Kinetics of Carbon Monoxide Binding to the Cooperative Dimeric Hemoglobin from Thyonella gemmata. Analysis of Carbon Monoxide Equilibrium Results†

Lawrence J Parkhurst, Robert C. Steinmeier

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Abstract

The CO association reaction for the dimeric hemoglobin of Thyonella gemmata is biphasic with rate constants of 2 x 103 and 1 x 104 M-1 s-1 among the slowest known for a hemoglobin. The kinetic heterogeneity almost certainly derives from differences in the α- and β-chain heme environments since 90% of the hemoglobin migrated as a single electrophoretic component and the more rapid kinetic phase contributed about 48% to the absorbance change. The activation enthalpies for the CO association reaction are 7.5 kcal/mol (rapid heme) and 9.0 kcal/mol (slow heme). The value for horse hemoglobin is 8.3-8.6 kcal/mol corresponding to a CO association constant of 1.7 x 105 M-1 s-1. These findings suggest that the CO binding site in Thyonella hemoglobin is even more restricted than that in horse hemoglobin. Stopped-flow and flash-photolysis results were identical. Although the hemoglobin is cooperative in CO binding, there is no evidence for a quickly reacting (Hb*) form of the protein. Tandem flash-photolysis experiments show that the heme that is rapidly reacting toward CO is also the more rapid in oxygen association and dissociation. Stopped-flow studies on partially CO-saturated hemoglobin show that intermediate liganded forms are present in significant amounts and allow all equilibrium constants to be determined in a heterogeneous Adair model. The Adair model can account satisfactorily for all existing CO equilibrium and kinetic results and predicts that the dissociation of CO from half-liganded forms should be about 10 times faster than that from Hb-(CO)2. A heterogeneous allosteric model using observed rate constants assumed to pertain to the R form gave L = 150 and c = 0.0207 and predicted a 48-fold enhancement in the CO off rate (T form vs. R form). If there exists a quickly reacting CO form (Hb*) with an association constant only 10 times that which is observed, but which reverts to the T form more rapidly than CO binds to Hb* (thus accounting for its nonappearance), L = 15 479, c = 0.00242, the enhancement in the CO dissociation rate is 41, and the equilibrium ratio of R to T for half-liganded hemoglobin is 0.02. A larger value for the hypothetical Hb* association constant leads to an even larger value for the allosteric constant L.

Original languageEnglish (US)
Pages (from-to)4651-4656
Number of pages6
JournalBiochemistry
Volume18
Issue number21
DOIs
StatePublished - Jan 1 1979

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Carbon Monoxide
Hemoglobins
Kinetics
Association reactions
Heme
Photolysis
Horses
Rate constants
L Forms
Equilibrium constants
Enthalpy
Chemical activation
Binding Sites

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kinetics of Carbon Monoxide Binding to the Cooperative Dimeric Hemoglobin from Thyonella gemmata. Analysis of Carbon Monoxide Equilibrium Results†. / Parkhurst, Lawrence J; Steinmeier, Robert C.

In: Biochemistry, Vol. 18, No. 21, 01.01.1979, p. 4651-4656.

