Sulfide binding properties of truncated hemoglobins

The truncated hemoglobins from Bacillus subtilis (Bs-trHb) and Thermobifida fusca (Tf-trHb) have been shown to form high-affinity complexes with hydrogen sulfide in their ferric state. The recombinant proteins, as extracted, from Escherichia coli cells after overexpression, are indeed partially satu...

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Autores principales:	Nicoletti, F.P., Comandini, A., Bonamore, A., Boechi, L., Boubeta, F.M., Feis, A., Smulevich, I., Boffi, A.
Formato:	JOUR
Materias:	Bacillus Subtilis Bearing-only Binding parameter Binding properties Bound ligands Classical molecular dynamics Cysteine biosynthesis Escherichia coli cells Experimental data Ferric iron High affinity Hydrogen bonding stabilization Kinetic barrier Kinetic study Lucina pectinata Order of magnitude Over-expression Partially saturated Recombinant protein Resonance Raman spectroscopy Second-order rate constants Stretching bands Sulfide species Thermobifida fusca Truncated hemoglobins Wild-type proteins Bacteriology Binding energy Biochemistry Coordination reactions Escherichia coli Hemoglobin Hydrogen Hydrogen bonds Molecular dynamics Raman spectroscopy Rate constants Spin dynamics Stabilization Sulfur determination Hydrogen sulfide bacterial protein cysteine ferric ion hydrogen sulfide recombinant protein truncated hemoglobin article Bacillus subtilis bacterial cell bacterium binding affinity biosynthesis homeostasis hydrogen bond kinetics molecular dynamics nonhuman point mutation priority journal protein analysis Raman spectrometry thermodynamics Actinomycetales Bacterial Proteins Kinetics Molecular Dynamics Simulation Protein Binding Protein Conformation Spectrophotometry, Ultraviolet Sulfides Thermodynamics Truncated Hemoglobins
Acceso en línea:	http://hdl.handle.net/20.500.12110/paper_00062960_v49_n10_p2269_Nicoletti
Aporte de:	Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) de Universidad de Buenos Aires

