Fluoride as a probe for h-bonding interactions in the active site of heme proteins: The case of thermobifida fusca hemoglobin
The structural and functional properties of the active site of the bacterial hemoglobin from Thermobifida fusca are largely determined by three polar amino acids: TrpG8, TyrCD1, and TyrB10. We have exploited the availability of a combinatorial set of mutants, in each of which these three amino acids...
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2011
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| Acceso en línea: | https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00027863_v133_n51_p20970_Nicoletti http://hdl.handle.net/20.500.12110/paper_00027863_v133_n51_p20970_Nicoletti |
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paper:paper_00027863_v133_n51_p20970_Nicoletti2023-06-08T14:22:50Z Fluoride as a probe for h-bonding interactions in the active site of heme proteins: The case of thermobifida fusca hemoglobin Active site Bacterial hemoglobin Charge transfer transitions Empirical correlations Excitation conditions Fluoride complexes Functional properties H-bonded H-bonding H-bonding interaction H-bonds Heme proteins Ligand dissociation Molecular dynamics simulations Polar amino acids Resonance Raman spectra Stopped-flow kinetics Stretching bands Thermobifida fusca Wave numbers Wild-type proteins Amino acids Charge transfer Hemoglobin Hydrogen bonds Ligands Molecular dynamics Porphyrins Proteins amino acid fluoride hemoglobin hemoprotein ligand phenylalanine article dissociation excitation flow kinetics hydrogen bond molecular dynamics mutant Raman spectrometry simulation Thermobifida fusca wild type Actinomycetales Bacterial Proteins Catalytic Domain Fluorides Hydrogen Bonding Molecular Dynamics Simulation Mutation Protein Binding Spectrophotometry Spectrum Analysis, Raman Truncated Hemoglobins The structural and functional properties of the active site of the bacterial hemoglobin from Thermobifida fusca are largely determined by three polar amino acids: TrpG8, TyrCD1, and TyrB10. We have exploited the availability of a combinatorial set of mutants, in each of which these three amino acids have been singly, doubly, or triply replaced by a Phe residue, to perform a detailed study on H-bonding interactions between the protein and heme-bound fluoride. By appropriate choice of the excitation conditions, ν(Fe-F) stretching bands have been detected in the resonance Raman spectra. In the wild-type protein and one of the mutants, two ν(Fe-F) bands have been observed and assigned to the presence of two protein conformers where fluoride is singly or doubly H-bonded. Furthermore, by plotting the CT1 charge-transfer transition energy vs the ν(Fe-F) wavenumbers, an empirical correlation has been found. The data are well fitted by a straight line with a positive slope. The position along the correlation line can be considered as a novel, general spectroscopic indicator of the extent of H-bonding in the active site of heme proteins. In agreement with the spectroscopic results, we have observed that the rate of ligand dissociation in stopped-flow kinetic measurements progressively increases upon substitution of the H-bonding amino acids. Molecular dynamics simulations have been performed on the fluoride complexes of native and mutated forms, indicating the prevalent interactions at the active site. All the techniques yield evidence that TrpG8 and TyrCD1 can form strong H bonds with fluoride, whereas TyrB10 plays only a minor role in the stabilization of the ligand. © 2011 American Chemical Society. 2011 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00027863_v133_n51_p20970_Nicoletti http://hdl.handle.net/20.500.12110/paper_00027863_v133_n51_p20970_Nicoletti |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
Active site Bacterial hemoglobin Charge transfer transitions Empirical correlations Excitation conditions Fluoride complexes Functional properties H-bonded H-bonding H-bonding interaction H-bonds Heme proteins Ligand dissociation Molecular dynamics simulations Polar amino acids Resonance Raman spectra Stopped-flow kinetics Stretching bands Thermobifida fusca Wave numbers Wild-type proteins Amino acids Charge transfer Hemoglobin Hydrogen bonds Ligands Molecular dynamics Porphyrins Proteins amino acid fluoride hemoglobin hemoprotein ligand phenylalanine article dissociation excitation flow kinetics hydrogen bond molecular dynamics mutant Raman spectrometry simulation Thermobifida fusca wild type Actinomycetales Bacterial Proteins Catalytic Domain Fluorides Hydrogen Bonding Molecular Dynamics Simulation Mutation Protein Binding Spectrophotometry Spectrum Analysis, Raman Truncated Hemoglobins |
| spellingShingle |
