4-nitroimidazole binding to horse metmyoglobin: evidence for preferential anion binding.
Abstract: The ionization of 4-nitroimidazole to 4-nitroimidazolate was investigated as a function of ionic strength. The apparent pKa varies from 8.99 to 9.50 between 0.001 and 1.0 M ionic strength, respectively, at 25 degrees C. The ionic strength dependence of this ionization is anomalous. The binding of 4-nitroimidazole by horse metmyoglobin was studied between pH 5.0 and 11.5 and as a function of ionic strength between 0.01 and 1.0 M. The association rate constant is pH-dependent, varying from 24 M(-1)s(-1) at pH 5 to a maximum value of 280 M(-1)s(-1) at pH 9.5 and then decreasing to 10 M(-1)s(-1) at pH 11.5 in 0.1 M ionic strength buffers. The dissociation rate constant has a much smaller pH dependence, varying from 0.082 s(-1) at low pH to 0.035 s(-1) at high pH, with an apparent pKa of 6.5. The binding affinity of 4-nitroimidazole to horse metmyoglobin is about 2.5 orders of magnitude stronger than that for imidazole and this increased affinity is attributed to the much slower dissociation rate for 4-nitroimidazole compared to that of imidazole. Although the ionic strength dependence of the binding rate is small and secondary kinetic salt effects can account for the ionic strength dependence of the association rate constant, the pH dependence of the rate constants and microscopic reversibility arguments indicate that the anionic form of the ligand binds more rapidly to all forms of metmyoglobin than does the neutral form of the ligand. However, the spectrum of the complex is similar to model complexes involving neutral imidazole and not imidazolate. The latter observation suggests that the initial metmyoglobin/4-nitroimidazolate complex rapidly binds a proton and the neutral form of the bound ligand is stabilized, probably through hydrogen binding with the distal histidine.
Publication Date: 2000-11-09 PubMed ID: 11068880DOI: 10.1006/abbi.2000.2039Google Scholar: Lookup
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- Journal Article
- Research Support
- U.S. Gov't
- Non-P.H.S.
- Research Support
- U.S. Gov't
- P.H.S.
Summary
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The research investigates the binding affinity of 4-nitroimidazole to horse metmyoglobin and demonstrates that its binding strength is significantly higher than that of imidazole due to its slower dissociation rate. The study also reveals that the anionic form of the ligand binds more rapidly to all forms of metmyoglobin than the neutral counterpart.
Ionization Investigation of 4-nitroimidazole
- The study commences by examining the ionization of 4-nitroimidazole to 4-nitroimidazolate. This process is monitored based on ionic strength, and the apparent pKa is observed to fluctuate between 8.99 and 9.50 for 0.001 and 1.0 M ionic strength respectively at 25 degrees Celsius. The study terms this ionic strength dependence as anomalous.
4-nitroimidazole Binding Study
- The main focus of the research is the examination of the binding of 4-nitroimidazole by horse metmyoglobin. This observation is performed between a pH range of 5.0 and 11.5 and an ionic strength magnitude between 0.01 and 1.0 M.
- The association rate constant has shown to be pH-dependent, changing from 24 M(-1)s(-1) at pH 5 reaching its peak value of 280 M(-1)s(-1) at pH 9.5 and then falls to 10 M(-1)s(-1) at pH 11.5 in 0.1 M ionic strength buffers. The dissociation rate constant that displays significantly less pH dependence, ranges from 0.082 s(-1) at low pH to 0.035 s(-1) at high pH, with an apparent pKa of 6.5.
Affinity of 4-nitroimidazole
- The research highlights that the binding affinity of 4-nitroimidazole to horse metmyoglobin is about two and a half times orders of magnitude stronger than that for imidazole. This increased affinity is related to the slower 4-nitroimidazole dissociation rate compared to imidazole.
- Despite the ionic strength dependence of the binding rate being small, and secondary kinetic salt effects being able to explain the ionic strength dependence of the association rate constant, the pH dependence of the rate constants and microscopic reversibility arguments indicate that the anionic form of the ligand binds more rapidly to all forms of metmyoglobin than the neutral form of the ligand.
Identification of Ligand Form
- The spectrum of the complex is similar to that involving neutral imidazole and not the imidazolate form. This observation suggests that the initial metmyoglobin/4-nitroimidazolate complex rapidly binds a proton, stabilizing the neutral form of the bound ligand, likely due to hydrogen binding with the distal histidine.
Cite This Article
APA
Taylor KC, Vitello LB, Erman JE.
(2000).
4-nitroimidazole binding to horse metmyoglobin: evidence for preferential anion binding.
Arch Biochem Biophys, 382(2), 284-295.
https://doi.org/10.1006/abbi.2000.2039 Publication
Researcher Affiliations
- Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb 60115, USA.
MeSH Terms
- Animals
- Anions
- Horses
- Hydrogen-Ion Concentration
- In Vitro Techniques
- Kinetics
- Metmyoglobin / chemistry
- Metmyoglobin / metabolism
- Nitroimidazoles / chemistry
- Nitroimidazoles / metabolism
- Osmolar Concentration
- Protein Binding
- Solubility
- Spectrophotometry
Grant Funding
- R15 GM59740-01 / NIGMS NIH HHS
Citations
This article has been cited 5 times.- Bidwai AK, Ahrendt AJ, Sullivan JS, Vitello LB, Erman JE. pH dependence of cyanide and imidazole binding to the heme domains of Sinorhizobium meliloti and Bradyrhizobium japonicum FixL.. J Inorg Biochem 2015 Dec;153:88-102.
- Erman JE, Chinchilla D, Studer J, Vitello LB. Binding of imidazole, 1-methylimidazole and 4-nitroimidazole to yeast cytochrome c peroxidase (CcP) and the distal histidine mutant, CcP(H52L).. Biochim Biophys Acta 2015 Aug;1854(8):869-81.
- Bidwai A, Ayala C, Vitello LB, Erman JE. Apolar distal pocket mutants of yeast cytochrome c peroxidase: Binding of imidazole, 1-methylimidazole and 4-nitroimidazole to the triAla, triVal, and triLeu variants.. Biochim Biophys Acta 2015 Aug;1854(8):919-29.
- Chinchilla D, Kilheeney H, Vitello LB, Erman JE. Kinetic and equilibrium studies of acrylonitrile binding to cytochrome c peroxidase and oxidation of acrylonitrile by cytochrome c peroxidase compound I.. Biochem Biophys Res Commun 2014 Jan 3;443(1):200-4.
- Bidwai AK, Ok EY, Erman JE. pH dependence of cyanide binding to the ferric heme domain of the direct oxygen sensor from Escherichia coli and the effect of alkaline denaturation.. Biochemistry 2008 Sep 30;47(39):10458-70.
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