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.
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ABSTRACT: CO recombination kinetics has been investigated in the type II truncated hemoglobin from Thermobifida fusca (Tf-trHb) over more than 10 time decades (from 1 ps to ∼100 ms) by combining femtosecond transient absorption, nanosecond laser flash photolysis and optoacoustic spectroscopy. Photolysis is followed by a rapid geminate recombination with a time constant of ∼2 ns representing almost 60% of the overall reaction. An additional, small amplitude geminate recombination was identified at ∼100 ns. Finally, CO pressure dependent measurements brought out the presence of two transient species in the second order rebinding phase, with time constants ranging from ∼3 to ∼100 ms. The available experimental evidence suggests that the two transients are due to the presence of two conformations which do not interconvert within the time frame of the experiment. Computational studies revealed that the plasticity of protein structure is able to define a branched pathway connecting the ligand binding site and the solvent. This allowed to build a kinetic model capable of describing the complete time course of the CO rebinding kinetics to Tf-trHb.PLoS ONE 01/2012; 7(7):e39884. · 3.73 Impact Factor
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ABSTRACT: In this work we review the application of classical and quantum-mechanical atomistic computer simulation tools to the investigation of small ligand interaction with globins. In the first part, studies of ligand migration, with its connection to kinetic association rate constants (kon), are presented. In the second part, we review studies for a variety of ligands such as O2, NO, CO, HS-, F-, and NO2- showing how the heme structure, proximal effects, and the interactions with the distal amino acids can modulate protein-ligand binding. The review presents mainly results derived from our previous works on the subject, in the context of other theoretical and experimental studies performed by others. The variety and extent of the presented data yields a clear example of how computer simulation tools have, in the last decade, contributed to our deeper understanding of small ligands interactions with globins. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.Biochimica et Biophysica Acta 03/2013; · 4.66 Impact Factor