Oxygen equilibrium and electron paramagnetic resonance studies on copper(II)‐iron(II) hybrid hemoglobins at room temperature
TL;DR: Electron paramagnetic resonance examinations on hybrid hemoglobins show that the interaction between copper(II) and the proximal histidine (F8) is specifically weakened in the α subunits within a low affinity conformation of hemoglobin, suggesting that copper (II) protoporphyrin IX is a useful EPR probe for investigating the deoxyheme environment in hemoglobin.
About: This article is published in FEBS Letters.The article was published on 1995-09-18 and is currently open access. It has received 11 citations till now. The article focuses on the topics: Copper & Electron paramagnetic resonance.
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TL;DR: These studies suggest the presence of two different environments of a tryptophan thereby revealing structural heterogeneity among the subunits and in support to previous findings by other spectroscopic studies on the same molecules.
69 citations
TL;DR: It is proposed that the classical T structure of deoxyhemoglobin in the crystals represents the lowest affinity state in solution, which is indicative of the existence of a distinct thermodynamic state which determines the lowest oxygen affinity of human hemoglobin.
47 citations
TL;DR: A new strategy is established for detecting chiral amino acids based on the electron transfer from hemoglobin Fe(II) to Cu( II) in copper complexes of the amino acids and shows a highly selective recognition of arginine enantiomers.
42 citations
TL;DR: Time-resolved resonance Raman spectroscopy was used to investigate intersubunit communication of hemoglobin using hybrid hemoglobin in which nickel was substituted for the heme iron in the β subunits, indicating the first real-time observation of the Perutz mechanism for allosteric control of oxygen binding in hemoglobin.
Abstract: Time-resolved resonance Raman spectroscopy was used to investigate intersubunit communication of hemoglobin using hybrid hemoglobin in which nickel was substituted for the heme iron in the β subunits. Changes in the resonance Raman spectra of the α heme and the β Ni–heme groups in the hybrid hemoglobin were observed upon CO photolysis in the α subunit using a probe pulse of 436 and 418 nm, respectively. Temporal evolution of the frequencies of the ν(Fe–His) and the γ7 band of α heme was similar to that of unsubstituted hemoglobin, suggesting that substitution with Ni–heme did not perturb the allosteric dynamics of the hemoglobin. In the β subunits, no structural change in the Ni–heme was observed until 1 μs. In the microsecond regime, temporal evolution of the frequencies of the ν(Ni–His) and the γ7 band of β Ni–heme was observed concomitant with an R → T quaternary change at about 20 μs. The changes in the ν(Fe–His) and ν(Ni–His) frequencies of the α and β subunits with the common time constant of ∼20 μs...
21 citations
TL;DR: From studies of dimer-tetramer equilibria of various pure and hybrid forms, it was concluded that a tetramer with two ligands bound on the same αβ dimer shows an enhanced tetramer stability, similar to singly liganded Hb, relative to the other three types of doubly liganding tetramers which resemble the triply liganded forms.
Abstract: While most researchers agree on the global features of cooperative ligand binding to haemoglobin (Hb), the internal mechanisms remain open to debate. This is not due to inaccurate measurements, but is rather a consequence of the cooperative ligand binding that decreases the equilibrium populations of the partially liganded states and makes observation of the transitions between these substates more difficult. For example, the equilibrium population of the doubly liganded tetramers is typically less than 5% of the total Hb. As a result many models with widely varying mechanisms may fit the oxygen equilibrium curve, but may not be consistent with observations of other parameters, such as ligand-binding kinetics or subunit association equilibria. The wide range of methods and models has led to divergent conclusions about the properties of specific substates. One notable debate concerns the properties of the doubly liganded forms. The simple two-state model predicts a shift in the allosteric equilibrium based on the number of ligands bound, but not on their distribution within the tetramer. From studies of dimer-tetramer equilibria of various pure and hybrid forms, it was concluded that a tetramer with two ligands bound on the same α
β dimer (species 21, an asymmetric hybrid) shows an enhanced tetramer stability, similar to singly liganded Hb, relative to the other three types of doubly liganded tetramers which resemble the triply liganded forms [Ackers et al. (1992), Science 255: 54–63]. The implications of this model and the relevant experiments will be reviewed here.
10 citations
References
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TL;DR: The oxygenation of haemoglobin is accompanied by structural changes in the subunits triggered by shifts of the iron atoms relative to the porphyrin and, in the β-subunits, also by the steric effect of oxygen itself.
