4-Nitroimidazole Binding to Horse Metmyoglobin: Evidence for Preferential Anion Binding

Taylor, K.C.; Vitello, Lidia B.; Erman, James E.

doi:10.1006/abbi.2000.2039

Cited by 8 publications

(18 citation statements)

References 24 publications

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“…The CcP(triVal) binds 4NI three orders of magnitude more strongly than yCcP and the spectrum of the CcP(triVal)/4NI complexes could be determined. The Soret band for the CcP(triVal)/4NI complex occurs at 416 nm with an extinction coefficients are 110 mM −1 cm −1 , Table 3, similar to the spectroscopic parameters for the metMb/4NI complex, Table 3 [19]. There is no evidence for bound imidazolate in any of the three triple mutants, with the largest increase in the difference spectra occurring between 418 and 420 nm, Figs.…”

Section: Discussionsupporting

confidence: 69%

Apolar distal pocket mutants of yeast cytochrome c peroxidase: Binding of imidazole, 1-methylimidazole and 4-nitroimidazole to the triAla, triVal, and triLeu variants

Ayala

Vitello

Erman

2015

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Imidazole binding to three apolar distal heme pocket mutants of yeast cytochrome c peroxidase (CcP) has been investigated between pH 4 and 8. The three CcP variants have Arg-48, Trp-51, and His-52 mutated to either all alanine, CcP(triAla), all valine, CcP(triVal), or all leucine residues, CcP(triLeu). The imidazole binding curves for all three mutants are biphasic indicating that each of the mutants exist in at least two conformational states with different affinities for imidazole. At pH 7, the high-affinity conformations of the three CcP mutants bind imidazole between 3.8 and 4.7 orders of magnitude stronger than that of wild-type CcP while the low-affinity conformations have binding affinities about 2.5 orders of magnitude larger than wild-type CcP. Imidazole binding to the three CcP mutants is pH dependent with the strongest binding observed at high pH. Apparent pKa values for the transition in binding vary between 5.6 and 7.5 for the high-affinity conformations and between 6.2 and 6.8 for the low-affinity conformations of the CcP triple mutants. The kinetics of imidazole binding are also biphasic. The fast phase of imidazole binding to CcP(triAla) and CcP(triLeu) is linearly dependent on the imidazole concentration while the slow phase is independent of imidazole concentration. Both phases of imidazole binding to CcP(triVal) have a hyperbolic dependence on the imidazole concentration. The apparent association rate constants vary between 30 and 170 M−1s−1 while the apparent dissociation rate constants vary between 0.05 and 0.43 s−1. The CcP triple mutants have higher binding affinities for 1-methylimidazole and 4-nitroimidazole than does wild-type CcP.

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Section: Discussionsupporting

confidence: 69%

Apolar distal pocket mutants of yeast cytochrome c peroxidase: Binding of imidazole, 1-methylimidazole and 4-nitroimidazole to the triAla, triVal, and triLeu variants

Ayala

Vitello

Erman

2015

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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“…4-Nitroimidazole (4NI) has limited solubility in aqueous buffer with saturated solutions having a concentration of less than 6 mM at neutral pH [7]. Adding ~5 mM 4NI to both yCcP and rCcP causes a small perturbation to the spectrum of yCcP, Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Metmyoglobin shows very little discrimination in the rates of imidazole and imidazolium ion binding [6,7,9,10]. When imidazolium ion binds to the heme iron, it must release the imidazolium proton.…”

Section: Discussionmentioning

confidence: 99%

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Binding of imidazole, 1-methylimidazole and 4-nitroimidazole to yeast cytochrome c peroxidase (CcP) and the distal histidine mutant, CcP(H52L)

Erman

Chinchilla

Studer

et al. 2015

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Imidazole, 1-methylimidazole and 4-nitroimidazole bind to yeast cytochrome c peroxidase (yCcP) with apparent equilibrium dissociation constants (KDnormalapp) of 3.3 ± 0.4, 0.85 ± 0.11, and ~0.2 M, respectively, at pH 7. 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 KDnormalapp values of 9.5 ± 4.5 and 0.13 ± 0.04 M, 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 10 s, 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.

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“…For the nitriles, any pH dependence of the ligand association and dissociation reactions can be attributed solely to properties of the protein. While there are many studies of imidazole binding to heme proteins [9,11,13–23], we have been unable to find any prior reports of nitrile binding to heme proteins, although binding of organonitriles to transition metals is well known [24,25]. …”

Section: Introductionmentioning

confidence: 78%

Kinetic and equilibrium studies of acrylonitrile binding to cytochrome c peroxidase and oxidation of acrylonitrile by cytochrome c peroxidase compound I

Chinchilla¹,

Kilheeney²,

Vitello³

et al. 2014

Biochemical and Biophysical Research Communications

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Ferric heme proteins bind weakly basic ligands and the binding affinity is often pH dependent due to protonation of the ligand as well as the protein. In an effort to find a small, neutral ligand without significant acid/base properties to probe ligand binding reactions in ferric heme proteins we were led to consider the organonitriles. Although organonitriles are known to bind to transition metals, we have been unable to find any prior studies of nitrile binding to heme proteins. In this communication we report on the equilibrium and kinetic properties of acrylonitrile binding to cytochrome c peroxidase (CcP) as well as the oxidation of acrylonitrile by CcP compound I. Acrylonitrile binding to CcP is independent of pH between pH 4 and 8. The association and dissociation rate constants are 0.32 ± 0.16 M−1s−1 and 0.34 ± 0.15 s−1, respectively, and the independently measured equilibrium dissociation constant for the complex is 1.1 ± 0.2 M. We have demonstrated for the first time that acrylonitrile can bind to a ferric heme protein. The binding mechanism appears to be a simple, one-step association of the ligand with the heme iron. We have also demonstrated that CcP can catalyze the oxidation of acrylonitrile, most likely to 2-cyanoethylene oxide in a “peroxygenase”-type reaction, with rates that are similar to rat liver microsomal cytochrome P450-catalyzed oxidation of acrylonitrile in the monooxygenase reaction. CcP compound I oxidizes acrylonitrile with a maximum turnover number of 0.61 min−1 at pH 6.0.

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4-Nitroimidazole Binding to Horse Metmyoglobin: Evidence for Preferential Anion Binding

Cited by 8 publications

References 24 publications

Apolar distal pocket mutants of yeast cytochrome c peroxidase: Binding of imidazole, 1-methylimidazole and 4-nitroimidazole to the triAla, triVal, and triLeu variants

Apolar distal pocket mutants of yeast cytochrome c peroxidase: Binding of imidazole, 1-methylimidazole and 4-nitroimidazole to the triAla, triVal, and triLeu variants

Binding of imidazole, 1-methylimidazole and 4-nitroimidazole to yeast cytochrome c peroxidase (CcP) and the distal histidine mutant, CcP(H52L)

Kinetic and equilibrium studies of acrylonitrile binding to cytochrome c peroxidase and oxidation of acrylonitrile by cytochrome c peroxidase compound I

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