title: Fluoride as a probe for H-bonding interactions in the active site of heme proteins: the case of Thermobifida fusca hemoglobin.
abstract: The structural and functional properties of the active site of the bacterial hemoglobin from Thermobifida fusca are largely determined by three polar amino acids: TrpG8, TyrCD1, and TyrB10. We have exploited the availability of a combinatorial set of mutants, in each of which these three amino acids have been singly, doubly, or triply replaced by a Phe residue, to perform a detailed study on H-bonding interactions between the protein and heme-bound fluoride. By appropriate choice of the excitation conditions, ν(Fe-F) stretching bands have been detected in the resonance Raman spectra. In the wild-type protein and one of the mutants, two ν(Fe-F) bands have been observed and assigned to the presence of two protein conformers where fluoride is singly or doubly H-bonded. Furthermore, by plotting the CT1 charge-transfer transition energy vs the ν(Fe-F) wavenumbers, an empirical correlation has been found. The data are well fitted by a straight line with a positive slope. The position along the correlation line can be considered as a novel, general spectroscopic indicator of the extent of H-bonding in the active site of heme proteins. In agreement with the spectroscopic results, we have observed that the rate of ligand dissociation in stopped-flow kinetic measurements progressively increases upon substitution of the H-bonding amino acids. Molecular dynamics simulations have been performed on the fluoride complexes of native and mutated forms, indicating the prevalent interactions at the active site. All the techniques yield evidence that TrpG8 and TyrCD1 can form strong H bonds with fluoride, whereas TyrB10 plays only a minor role in the stabilization of the ligand.
authors.affiliation: Dipartimento di Chimica Ugo Schiff, Università di Firenze, Via della Lastruccia 3-13, I-50019 Sesto Fiorentino (FI), Italy.
authors.forename: Francesco P
authors.initials: FP
authors.lastname: Nicoletti
authors.forename: Enrica
authors.initials: E
authors.lastname: Droghetti
authors.forename: Leonardo
authors.initials: L
authors.lastname: Boechi
authors.forename: Alessandra
authors.initials: A
authors.lastname: Bonamore
authors.forename: Natascia
authors.initials: N
authors.lastname: Sciamanna
authors.forename: Darío A
authors.initials: DA
authors.lastname: Estrin
authors.forename: Alessandro
authors.initials: A
authors.lastname: Feis
authors.forename: Alberto
authors.initials: A
authors.lastname: Boffi
authors.forename: Giulietta
authors.initials: G
authors.lastname: Smulevich
mesh_terms: D000192:Actinomycetales / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D005459:Fluorides / Q000378:metabolism*; D006860:Hydrogen Bonding; D056004:Molecular Dynamics Simulation; D009154:Mutation; D011485:Protein Binding; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman; D054793:Truncated Hemoglobins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D054793:Truncated Hemoglobins; D005459:Fluorides