title: Binding of imidazole, 1-methylimidazole and 4-nitroimidazole to yeast cytochrome c peroxidase (CcP) and the distal histidine mutant, CcP(H52L).
abstract: Imidazole, 1-methylimidazole and 4-nitroimidazole bind to yeast cytochrome c peroxidase (yCcP) with apparent equilibrium dissociation constants (KD(app)) of 3.3±0.4, 0.85±0.11, and ~0.2M, respectively, at pH7. This is the weakest imidazole binding to a heme protein reported to date and it is about 120 times weaker than imidazole binding to metmyoglobin. Spectroscopic changes associated with imidazole and 1-methylimidazole binding to yCcP suggest partial ionization of bound imidazole to imidazolate. The pKa for ionization of bound imidazole is estimated to be 7.4±0.2, about 7 units lower than that of free imidazole and about 3 units lower than imidazole bound to metmyoglobin. Equilibrium binding of imidazole to CcP(H52L) is biphasic with low- and high-affinity phases having KD(app) values of 9.5±4.5 and 0.13±0.04M, respectively. CcP(H52L) binding of 1-methylimidazole is monophasic with an affinity similar to those of yCcP and rCcP. Binding of 1-methylimidazole to rCcP is associated with two kinetic phases, the initial binding complete within 10s, followed by a process that is consistent with 1-methylimidazole binding to a cavity created by movement of Trp-191 from the interior of the protein to the surface. Both the equilibrium binding and kinetics of 1-methylimidazole binding to yCcP are pH dependent. yCcP has a four-fold increase in 1-methylimidazole binding affinity on decreasing the pH from 7.5 to 4.0, an observation that is unique among the many studies on binding of imidazole and imidazole derivatives to heme proteins.
authors.affiliation: Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL 60115, USA. Electronic address: jerman@niu.edu.
authors.forename: James E
authors.initials: JE
authors.lastname: Erman
authors.affiliation: Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL 60115, USA.
authors.forename: Diana
authors.initials: D
authors.lastname: Chinchilla
authors.affiliation: Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL 60115, USA.
authors.forename: Jason
authors.initials: J
authors.lastname: Studer
authors.affiliation: Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL 60115, USA.
authors.forename: Lidia B
authors.initials: LB
authors.lastname: Vitello
mesh_terms: D019943:Amino Acid Substitution; D003578:Cytochrome-c Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000737:chemistry* / Q000378:metabolism; D020125:Mutation, Missense; D009593:Nitroimidazoles / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D007093:Imidazoles; D009593:Nitroimidazoles; D029701:Saccharomyces cerevisiae Proteins; D003578:Cytochrome-c Peroxidase; C018100:1-methylimidazole; C052587:4-nitroimidazole
keywords: 1-Methylimidazole; 4-Nitroimidazole; CcP(H52L); Cytochrome c peroxidase; Imidazole