title: New insights into the role of distal histidine flexibility in ligand stabilization of dehaloperoxidase-hemoglobin from Amphitrite ornata.
abstract: The present work highlights the important role played by the distal histidine in controlling the binding of heme ligands in dehaloperoxidase (DHP) as compared to myoglobin and peroxidases. In DHP the distal histidine is highly mobile and undergoes a conformational change that places it within hydrogen-bonding distance of anionic ligands and water, where strong hydrogen bonding can occur. The detailed resonance Raman (RR) analysis at room temperature shows the presence of an equilibrium between a 5-coordinate and a 6-coordinate (aquo) high-spin form. The equilibrium shifts toward the aquo form at 12 K. These two forms are consistent with the existing X-ray structures where a closed conformation, with His55 positioned in the distal pocket and H-bonded with the distal water molecule (6-coordinate), and an open solvent-exposed conformation, with the His55 displaced from the distal pocket (5-coordinate form), are in equilibrium. Moreover, the comparison between the Raman data at 298 and 12 K and the results obtained by EPR of DHP in the presence of 4-iodophenol highlights the formation of a pure 5-coordinate high-spin form (open conformation). The data reported herein support the role of His55 in facilitating the interaction of substrate and inhibitor in the regulation of enzyme function, as previously suggested. The two conformations of His55 in equilibrium at room temperature provide a level of control that permits the distal histidine to act as both the acid-base catalyst in the peroxidase mechanism and the stabilizing amino acid for exogenous heme-coordinated ligands.
authors.affiliation: Dipartimento di Chimica, Università di Firenze, Via della Lastruccia 3, I-50019 Sesto Fiorentino (FI), Italy.
authors.forename: Francesco P
authors.initials: FP
authors.lastname: Nicoletti
authors.forename: Matthew K
authors.initials: MK
authors.lastname: Thompson
authors.forename: Barry D
authors.initials: BD
authors.lastname: Howes
authors.forename: Stefan
authors.initials: S
authors.lastname: Franzen
authors.forename: Giulietta
authors.initials: G
authors.lastname: Smulevich
mesh_terms: D000818:Animals; D004795:Enzyme Stability; D005290:Ferric Compounds / Q000737:chemistry; D006454:Hemoglobins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D006639:Histidine / Q000737:chemistry*; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D010544:Peroxidases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D011077:Polychaeta / Q000201:enzymology*; D011485:Protein Binding; D011487:Protein Conformation; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman; D013379:Substrate Specificity
chemical_list: D005290:Ferric Compounds; D006454:Hemoglobins; D008024:Ligands; D006639:Histidine; C098661:DHP I dehaloperoxidase; D010544:Peroxidases