id	todo:paper_00062960_v49_n10_p2269_Nicoletti
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spelling	todo:paper_00062960_v49_n10_p2269_Nicoletti2023-10-03T14:04:30Z Sulfide binding properties of truncated hemoglobins Nicoletti, F.P. Comandini, A. Bonamore, A. Boechi, L. Boubeta, F.M. Feis, A. Smulevich, I. Boffi, A. Bacillus Subtilis Bearing-only Binding parameter Binding properties Bound ligands Classical molecular dynamics Cysteine biosynthesis Escherichia coli cells Experimental data Ferric iron High affinity Hydrogen bonding stabilization Kinetic barrier Kinetic study Lucina pectinata Order of magnitude Over-expression Partially saturated Recombinant protein Resonance Raman spectroscopy Second-order rate constants Stretching bands Sulfide species Thermobifida fusca Truncated hemoglobins Wild-type proteins Bacteriology Binding energy Biochemistry Coordination reactions Escherichia coli Hemoglobin Hydrogen Hydrogen bonds Molecular dynamics Raman spectroscopy Rate constants Spin dynamics Stabilization Sulfur determination Hydrogen sulfide bacterial protein cysteine ferric ion hydrogen sulfide recombinant protein truncated hemoglobin article Bacillus subtilis bacterial cell bacterium binding affinity biosynthesis Escherichia coli homeostasis hydrogen bond kinetics molecular dynamics nonhuman point mutation priority journal protein analysis Raman spectrometry Thermobifida fusca thermodynamics Actinomycetales Bacillus subtilis Bacterial Proteins Kinetics Molecular Dynamics Simulation Protein Binding Protein Conformation Spectrophotometry, Ultraviolet Sulfides Thermodynamics Truncated Hemoglobins Bacillus subtilis Escherichia coli Lucina pectinata Thermobifida fusca The truncated hemoglobins from Bacillus subtilis (Bs-trHb) and Thermobifida fusca (Tf-trHb) have been shown to form high-affinity complexes with hydrogen sulfide in their ferric state. The recombinant proteins, as extracted, from Escherichia coli cells after overexpression, are indeed partially saturated with sulfide, and even highly purified samples still contain a small but significant amount of iron-bound sulfide. Thus, a complete thermodynamic and kinetic study has been undertaken by means of equilibrium and kinetic displacement experiments to assess the relevant sulfide binding parameters. The body of experimental data indicates that both proteins possess a high, affinity for hydrogen sulfide (K= 5.0 × 106 and 28 × 106 M-1 for Bs-trHb and Tf-trHb, respectively, at pH 7.0), though, lower with, respect to that reported previously for the sulfide avid Lucina pectinata I hemoglobins (2.9 × 108 M-1). From the kinetic point of view, the overall high affinity resides in the slow rate of sulfide release, attributed to hydrogen bonding stabilization of the bound ligand by distal residue WG8. A set of point mutants in which these residues have been replaced with Phe indicates that the WG8 residue represents the major kinetic barrier to the escape of the bound sulfide species. Accordingly, classical molecular dynamics simulations of SH.....-bound ferric Tf-trHb show that WG8 plays a key role in the stabilization of coordinated SH -whereas the YCD1 and. YB10 contributions are negligible. Interestingly, the triple Tf-trHb mutant bearing only Phe residues in the relevant B10, G8, and CD1 positions is endowed with a higher overall affinity for sulfide characterized, by a very fast second-order rate constant and 2 order of magnitude faster kinetics of sulfide release with respect to the wild-type protein. Resonance Raman spectroscopy data indicate that the sulfide adducts are typical of a ferric iron, low-spin derivative. In analogy with other low-spin ferric sulfide adducts, the strong band at 375 cm-1 is tentatively assigned to a Fe-S stretching band. The high affinity for hydrogen, sulfide is thought to have a possible physiological significance as H2S is produced in bacteria at metabolic steps involved in cysteine biosynthesis and hence in thiol redox homeostasis. © 2010 American Chemical Society. JOUR info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar http://hdl.handle.net/20.500.12110/paper_00062960_v49_n10_p2269_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	Bacillus Subtilis Bearing-only Binding parameter Binding properties Bound ligands Classical molecular dynamics Cysteine biosynthesis Escherichia coli cells Experimental data Ferric iron High affinity Hydrogen bonding stabilization Kinetic barrier Kinetic study Lucina pectinata Order of magnitude Over-expression Partially saturated Recombinant protein Resonance Raman spectroscopy Second-order rate constants Stretching bands Sulfide species Thermobifida fusca Truncated hemoglobins Wild-type proteins Bacteriology Binding energy Biochemistry Coordination reactions Escherichia coli Hemoglobin Hydrogen Hydrogen bonds Molecular dynamics Raman spectroscopy Rate constants Spin dynamics Stabilization Sulfur determination Hydrogen sulfide bacterial protein cysteine ferric ion hydrogen sulfide recombinant protein truncated hemoglobin article Bacillus subtilis bacterial cell bacterium binding affinity biosynthesis Escherichia coli homeostasis hydrogen bond kinetics molecular dynamics nonhuman point mutation priority journal protein analysis Raman spectrometry Thermobifida fusca thermodynamics Actinomycetales Bacillus subtilis Bacterial Proteins Kinetics Molecular Dynamics Simulation Protein Binding Protein Conformation Spectrophotometry, Ultraviolet Sulfides Thermodynamics Truncated Hemoglobins Bacillus subtilis Escherichia coli Lucina pectinata Thermobifida fusca
