Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
H-bonding networks of the distal residues and water molecules in the active site of Thermobifida fusca hemoglobin☆
Introduction
Ligand binding in heme-containing proteins is determined by a number of factors, including the nature and conformation of the distal residues and their capability to stabilize the heme-bound ligand via hydrogen-bonding and electrostatic interactions. The truncated hemoglobin II from Thermobifida fusca (Tf-trHb) contains three polar residues in the distal site: TrpG8, TyrCD1 and TyrB10. Whereas TrpG8 can act as a potential hydrogen-bond donor, the tyrosines can act as donors or acceptors.
The ferric and ferrous derivatives of truncated hemoglobins, analogous to mammalian globins, bind a variety of small molecules, such as H2O, NO, CN−, F−, and CO. Previous studies carried out on the CO [1], F− [2], [3] and HS− [4] adducts formed with the native Tf-trHb and a combinatorial set of mutants, in which the three distal amino acids have been singly, doubly, or triply replaced by a Phe residue, revealed that all the ligands are stabilized by TrpG8 via a strong H-bond. TyrCD1 is able to interact with CO and fluoride, whereas TyrB10 is not directly involved in ligand stabilization and plays only a minor role.
In the present work we have extended the analysis to the ferric form, studying the behavior of the ferric native protein and its triple mutant WG8F-YB10F-YCD1F at neutral and alkaline pH, and in the presence of CN−. Since both the RR Fe–OH− and Fe–CN− frequencies are very sensitive to the distal environment, detailed information on structural variations can be obtained. In particular, the comparison of the spectroscopic signature of the OH− ligated proteins at alkaline pH with those of the cyanide adducts is expected to provide information on the effects of H-bond interactions between the ligand and the distal residues as well on the Fe–CN geometry.
Section snippets
Sample preparation
Wild type (WT) Tf-trHb was expressed as a recombinant protein in Escherichia coli cells and purified as described previously [5]. As previously reported [1] the acidic surface variant (ASV) of Tf-trHb differs from the WT protein by mutation of both Phe107 and Arg91 to glutamic acid, which increases protein solubility during recombinant expression, without affecting thermostability or ligand binding properties [1], [2], [3]. Therefore, ASV was taken as an engineered scaffold of the WT protein
Hydroxyl ligand
Fig. 1 shows the UV–vis titration and the RR spectra in the high frequency region of Tf-trHb between pH 6.1 and 10.1, together with the corresponding spectra of the triple mutant at neutral pH. In this pH range Tf-trHb undergoes coordination and spin state changes. At acidic pH the absorption spectrum shows a Soret band at 407 nm (409 nm at neutral pH), Q bands at 498, 541 and 577 nm, and a broad CT band centered at 634 nm. The corresponding RR spectrum clearly indicates that at low pH the protein
Discussion
Extensive work on heme proteins has demonstrated that the frequency of the ν(Fe–OH) RR mode depends on the Fe ion spin state, the distal environment, and especially on the number and strength of H-bonding interactions between the hydroxide and the distal polar residues. [9], [13], [14], [15]. Typical ν(Fe–OH) wavenumbers are around 490 and 550 cm− 1 for 6cHS and 6cLS species, respectively. H-bonding can lower the stretching frequency by decreasing the Fe–O electron density and, as a consequence,
Conclusions and perspectives
In conclusion, we have demonstrated how complementary structural information can be obtained by a combination of RR spectra, EPR spectra and MD simulations of the cyanide and hydroxide complexes of Tf-trHb. Whereas the effect of distal H-bonding on the ν(Fe–OH) band has been rationalized, further studies are necessary to highlight the correlation between RR Fe–CN frequencies, X-ray structures, and MD simulations, taking into account the effect of both steric effects and H-bonding interactions.
Acknowledgments
This work was supported by the Institute Pasteur Fondazione Cenci Bolognetti (A. Boffi), MIUR FIRB RBFR08F41U_002 (A. Bonamore), MIUR PRIN 2008BFj34 (A. Feis and A. Boffi), University of Buenos Aires (grant X074), CONICET and European Union FP7 project NOStress (D. Estrin), and the Italian Ministero dell'Istruzione, dell'Università e della Ricerca (MIUR), Direzione Generale per l'Internazionalizzazione della Ricerca, Progetti di Grande Rilevanza Italia-Argentina. We thank Dr Maria Fittipaldi
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This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.