Interplay of the H‑Bond Donor−Acceptor Role of the Distal Residues in Hydroxyl Ligand Stabilization of Thermobif ida f usca Truncated Hemoglobin

International audienceThe unique architecture of the active site of Thermobifida fusca truncated hemoglobin (Tf-trHb) and other globins belonging to the same family has stimulated extensive studies aimed at understanding the interplay between iron-bound ligands and distal amino acids. The behavior of the heme-bound hydroxyl, in particular, has generated much interest in view of the relationships between the spin-state equilibrium of the ferric iron atom and hydrogen-bonding capabilities (as either acceptor or donor) of the OH − group itself. The present investigation offers a detailed molecular dynamics and spectroscopic picture of the hydroxyl complexes of the WT protein and a combinatorial set of mutants, in which the distal polar residues, TrpG8, TyrCD1, and TyrB10, have been singly, doubly, or triply replaced by a Phe residue. Each mutant is characterized by a complex interplay of interactions in which the hydroxyl ligand may act both as a H-bond donor or acceptor. The resonance Raman stretching frequencies of the Fe−OH moiety, together with electron paramagnetic resonance spectra and MD simulations on each mutant, have enabled the identification of specific contributions to the unique ligand-inclusive H-bond network typical of this globin family. H eme proteins are a ubiquitous family of metallo-proteins containing heme as a prosthetic group. In spite of the fact that all heme proteins share the same active site, they display an amazing variety of behaviors that makes them an excellent benchmark for the investigation of the relationship between protein structure and function. In this context, the currently available heme spectroscopic data provide the scientific foundation upon which functional and biological paradigms of heme proteins can be validated or challenged. Understanding heme protein spectroscopy thus allows one to translate physicochemical properties of the active site into the observed functional behavior. X-ray crystallography has provided fundamental snapshots of active site structural frameworks and highlighted the network of interactions that contributes to modulate the functional properties of the macromolecule around the metal binding site. On the basis of X-ray diffraction data and static or dynamic spectroscopic observations, it is now clear that iron-bound ligands in the heme binding pocket experience a network of electrostatic fields and hydrogen-bonding interactions exerted by nearby amino acid side chains