NfsA is a dimeric flavoprotein that catalyses the reduction in nitroaromatics and quinones by NADPH. This reduction is required for the activity of nitrofuran antibiotics. The crystal structure of free Escherichia coli NfsA and several homologues have been determined previously, but there is no structure of the enzyme with ligands. We present here crystal structures of oxidised E. coli NfsA in the presence of several ligands, including the antibiotic nitrofurantoin. Nitrofurantoin binds with the furan ring, rather than the nitro group that is reduced, near the N5 of the FMN. Molecular dynamics simulations show that this orientation is only favourable in the oxidised enzyme, while potentiometry suggests that little semiquinone is formed in the free protein. This suggests that the reduction occurs by direct hydride transfer from FMNH − to nitrofurantoin bound in the reverse orientation to that in the crystal structure. We present a model of nitrofurantoin bound to reduced NfsA in a viable hydride transfer orientation. The substrate 1,4-benzoquinone and the product hydroquinone are positioned close to the FMN N5 in the respective crystal structures with NfsA, suitable for reaction, but are mobile within the active site. The structure with a second FMN, bound as a ligand, shows that a mobile loop in the free protein forms a phosphate-binding pocket. NfsA is specific for NADPH and a similar conformational change, forming a phosphate-binding pocket, is likely to also occur with the natural cofactor.

The regulation of the 2-oxoglutarate dehydrogenase complex is central to intramitochondrial energy metabolism. In the present study, the active full-length E1 subunit of the human complex has been expressed and shown to be regulated by Ca 2+ , adenine nucleotides and NADH, with NADH exerting a major influence on the K 0.5 value for Ca 2+ . We investigated two potential Ca 2+ -binding sites on E1, which we term site 1 (D 114 ADLD) and site 2 (E 139 SDLD). Comparison of sequences from vertebrates with those from Ca 2+ -insensitive non-vertebrate complexes suggest that site 1 may be the more important. Consistent with this view, a mutated form of E1, D114A, shows a 6-fold decrease in sensitivity for Ca 2+ , whereas variant ∆site1 (in which the sequence of site 1 is replaced by A 114 AALA) exhibits an almost complete loss of Ca 2+ activation. Variant ∆site2 (in which the sequence is replaced with A 139 SALA) shows no measurable change in Ca 2+ sensitivity. We conclude that site 1, but not site 2, forms part of a regulatory Ca 2+ -binding site, which is distinct from other previously described Ca 2+ -binding sites.