title: Measurement of internal substrate binding in dehaloperoxidase-hemoglobin by competition with the heme-fluoride binding equilibrium.
abstract: The application of fluoride anion as a probe for investigating the internal substrate binding has been developed and applied to dehaloperoxidase-hemoglobin (DHP) from Amphitrite ornata. By applying the fluoride titration strategy using UV-vis spectroscopy, we have studied series of halogenated phenols, other substituted phenols, halogenated indoles, and several natural amino acids that bind internally (and noncovalently) in the distal binding pocket of the heme. This approach has identified 2,4-dibromophenol (2,4-DBP) as the tightest binding substrate discovered thus far, with approximately 20-fold tighter binding affinity than that of 4-bromophenol (4-BP), a known internally binding inhibitor in DHP. Combined with resonance Raman spectroscopy, we have confirmed that competitive binding equilibria exist between fluoride anion and internally bound molecules. We have further investigated the hydrogen bonding network of the active site of DHP that stabilizes the exogenous fluoride ligand. These measurements demonstrate a general method for determination of differences in substrate binding affinity based on detection of a competitive fluoride binding equilibrium. The significance of the binding that 2,4-dibromophenol binds more tightly than any other substrate is evident when the structural and mechanistic data are taken into consideration.
authors.affiliation: Department of Chemistry, North Carolina State University , Raleigh, North Carolina 27695, United States.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhao
authors.forename: Justin
authors.initials: J
authors.lastname: Moretto
authors.forename: Peter
authors.initials: P
authors.lastname: Le
authors.forename: Stefan
authors.initials: S
authors.lastname: Franzen
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D000838:Anions / Q000737:chemistry; D001665:Binding Sites; D001667:Binding, Competitive; D005459:Fluorides / Q000737:chemistry*; D054879:Halogenation; D006418:Heme / Q000737:chemistry*; D006454:Hemoglobins / Q000737:chemistry*; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007211:Indoles / Q000737:chemistry; D010544:Peroxidases / Q000737:chemistry*; D010636:Phenols / Q000737:chemistry; D011077:Polychaeta; D011485:Protein Binding; D013057:Spectrum Analysis
chemical_list: D000596:Amino Acids; D000838:Anions; D006454:Hemoglobins; D007211:Indoles; D010636:Phenols; D006418:Heme; D010544:Peroxidases; C013930:2,4-dibromophenol; C032037:4-bromophenol; D005459:Fluorides