NMR assignments, secondary structure and hydration of oxidized Escherichia coli flavodoxin

Recombinant flavodoxin from Escherichia coli was uniformly enriched with ¹⁵N and ¹³C isotopes and its oxidized form in aqueous solution investigated by three-dimensional NMR spectroscopy. Nearly complete ¹H, ¹⁵N and ¹³C resonance assignments were obtained. The secondary structure was determined from chemical shift, NOE and ³J(HNHα) coupling constant data. Like homologous long-chain flavodoxins, E. coli flavodoxin contains a five-stranded parallel β-sheet and five helices. The β-strands were found to comprise the residues 3-8, 29-34, 48-56, 80-89, 114-116 and 141-145. The helices comprise residues 12-25, 40-45, 62-73, 98-108 and 152-166. The FMN-binding site was determined by intermolecular NOEs and low-field shifted amide proton resonances induced by the phosphoester group of the cofactor. The data are in good agreement with a previously predicted model of E. coli flavodoxin [Havel, T.F. (1993) Mol. Sim. 10, 175-210]. The analysis of of water-flavodoxin NOEs revealed the presence of two, possibly three, buried hydration water molecules which are located at sites, where homologous flavodoxins from Anacystis nidulans and Anabena 7120 contain conserved hydration water molecules. One of these water molecules mediates hydrogen bonds between the protein backbone and the ribityl chain of the FMN cofactor.