Oxygen and Carbon Monoxide Equilibria and the Kinetics of Oxygen Binding by the Cooperative Dimeric Hemoglobin of Thyonella gemmated†

R. C. Steinmeier, Lawrence J Parkhurst

Research output: Contribution to journalArticle

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Abstract

Oxygen and carbon monoxide equilibrium measurements have been made for the dimeric hemoglobin of Thyonella gemmata. For both ligands, the Hill number is ~ 1.4. The affinity for CO, however, is quite low, the median ligand activity at 20 °C, pH 7, being 1.3 μM. There is no apparent Bohr effect from pH 7 to pH 9. The replacement of 02 by CO gives an apparent value for M of 2.4. More unusual is the finding that this replacement reaction is also cooperative, the apparent Hill number being 1.4, suggesting that, for this hemoglobin, the HbO2 and HbCO conformations differ. HbO2 - HbCO difference spectra in the 290-nm region suggest that replacement of O2 by CO at the heme site is associated with the movement of a tryptophan from a nonpolar to a polar environment. The kinetics of oxygen dissociation from the hemoglobin of T. gemmata are biphasic. The dissociation rate constants for the hemes in the two polypeptide chains differ by a factor of about 5. Oxygen dissociation in the presence of CO is somewhat slower, but measurements at the HbO2-HbCO isosbestic wavelength show that a maximum of nearly 1/2 of the hemes are unliganded during the reaction. This shows that the predominant half-liganded oxyhemoglobin species does not provide a quickly reacting form for CO binding, since the maximum CO association constant can be only ~2 X 10-4 M-1 s_1. Oxygen-pulse experiments provide the clearest kinetic evidence for cooperativity in oxygen binding. The rate constants for oxygen dissociation from singly oxygenated forms must be greater than 5 times those for the fully oxygenated hemoglobin. The oxygen dissociation and oxygen-pulse measurements cannot be accommodated within a heterogeneous Adair model. In terms of an allosteric model, the rate constant for R→ T or for T→R for intermediates such as αO2-β is small (ti/2 is tens of milliseconds), or the R⇄ T equilibrium constant for the half-liganded form is ~ 1. The kinetics of oxygen association following HbCO photolysis are unusual in that the rate of oxygen binding is less than first power in oxygen. A model is proposed to account for these results. In this model, binding of oxygen to the initial pho-toproduct is some 30-40-fold slower than that to a second form, derived from the first by a conformational change, the t1/2 for which is ~1 ms.

Original languageEnglish (US)
Pages (from-to)4645-4651
Number of pages7
JournalBiochemistry
Volume18
Issue number21
DOIs
StatePublished - Jan 1 1979

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Carbon Monoxide
Hemoglobins
Oxygen
Kinetics
Heme
Rate constants
Association reactions
Ligands
Oxyhemoglobins
Photolysis
Equilibrium constants
Theophylline
Tryptophan
Conformations

ASJC Scopus subject areas

  • Biochemistry

Cite this

Oxygen and Carbon Monoxide Equilibria and the Kinetics of Oxygen Binding by the Cooperative Dimeric Hemoglobin of Thyonella gemmated†. / Steinmeier, R. C.; Parkhurst, Lawrence J.

In: Biochemistry, Vol. 18, No. 21, 01.01.1979, p. 4645-4651.

Research output: Contribution to journalArticle

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abstract = "Oxygen and carbon monoxide equilibrium measurements have been made for the dimeric hemoglobin of Thyonella gemmata. For both ligands, the Hill number is ~ 1.4. The affinity for CO, however, is quite low, the median ligand activity at 20 °C, pH 7, being 1.3 μM. There is no apparent Bohr effect from pH 7 to pH 9. The replacement of 02 by CO gives an apparent value for M of 2.4. More unusual is the finding that this replacement reaction is also cooperative, the apparent Hill number being 1.4, suggesting that, for this hemoglobin, the HbO2 and HbCO conformations differ. HbO2 - HbCO difference spectra in the 290-nm region suggest that replacement of O2 by CO at the heme site is associated with the movement of a tryptophan from a nonpolar to a polar environment. The kinetics of oxygen dissociation from the hemoglobin of T. gemmata are biphasic. The dissociation rate constants for the hemes in the two polypeptide chains differ by a factor of about 5. Oxygen dissociation in the presence of CO is somewhat slower, but measurements at the HbO2-HbCO isosbestic wavelength show that a maximum of nearly 1/2 of the hemes are unliganded during the reaction. This shows that the predominant half-liganded oxyhemoglobin species does not provide a quickly reacting form for CO binding, since the maximum CO association constant can be only ~2 X 10-4 M-1 s_1. Oxygen-pulse experiments provide the clearest kinetic evidence for cooperativity in oxygen binding. The rate constants for oxygen dissociation from singly oxygenated forms must be greater than 5 times those for the fully oxygenated hemoglobin. The oxygen dissociation and oxygen-pulse measurements cannot be accommodated within a heterogeneous Adair model. In terms of an allosteric model, the rate constant for R→ T or for T→R for intermediates such as αO2-β is small (ti/2 is tens of milliseconds), or the R⇄ T equilibrium constant for the half-liganded form is ~ 1. The kinetics of oxygen association following HbCO photolysis are unusual in that the rate of oxygen binding is less than first power in oxygen. A model is proposed to account for these results. In this model, binding of oxygen to the initial pho-toproduct is some 30-40-fold slower than that to a second form, derived from the first by a conformational change, the t1/2 for which is ~1 ms.",
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N2 - Oxygen and carbon monoxide equilibrium measurements have been made for the dimeric hemoglobin of Thyonella gemmata. For both ligands, the Hill number is ~ 1.4. The affinity for CO, however, is quite low, the median ligand activity at 20 °C, pH 7, being 1.3 μM. There is no apparent Bohr effect from pH 7 to pH 9. The replacement of 02 by CO gives an apparent value for M of 2.4. More unusual is the finding that this replacement reaction is also cooperative, the apparent Hill number being 1.4, suggesting that, for this hemoglobin, the HbO2 and HbCO conformations differ. HbO2 - HbCO difference spectra in the 290-nm region suggest that replacement of O2 by CO at the heme site is associated with the movement of a tryptophan from a nonpolar to a polar environment. The kinetics of oxygen dissociation from the hemoglobin of T. gemmata are biphasic. The dissociation rate constants for the hemes in the two polypeptide chains differ by a factor of about 5. Oxygen dissociation in the presence of CO is somewhat slower, but measurements at the HbO2-HbCO isosbestic wavelength show that a maximum of nearly 1/2 of the hemes are unliganded during the reaction. This shows that the predominant half-liganded oxyhemoglobin species does not provide a quickly reacting form for CO binding, since the maximum CO association constant can be only ~2 X 10-4 M-1 s_1. Oxygen-pulse experiments provide the clearest kinetic evidence for cooperativity in oxygen binding. The rate constants for oxygen dissociation from singly oxygenated forms must be greater than 5 times those for the fully oxygenated hemoglobin. The oxygen dissociation and oxygen-pulse measurements cannot be accommodated within a heterogeneous Adair model. In terms of an allosteric model, the rate constant for R→ T or for T→R for intermediates such as αO2-β is small (ti/2 is tens of milliseconds), or the R⇄ T equilibrium constant for the half-liganded form is ~ 1. The kinetics of oxygen association following HbCO photolysis are unusual in that the rate of oxygen binding is less than first power in oxygen. A model is proposed to account for these results. In this model, binding of oxygen to the initial pho-toproduct is some 30-40-fold slower than that to a second form, derived from the first by a conformational change, the t1/2 for which is ~1 ms.

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