The X-ray structure of ferric Escherichia coli flavohemoglobin reveals an unexpected geometry of the distal heme pocket.

The x-ray structure of ferric unliganded lipid-free Escherichia coli flavohemoglobin has been solved to a resolution of 2.2 A and refined to an R-factor of 19%. The overall fold is similar to that of ferrous lipid-bound Alcaligenes eutrophus flavohemoglobin with the notable exception of the E helix positioning within the globin domain and a rotation of the NAD binding module with respect to the FAD-binding domain accompanied by a substantial rearrangement of the C-terminal region. An inspection of the heme environment in E. coli flavohemoglobin reveals an unexpected architecture of the distal pocket. In fact, the distal site is occupied by the isopropyl side chain Leu-E11 that shields the heme iron from the residues in the topological positions predicted to interact with heme iron-bound ligands, namely Tyr-B10 and Gln-E7, and stabilizes a pentacoordinate ferric iron species. Ligand binding properties are consistent with the presence of a pentacoordinate species in solution as indicated by a very fast second order combination rates with imidazole and azide. Surprisingly, imidazole, cyanide, and azide binding profiles at equilibrium are not accounted for by a single site titration curve but are biphasic and strongly suggest the presence of two distinct conformers within the liganded species.