichia coli BioB affect the formation and stability of the [4Fe-4S](1+) cluster on reduction with dithionite and report the observation of a new [2Fe-2S](1+) cluster. These results serve to illustrate the dynamic nature of iron-sulfur clusters in biotin synthase and the role played by the protein in cluster interconversion.