Abstract: The oxygenation of haemoglobin is accompanied by structural changes in the subunits triggered by shifts of the iron atoms relative to the porphyrin and, in the β-subunits, also by the steric effect of oxygen itself. The oxygen-free form is constrained by salt-bridges which are broken by the energy of haem–haem interaction with the release of H+. 2,3-Diphosphoglycerate may add to the constraints by being stereochemically complementary to a site between the β-chains ; this complementarity is lost on oxygenation.
2,625 citations
TL;DR: An apparatus with which the continuous oxygen equilibrium curve of hemo globin can be recorded automatically was constructed and its performance was examined, finding it suitable for studies on the function of abnormal hemoglobins.
333 citations
TL;DR: Study of the effect of inositol hexaphosphate on the three low spin ferrous compounds of hemoglobin with O2, CO, and NO shows that the R leads to T transition causes either a rupture or at least a very dramatic stretching of the bond from the iron to the heme-linked histidine, such that an equilibrium is set up between five- and six-coordinated hemes.
Abstract: Studies of high spin ferrous and ferric derivatives led us to conclude that in the quaternary R structure the state of the hemes is similar to that in the free alpha and beta subunits, but in the T structure a tension acts on the hemes which tries to pull the iron and the proximal histidine further from the plane of the porphyrin. We have now studied the effect of inositol hexaphosphate (IHP) on the three low spin ferrous compounds of hemoglobin with O2, CO, and NO. IHP failed to switch the quaternary structure of carbonmonoxy- and oxyhemoglobin A to the T state, but merely caused a transition to an as yet undefined modification of the R structure. IHP is known to cause a switch to the T structure in hemoglobin Kansas. We have found that this switch induces red shifts of the visible alpha and beta absorption bands and the appearance of a shoulder on the red side of the alpha band; these changes are very weak in carbonmonoxy- and slightly stronger in oxyhemoglobin Kansas. As already noted by previous authors, addition of IHP to nitrosylhemoglobin A induces all the changes in uv absorption and CD spectra, sulfhydryl reactivities, and exchangeable proton resonances normally associated with the R leads to T transition, and is accompanied by large changes in the Soret and visible absorption bands. Experiments with nitrosyl hybrids show that these changes in absorption are caused predominantly by the hemes in the alpha subunits. In the accompanying paper Maxwell and Caughey (J. C. Maxwell and W. S. Caughey (1976), Biochemistry, following paper in this issue) report that the NO in nitrosylhemoglobin without IHP gives a single ir stretching frequency characteristic for six-coordinated nitrosyl hemes; addition of IHP causes the appearance of a second ir band, of intensity equal to that of the first, which is characteristic for five-coordinated nitrosyl hemes. Taken together, these results show that the R leads to T transition causes either a rupture or at least a very dramatic stretching of the bond from the iron to the heme-linked histidine, such that an equilibrium is set up between five- and six-coordinated hemes, biased toward five-coordinated hemes in the alpha and six-coordinated ones in the beta subunits. The reason why IHP can switch nitrosyl-, but not carbonmonoxy- or oxyhemoglobin A, from the R to the T structure is to be found in the weakening of the iron-histidine bond by the unpaired NO electron and by the very short Fe-NO bond length.
219 citations
205 citations
TL;DR: The results reported confirm the hypothesis that, under physiological conditions, haemoglobin binds CO(2) through the four terminal alpha-amino groups and that the two terminalalpha-aminos groups of alpha-chains are involved in the Bohr effect.
Abstract: 1. Three modified horse haemoglobins have been prepared: (i) αc2βc2, in which both the α-amino groups of the α- and β-chains have reacted with cyanate, (ii) αc2β2, in which the α-amino groups of the α-chains have reacted with cyanate, and (iii) α2βc2, in which the two α-amino groups of the β-chain have reacted with cyanate. 2. The values of n (the Hill constant) for αc2βc2, α2βc2 and αc2β2 were (respectively) 2.5, 2.0 and 2.6, indicating the presence of co-operative interactions between the haem groups for all derivatives. 3. In the alkaline pH range (about pH8.0) all the derivatives show the same charge as normal haemoglobin whereas in the acid pH range (about pH6.0) αc2βc2 differs by four protonic charges and αc2β2, α2βc2 by two protonic charges from normal haemoglobin, indicating that the expected number of ionizing groups have been removed. 4. αc2β2 and αc2βc2 show a 25% decrease in the alkaline Bohr effect, in contrast with α2βc2, which has the same Bohr effect as normal haemoglobin. 5. The deoxy form of αc2βc2 does not bind more CO2 than the oxy form of αc2βc2, whereas αc2β2 and α2βc2 show intermediate binding. 6. The results reported confirm the hypothesis that, under physiological conditions, haemoglobin binds CO2 through the four terminal α-amino groups and that the two terminal α-amino groups of α-chains are involved in the Bohr effect.
148 citations