Recombinant Human Hemoglobin: Modification of the Polarity of the β-Heme Pocket by a Valine67(E11) → Threonine Mutation

Clara Fronticelli, William S. Brinigar, John S. Olson, Enrico Bucci, Zygmunt Gryczynski, J. Kevin O'Donnell, Janusz Kowalczyk

Research output: Contribution to journalArticle

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Abstract

Using the mutagenesis and a gene expression system previously described [Fronticelli et al. (1991) J. Protein Chem. 10, 495-501], we have replaced Val67E11 in the distal heme pocket of the β-chains of hemoglobin with Thr. The valine to threonine substitution is isosteric and only modifies the polarity of the β-heme environment. The absorption and CD spectra of the resultant mutant hemoglobin were essentially the same as that of wild-type protein, indicating that the mutation did not cause any large conformational changes and that a water molecule was not coordinated to the ferrous iron atom. Equilibrium measurements of oxygen binding to the mutant indicate a 2-fold decrease in overall affinity relative to native or wild-type human hemoglobin. Thermodynamic analyses of O2 binding curves, based either on the sequential Adair model or on the MWC two-state model, indicated that the overall decrease of O2 affinity in the system was due to a lower association equilibrium constant for the intermediates of oxygenation, particularly those involved at the third ligation step. The functional characteristics of the mutant hemoglobin in either the T- or R-state were not modified greatly by the mutation; however, the Bohr effect and sensitivity to Cl were increased, suggesting a role of the intermediates of oxygenation in the modulation of these parameters. Kinetic measurements of the last step (Hb4X3 + X → Hb4X4) in ligand binding showed that the βVal67(E11) → Thr mutation decreases the O2 association rate constant roughly 2-fold, has no effect on the O2 dissociation constant, has no effect on the CO association rate constant, and increases the CO dissociation rate constant roughly 2-fold. The net result of these effects is a 2-fold decrease in the equilibrium constant for both O2 and CO binding to β subunits in the triliganded intermediate. Unexpectedly, the βVal67(E11) → Thr mutation caused a 3-fold increase in the rate of CO binding to β-subunits within T-state deoxyhemoglobin.

Original languageEnglish
Pages (from-to)1235-1242
Number of pages8
JournalBiochemistry
Volume32
Issue number5
DOIs
StatePublished - 1 Jan 1993

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Threonine
Carbon Monoxide
Heme
Hemoglobins
Rate constants
Mutation
Oxygenation
Equilibrium constants
Association reactions
Mutagenesis
Valine
Thermodynamics
Gene expression
Ligation
Proteins
Substitution reactions
Iron
Modulation
Oxygen
Ligands

Cite this

Fronticelli, Clara ; Brinigar, William S. ; Olson, John S. ; Bucci, Enrico ; Gryczynski, Zygmunt ; O'Donnell, J. Kevin ; Kowalczyk, Janusz. / Recombinant Human Hemoglobin : Modification of the Polarity of the β-Heme Pocket by a Valine67(E11) → Threonine Mutation. In: Biochemistry. 1993 ; Vol. 32, No. 5. pp. 1235-1242.
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title = "Recombinant Human Hemoglobin: Modification of the Polarity of the β-Heme Pocket by a Valine67(E11) → Threonine Mutation",
abstract = "Using the mutagenesis and a gene expression system previously described [Fronticelli et al. (1991) J. Protein Chem. 10, 495-501], we have replaced Val67E11 in the distal heme pocket of the β-chains of hemoglobin with Thr. The valine to threonine substitution is isosteric and only modifies the polarity of the β-heme environment. The absorption and CD spectra of the resultant mutant hemoglobin were essentially the same as that of wild-type protein, indicating that the mutation did not cause any large conformational changes and that a water molecule was not coordinated to the ferrous iron atom. Equilibrium measurements of oxygen binding to the mutant indicate a 2-fold decrease in overall affinity relative to native or wild-type human hemoglobin. Thermodynamic analyses of O2 binding curves, based either on the sequential Adair model or on the MWC two-state model, indicated that the overall decrease of O2 affinity in the system was due to a lower association equilibrium constant for the intermediates of oxygenation, particularly those involved at the third ligation step. The functional characteristics of the mutant hemoglobin in either the T- or R-state were not modified greatly by the mutation; however, the Bohr effect and sensitivity to Cl− were increased, suggesting a role of the intermediates of oxygenation in the modulation of these parameters. Kinetic measurements of the last step (Hb4X3 + X → Hb4X4) in ligand binding showed that the βVal67(E11) → Thr mutation decreases the O2 association rate constant roughly 2-fold, has no effect on the O2 dissociation constant, has no effect on the CO association rate constant, and increases the CO dissociation rate constant roughly 2-fold. The net result of these effects is a 2-fold decrease in the equilibrium constant for both O2 and CO binding to β subunits in the triliganded intermediate. Unexpectedly, the βVal67(E11) → Thr mutation caused a 3-fold increase in the rate of CO binding to β-subunits within T-state deoxyhemoglobin.",
author = "Clara Fronticelli and Brinigar, {William S.} and Olson, {John S.} and Enrico Bucci and Zygmunt Gryczynski and O'Donnell, {J. Kevin} and Janusz Kowalczyk",
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Fronticelli, C, Brinigar, WS, Olson, JS, Bucci, E, Gryczynski, Z, O'Donnell, JK & Kowalczyk, J 1993, 'Recombinant Human Hemoglobin: Modification of the Polarity of the β-Heme Pocket by a Valine67(E11) → Threonine Mutation', Biochemistry, vol. 32, no. 5, pp. 1235-1242. https://doi.org/10.1021/bi00056a006