Active site Bacterial hemoglobin Charge transfer transitions Empirical correlations Excitation conditions Fluoride complexes Functional properties H-bonded H-bonding H-bonding interaction H-bonds Heme proteins Ligand dissociation Molecular dynamics simulations Polar amino acids Resonance Raman spectra Stopped-flow kinetics Stretching bands Thermobifida fusca Wave numbers Wild-type proteins Amino acids Charge transfer Hemoglobin Hydrogen bonds Ligands Molecular dynamics Porphyrins Proteins amino acid fluoride hemoglobin hemoprotein ligand phenylalanine article dissociation excitation flow kinetics hydrogen bond molecular dynamics mutant Raman spectrometry simulation Thermobifida fusca wild type Actinomycetales Bacterial Proteins Catalytic Domain Fluorides Hydrogen Bonding Molecular Dynamics Simulation Mutation Protein Binding Spectrophotometry Spectrum Analysis, Raman Truncated Hemoglobins Fluoride as a probe for h-bonding interactions in the active site of heme proteins: The case of thermobifida fusca hemoglobin |
| topic_facet |
Active site Bacterial hemoglobin Charge transfer transitions Empirical correlations Excitation conditions Fluoride complexes Functional properties H-bonded H-bonding H-bonding interaction H-bonds Heme proteins Ligand dissociation Molecular dynamics simulations Polar amino acids Resonance Raman spectra Stopped-flow kinetics Stretching bands Thermobifida fusca Wave numbers Wild-type proteins Amino acids Charge transfer Hemoglobin Hydrogen bonds Ligands Molecular dynamics Porphyrins Proteins amino acid fluoride hemoglobin hemoprotein ligand phenylalanine article dissociation excitation flow kinetics hydrogen bond molecular dynamics mutant Raman spectrometry simulation Thermobifida fusca wild type Actinomycetales Bacterial Proteins Catalytic Domain Fluorides Hydrogen Bonding Molecular Dynamics Simulation Mutation Protein Binding Spectrophotometry Spectrum Analysis, Raman Truncated Hemoglobins |
| description |
The structural and functional properties of the active site of the bacterial hemoglobin from Thermobifida fusca are largely determined by three polar amino acids: TrpG8, TyrCD1, and TyrB10. We have exploited the availability of a combinatorial set of mutants, in each of which these three amino acids have been singly, doubly, or triply replaced by a Phe residue, to perform a detailed study on H-bonding interactions between the protein and heme-bound fluoride. By appropriate choice of the excitation conditions, ν(Fe-F) stretching bands have been detected in the resonance Raman spectra. In the wild-type protein and one of the mutants, two ν(Fe-F) bands have been observed and assigned to the presence of two protein conformers where fluoride is singly or doubly H-bonded. Furthermore, by plotting the CT1 charge-transfer transition energy vs the ν(Fe-F) wavenumbers, an empirical correlation has been found. The data are well fitted by a straight line with a positive slope. The position along the correlation line can be considered as a novel, general spectroscopic indicator of the extent of H-bonding in the active site of heme proteins. In agreement with the spectroscopic results, we have observed that the rate of ligand dissociation in stopped-flow kinetic measurements progressively increases upon substitution of the H-bonding amino acids. Molecular dynamics simulations have been performed on the fluoride complexes of native and mutated forms, indicating the prevalent interactions at the active site. All the techniques yield evidence that TrpG8 and TyrCD1 can form strong H bonds with fluoride, whereas TyrB10 plays only a minor role in the stabilization of the ligand. © 2011 American Chemical Society. |
| title |
Fluoride as a probe for h-bonding interactions in the active site of heme proteins: The case of thermobifida fusca hemoglobin |
| title_short |
Fluoride as a probe for h-bonding interactions in the active site of heme proteins: The case of thermobifida fusca hemoglobin |
| title_full |
Fluoride as a probe for h-bonding interactions in the active site of heme proteins: The case of thermobifida fusca hemoglobin |
| title_fullStr |
Fluoride as a probe for h-bonding interactions in the active site of heme proteins: The case of thermobifida fusca hemoglobin |
| title_full_unstemmed |
Fluoride as a probe for h-bonding interactions in the active site of heme proteins: The case of thermobifida fusca hemoglobin |
| title_sort |
fluoride as a probe for h-bonding interactions in the active site of heme proteins: the case of thermobifida fusca hemoglobin |
| publishDate |
2011 |
| url |
https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00027863_v133_n51_p20970_Nicoletti http://hdl.handle.net/20.500.12110/paper_00027863_v133_n51_p20970_Nicoletti |
| _version_ |
1768542011696087040 |