H-bonding networks of the distal residues and water molecules in the active site of Thermobifida fusca hemoglobin.
The ferric form of truncated hemoglobin II from Thermobifida fusca (Tf-trHb) and its triple mutant WG8F-YB10F-YCD1F at neutral and alkaline pH, and in the presence of CN(-) have been characterized by resonance Raman spectroscopy, electron paramagnetic resonance spectroscopy, and molecular dynamics simulations. Tf-trHb contains three polar residues in the distal site, namely TrpG8, TyrCD1 and TyrB10. Whereas TrpG8 can act as a potential hydrogen-bond donor, the tyrosines can act as donors or acceptors. 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. Since both the RR Fe-OH(-) and Fe-CN(-) frequencies are very sensitive to the distal environment, detailed information on structural variations has been obtained. The hydroxyl ligand binds only the WT protein giving rise to two different conformers. In form 1 the anion is stabilized by H-bonds with TrpG8, TyrCD1 and a water molecule, in turn H-bonded to TyrB10. In form 2, H-bonding with TyrCD1 is mediated by a water molecule. Unlike the OH(-) ligand, CN(-) binds both WT and the triple mutant giving rise to two forms with similar spectroscopic characteristics. The overall results clearly indicate that H-bonding interactions both with distal residues and water molecules are important structural determinants in the active site of Tf-trHb. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.
['Nicoletti|Francesco P|FP|', 'Droghetti|Enrica|E|', 'Howes|Barry D|BD|', 'Bustamante|Juan P|JP|', 'Bonamore|Alessandra|A|', 'Sciamanna|Natascia|N|', 'Estrin|Darío A|DA|', 'Feis|Alessandro|A|', 'Boffi|Alberto|A|', 'Smulevich|Giulietta|G|']
D000192:Actinomycetales D020134:Catalytic Domain D003486:Cyanides D006418:Heme D006454:Hemoglobins D006860:Hydrogen Bonding D006863:Hydrogen-Ion Concentration D006878:Hydroxides D056004:Molecular Dynamics Simulation D011485:Protein Binding D013059:Spectrum Analysis, Raman D014443:Tyrosine D014867:Water
Thermobifida fusca hemoglobin Truncated hemoglobin Truncated hemoglobin Resonance Raman Resonance Raman electron paramagnetic resonance molecular dynamics Hydrogen-bond RR Hydroxyl ligand WT 2-(N-morpholino)ethanesulfonic acid 5c 6c ASV Cyanide ligand EPR HS Hb LS MD MES Mb Tf acidic surface variant of Tf-trHb containing two single site mutations Phe107Glu and Arg91Glu five-coordinate high spin low spin myoglobin resonance Raman six-coordinate trHb truncated hemoglobin wild type