Recombinant Human Hemoglobin : Modification of the Polarity of the β-Heme Pocket by a Valine67(E11) → Threonine Mutation. / Fronticelli, Clara; Brinigar, William S.; Olson, John S.; Bucci, Enrico; Gryczynski, Zygmunt; O'Donnell, J. Kevin; Kowalczyk, Janusz.

In: Biochemistry, Vol. 32, No. 5, 01.01.1993, p. 1235-1242.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Recombinant Human Hemoglobin

T2 - Modification of the Polarity of the β-Heme Pocket by a Valine67(E11) → Threonine Mutation

AU - Fronticelli, Clara

AU - Brinigar, William S.

AU - Olson, John S.

AU - Bucci, Enrico

AU - Gryczynski, Zygmunt

AU - O'Donnell, J. Kevin

AU - Kowalczyk, Janusz

PY - 1993/1/1

Y1 - 1993/1/1

N2 - Using the mutagenesis and a gene expression system previously described [Fronticelli et al. (1991) J. Protein Chem. 10, 495-501], we have replaced Val67E11 in the distal heme pocket of the β-chains of hemoglobin with Thr. The valine to threonine substitution is isosteric and only modifies the polarity of the β-heme environment. The absorption and CD spectra of the resultant mutant hemoglobin were essentially the same as that of wild-type protein, indicating that the mutation did not cause any large conformational changes and that a water molecule was not coordinated to the ferrous iron atom. Equilibrium measurements of oxygen binding to the mutant indicate a 2-fold decrease in overall affinity relative to native or wild-type human hemoglobin. Thermodynamic analyses of O2 binding curves, based either on the sequential Adair model or on the MWC two-state model, indicated that the overall decrease of O2 affinity in the system was due to a lower association equilibrium constant for the intermediates of oxygenation, particularly those involved at the third ligation step. The functional characteristics of the mutant hemoglobin in either the T- or R-state were not modified greatly by the mutation; however, the Bohr effect and sensitivity to Cl− were increased, suggesting a role of the intermediates of oxygenation in the modulation of these parameters. Kinetic measurements of the last step (Hb4X3 + X → Hb4X4) in ligand binding showed that the βVal67(E11) → Thr mutation decreases the O2 association rate constant roughly 2-fold, has no effect on the O2 dissociation constant, has no effect on the CO association rate constant, and increases the CO dissociation rate constant roughly 2-fold. The net result of these effects is a 2-fold decrease in the equilibrium constant for both O2 and CO binding to β subunits in the triliganded intermediate. Unexpectedly, the βVal67(E11) → Thr mutation caused a 3-fold increase in the rate of CO binding to β-subunits within T-state deoxyhemoglobin.

AB - Using the mutagenesis and a gene expression system previously described [Fronticelli et al. (1991) J. Protein Chem. 10, 495-501], we have replaced Val67E11 in the distal heme pocket of the β-chains of hemoglobin with Thr. The valine to threonine substitution is isosteric and only modifies the polarity of the β-heme environment. The absorption and CD spectra of the resultant mutant hemoglobin were essentially the same as that of wild-type protein, indicating that the mutation did not cause any large conformational changes and that a water molecule was not coordinated to the ferrous iron atom. Equilibrium measurements of oxygen binding to the mutant indicate a 2-fold decrease in overall affinity relative to native or wild-type human hemoglobin. Thermodynamic analyses of O2 binding curves, based either on the sequential Adair model or on the MWC two-state model, indicated that the overall decrease of O2 affinity in the system was due to a lower association equilibrium constant for the intermediates of oxygenation, particularly those involved at the third ligation step. The functional characteristics of the mutant hemoglobin in either the T- or R-state were not modified greatly by the mutation; however, the Bohr effect and sensitivity to Cl− were increased, suggesting a role of the intermediates of oxygenation in the modulation of these parameters. Kinetic measurements of the last step (Hb4X3 + X → Hb4X4) in ligand binding showed that the βVal67(E11) → Thr mutation decreases the O2 association rate constant roughly 2-fold, has no effect on the O2 dissociation constant, has no effect on the CO association rate constant, and increases the CO dissociation rate constant roughly 2-fold. The net result of these effects is a 2-fold decrease in the equilibrium constant for both O2 and CO binding to β subunits in the triliganded intermediate. Unexpectedly, the βVal67(E11) → Thr mutation caused a 3-fold increase in the rate of CO binding to β-subunits within T-state deoxyhemoglobin.

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U2 - 10.1021/bi00056a006

DO - 10.1021/bi00056a006

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