spellingShingle	Bacillus Subtilis Bearing-only Binding parameter Binding properties Bound ligands Classical molecular dynamics Cysteine biosynthesis Escherichia coli cells Experimental data Ferric iron High affinity Hydrogen bonding stabilization Kinetic barrier Kinetic study Lucina pectinata Order of magnitude Over-expression Partially saturated Recombinant protein Resonance Raman spectroscopy Second-order rate constants Stretching bands Sulfide species Thermobifida fusca Truncated hemoglobins Wild-type proteins Bacteriology Binding energy Biochemistry Coordination reactions Escherichia coli Hemoglobin Hydrogen Hydrogen bonds Molecular dynamics Raman spectroscopy Rate constants Spin dynamics Stabilization Sulfur determination Hydrogen sulfide bacterial protein cysteine ferric ion hydrogen sulfide recombinant protein truncated hemoglobin article Bacillus subtilis bacterial cell bacterium binding affinity biosynthesis Escherichia coli homeostasis hydrogen bond kinetics molecular dynamics nonhuman point mutation priority journal protein analysis Raman spectrometry Thermobifida fusca thermodynamics Actinomycetales Bacillus subtilis Bacterial Proteins Kinetics Molecular Dynamics Simulation Protein Binding Protein Conformation Spectrophotometry, Ultraviolet Sulfides Thermodynamics Truncated Hemoglobins Bacillus subtilis Escherichia coli Lucina pectinata Thermobifida fusca Nicoletti, F.P. Comandini, A. Bonamore, A. Boechi, L. Boubeta, F.M. Feis, A. Smulevich, I. Boffi, A. Sulfide binding properties of truncated hemoglobins
topic_facet	Bacillus Subtilis Bearing-only Binding parameter Binding properties Bound ligands Classical molecular dynamics Cysteine biosynthesis Escherichia coli cells Experimental data Ferric iron High affinity Hydrogen bonding stabilization Kinetic barrier Kinetic study Lucina pectinata Order of magnitude Over-expression Partially saturated Recombinant protein Resonance Raman spectroscopy Second-order rate constants Stretching bands Sulfide species Thermobifida fusca Truncated hemoglobins Wild-type proteins Bacteriology Binding energy Biochemistry Coordination reactions Escherichia coli Hemoglobin Hydrogen Hydrogen bonds Molecular dynamics Raman spectroscopy Rate constants Spin dynamics Stabilization Sulfur determination Hydrogen sulfide bacterial protein cysteine ferric ion hydrogen sulfide recombinant protein truncated hemoglobin article Bacillus subtilis bacterial cell bacterium binding affinity biosynthesis Escherichia coli homeostasis hydrogen bond kinetics molecular dynamics nonhuman point mutation priority journal protein analysis Raman spectrometry Thermobifida fusca thermodynamics Actinomycetales Bacillus subtilis Bacterial Proteins Kinetics Molecular Dynamics Simulation Protein Binding Protein Conformation Spectrophotometry, Ultraviolet Sulfides Thermodynamics Truncated Hemoglobins Bacillus subtilis Escherichia coli Lucina pectinata Thermobifida fusca
description	The truncated hemoglobins from Bacillus subtilis (Bs-trHb) and Thermobifida fusca (Tf-trHb) have been shown to form high-affinity complexes with hydrogen sulfide in their ferric state. The recombinant proteins, as extracted, from Escherichia coli cells after overexpression, are indeed partially saturated with sulfide, and even highly purified samples still contain a small but significant amount of iron-bound sulfide. Thus, a complete thermodynamic and kinetic study has been undertaken by means of equilibrium and kinetic displacement experiments to assess the relevant sulfide binding parameters. The body of experimental data indicates that both proteins possess a high, affinity for hydrogen sulfide (K= 5.0 × 106 and 28 × 106 M-1 for Bs-trHb and Tf-trHb, respectively, at pH 7.0), though, lower with, respect to that reported previously for the sulfide avid Lucina pectinata I hemoglobins (2.9 × 108 M-1). From the kinetic point of view, the overall high affinity resides in the slow rate of sulfide release, attributed to hydrogen bonding stabilization of the bound ligand by distal residue WG8. A set of point mutants in which these residues have been replaced with Phe indicates that the WG8 residue represents the major kinetic barrier to the escape of the bound sulfide species. Accordingly, classical molecular dynamics simulations of SH.....-bound ferric Tf-trHb show that WG8 plays a key role in the stabilization of coordinated SH -whereas the YCD1 and. YB10 contributions are negligible. Interestingly, the triple Tf-trHb mutant bearing only Phe residues in the relevant B10, G8, and CD1 positions is endowed with a higher overall affinity for sulfide characterized, by a very fast second-order rate constant and 2 order of magnitude faster kinetics of sulfide release with respect to the wild-type protein. Resonance Raman spectroscopy data indicate that the sulfide adducts are typical of a ferric iron, low-spin derivative. In analogy with other low-spin ferric sulfide adducts, the strong band at 375 cm-1 is tentatively assigned to a Fe-S stretching band. The high affinity for hydrogen, sulfide is thought to have a possible physiological significance as H2S is produced in bacteria at metabolic steps involved in cysteine biosynthesis and hence in thiol redox homeostasis. © 2010 American Chemical Society.
format	JOUR
author	Nicoletti, F.P. Comandini, A. Bonamore, A. Boechi, L. Boubeta, F.M. Feis, A. Smulevich, I. Boffi, A.
author_facet	Nicoletti, F.P. Comandini, A. Bonamore, A. Boechi, L. Boubeta, F.M. Feis, A. Smulevich, I. Boffi, A.
author_sort	Nicoletti, F.P.
title	Sulfide binding properties of truncated hemoglobins
title_short	Sulfide binding properties of truncated hemoglobins
title_full	Sulfide binding properties of truncated hemoglobins
title_fullStr	Sulfide binding properties of truncated hemoglobins
title_full_unstemmed	Sulfide binding properties of truncated hemoglobins
title_sort	sulfide binding properties of truncated hemoglobins
url	http://hdl.handle.net/20.500.12110/paper_00062960_v49_n10_p2269_Nicoletti
work_keys_str_mv	AT nicolettifp sulfidebindingpropertiesoftruncatedhemoglobins AT comandinia sulfidebindingpropertiesoftruncatedhemoglobins AT bonamorea sulfidebindingpropertiesoftruncatedhemoglobins AT boechil sulfidebindingpropertiesoftruncatedhemoglobins AT boubetafm sulfidebindingpropertiesoftruncatedhemoglobins AT feisa sulfidebindingpropertiesoftruncatedhemoglobins AT smulevichi sulfidebindingpropertiesoftruncatedhemoglobins AT boffia sulfidebindingpropertiesoftruncatedhemoglobins
_version_	1807323634183700480

Sulfide binding properties of truncated hemoglobins

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