Research output: Contribution to journalArticle

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abstract = "The CO association reaction for the dimeric hemoglobin of Thyonella gemmata is biphasic with rate constants of 2 x 103 and 1 x 104 M-1 s-1 among the slowest known for a hemoglobin. The kinetic heterogeneity almost certainly derives from differences in the α- and β-chain heme environments since 90{\%} of the hemoglobin migrated as a single electrophoretic component and the more rapid kinetic phase contributed about 48{\%} to the absorbance change. The activation enthalpies for the CO association reaction are 7.5 kcal/mol (rapid heme) and 9.0 kcal/mol (slow heme). The value for horse hemoglobin is 8.3-8.6 kcal/mol corresponding to a CO association constant of 1.7 x 105 M-1 s-1. These findings suggest that the CO binding site in Thyonella hemoglobin is even more restricted than that in horse hemoglobin. Stopped-flow and flash-photolysis results were identical. Although the hemoglobin is cooperative in CO binding, there is no evidence for a quickly reacting (Hb*) form of the protein. Tandem flash-photolysis experiments show that the heme that is rapidly reacting toward CO is also the more rapid in oxygen association and dissociation. Stopped-flow studies on partially CO-saturated hemoglobin show that intermediate liganded forms are present in significant amounts and allow all equilibrium constants to be determined in a heterogeneous Adair model. The Adair model can account satisfactorily for all existing CO equilibrium and kinetic results and predicts that the dissociation of CO from half-liganded forms should be about 10 times faster than that from Hb-(CO)2. A heterogeneous allosteric model using observed rate constants assumed to pertain to the R form gave L = 150 and c = 0.0207 and predicted a 48-fold enhancement in the CO off rate (T form vs. R form). If there exists a quickly reacting CO form (Hb*) with an association constant only 10 times that which is observed, but which reverts to the T form more rapidly than CO binds to Hb* (thus accounting for its nonappearance), L = 15 479, c = 0.00242, the enhancement in the CO dissociation rate is 41, and the equilibrium ratio of R to T for half-liganded hemoglobin is 0.02. A larger value for the hypothetical Hb* association constant leads to an even larger value for the allosteric constant L.",
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N2 - The CO association reaction for the dimeric hemoglobin of Thyonella gemmata is biphasic with rate constants of 2 x 103 and 1 x 104 M-1 s-1 among the slowest known for a hemoglobin. The kinetic heterogeneity almost certainly derives from differences in the α- and β-chain heme environments since 90% of the hemoglobin migrated as a single electrophoretic component and the more rapid kinetic phase contributed about 48% to the absorbance change. The activation enthalpies for the CO association reaction are 7.5 kcal/mol (rapid heme) and 9.0 kcal/mol (slow heme). The value for horse hemoglobin is 8.3-8.6 kcal/mol corresponding to a CO association constant of 1.7 x 105 M-1 s-1. These findings suggest that the CO binding site in Thyonella hemoglobin is even more restricted than that in horse hemoglobin. Stopped-flow and flash-photolysis results were identical. Although the hemoglobin is cooperative in CO binding, there is no evidence for a quickly reacting (Hb*) form of the protein. Tandem flash-photolysis experiments show that the heme that is rapidly reacting toward CO is also the more rapid in oxygen association and dissociation. Stopped-flow studies on partially CO-saturated hemoglobin show that intermediate liganded forms are present in significant amounts and allow all equilibrium constants to be determined in a heterogeneous Adair model. The Adair model can account satisfactorily for all existing CO equilibrium and kinetic results and predicts that the dissociation of CO from half-liganded forms should be about 10 times faster than that from Hb-(CO)2. A heterogeneous allosteric model using observed rate constants assumed to pertain to the R form gave L = 150 and c = 0.0207 and predicted a 48-fold enhancement in the CO off rate (T form vs. R form). If there exists a quickly reacting CO form (Hb*) with an association constant only 10 times that which is observed, but which reverts to the T form more rapidly than CO binds to Hb* (thus accounting for its nonappearance), L = 15 479, c = 0.00242, the enhancement in the CO dissociation rate is 41, and the equilibrium ratio of R to T for half-liganded hemoglobin is 0.02. A larger value for the hypothetical Hb* association constant leads to an even larger value for the allosteric constant L.

AB - The CO association reaction for the dimeric hemoglobin of Thyonella gemmata is biphasic with rate constants of 2 x 103 and 1 x 104 M-1 s-1 among the slowest known for a hemoglobin. The kinetic heterogeneity almost certainly derives from differences in the α- and β-chain heme environments since 90% of the hemoglobin migrated as a single electrophoretic component and the more rapid kinetic phase contributed about 48% to the absorbance change. The activation enthalpies for the CO association reaction are 7.5 kcal/mol (rapid heme) and 9.0 kcal/mol (slow heme). The value for horse hemoglobin is 8.3-8.6 kcal/mol corresponding to a CO association constant of 1.7 x 105 M-1 s-1. These findings suggest that the CO binding site in Thyonella hemoglobin is even more restricted than that in horse hemoglobin. Stopped-flow and flash-photolysis results were identical. Although the hemoglobin is cooperative in CO binding, there is no evidence for a quickly reacting (Hb*) form of the protein. Tandem flash-photolysis experiments show that the heme that is rapidly reacting toward CO is also the more rapid in oxygen association and dissociation. Stopped-flow studies on partially CO-saturated hemoglobin show that intermediate liganded forms are present in significant amounts and allow all equilibrium constants to be determined in a heterogeneous Adair model. The Adair model can account satisfactorily for all existing CO equilibrium and kinetic results and predicts that the dissociation of CO from half-liganded forms should be about 10 times faster than that from Hb-(CO)2. A heterogeneous allosteric model using observed rate constants assumed to pertain to the R form gave L = 150 and c = 0.0207 and predicted a 48-fold enhancement in the CO off rate (T form vs. R form). If there exists a quickly reacting CO form (Hb*) with an association constant only 10 times that which is observed, but which reverts to the T form more rapidly than CO binds to Hb* (thus accounting for its nonappearance), L = 15 479, c = 0.00242, the enhancement in the CO dissociation rate is 41, and the equilibrium ratio of R to T for half-liganded hemoglobin is 0.02. A larger value for the hypothetical Hb* association constant leads to an even larger value for the allosteric constant L.

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