Hemoglobin allostery: Variations on the theme

AbstractThe two-state allosteric model of Monod, Wyman and Changeux (1965) offers a simple and elegant, yet very powerful and comprehensive, description of the functional behavior of hemoglobin. Although the extensive body of structural and functional information available is by-and-large consistent with this conceptual framework, some discrepancies between theory and experiment have been extensively discussed and considered to demand modifications of the original hypothesis. More recently the role of tertiary structural changes has been re-analyzed leading to extended kinetic models or indicating that powerful heterotropic effectors may be of paramount importance in controlling the function of human hemoglobin. The aim of this review is to analyze, and possibly reconcile, some discrepancies. We always felt that by looking at hemoglobins other than human HbA, the relative role of tertiary and quaternary allosteric effects may be better understood. The model systems illustrated below are the different hemoglobins from trout's blood, since they are characterized by the most striking variability of heterotropic effects, ranging from totally absent to very extreme with dominant contributions of tertiary effects. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins

Kinetics of the Reaction of Hemoglobin with Ethylisocyanide: INTERPRETATION OF THE RESULTS WITHIN A DIMER SCHEME

Abstract The kinetics of the reaction of human deoxyhemoglobin with ethylisocyanide has been studied, by rapid mixing, over a 50- to 100-fold range of ligand concentration, both as a function of protein concentration (from 3 to 30 x 10-6 m) and ionic strength (from 0.2 to 2.2 m). The results show that the progress curve, which is autocatalytic at high ligand concentration, tends to change shape as the ethylisocyanide concentration is decreased, and finally becomes markedly diphasic. The experimental results can be fitted satisfactorily with a simple dimer scheme, with only two combination and two dissociation velocity constants. Consideration of these results, in conjunction with other data, allows us to arrive at important conclusions concerning the kinetic origin of co-operativity as observed at equilibrium. The most significant of these is that, to a major degree, cooperative ligand binding finds its kinetic justification in a large decrease of the dissociation velocity constant as the reaction proceeds

Kinetics of the reaction with oxygen of mixtures of oxy- and carbon monoxide hemoglobin.

Abstract The paper reports rapid mixing and relaxation experiments performed on mixtures of oxy- (HbO2) and carbon monoxide (HbCO) human hemoglobin. On the (well justified) assumption that the two ligands will distribute at random between the available sites, intermediates containing different proportions of O2 and CO will be formed. In the stopped flow experiments mixtures containing different proportions of the two ligands have been mixed with sodium dithionite (Na2S2O4), which rapidly reduces to zero the O2 concentration in the system. The apparent dissociation velocity constant for O2 (koff) measured under these conditions decreases progressively as the fraction of HbCO in the mixture increases, in agreement with previous observations on sheep hemoglobin. Temperature jump experiments performed on mixtures of HbCO and HbO2 show that the amplitude of the faster relaxation time (τf) relative to that of the slower one (τf) increases as the percentage of HbCO in the mixture is progressively increased. At high enough percentage of HbCO (≥70%), the amplitude of the faster relaxation time becomes dominant. The reciprocal relaxation time (τf-1), measured under these conditions, is linearly dependent on oxygen concentration, while it is independent of protein concentration (so long as O2 is buffered). The apparent second order velocity constant is kon = 4.8 x 107 m-1 s-1 at 25°. Simple considerations indicate that the kinetics of the reaction with oxygen of mixtures containing high enough percentages of HbCO should represent the combination and dissociation velocity constants of high affinity forms of hemoglobin

Modulation of allosteric control and evolution of hemoglobin

Allostery arises when a ligand-induced change in shape of a binding site of a protein is coupled to a tertiary/quaternary conformational change with a consequent modulation of functional properties. The two-state allosteric model of Monod, Wyman and Changeux [J. Mol. Biol. 1965; 12, 88-118] is an elegant and effective theory to account for protein regulation and control. Tetrameric hemoglobin (Hb), the oxygen transporter of all vertebrates, has been for decades the ideal system to test for the validity of the MWC theory. The small ligands affecting Hb's behavior (organic phosphates, protons, bicarbonate) are produced by the red blood cell during metabolism. By binding to specific sites, these messengers make Hb sensing the environment and reacting consequently. HbI and HbIV from trout and human HbA are classical cooperative models, being similar yet different. They share many fundamental features, starting with the globin fold and the quaternary assembly, and reversible cooperative O2 binding. Nevertheless, they differ in ligand affinity, binding of allosteric effectors, and stability of the quaternary assembly. Here, we recollect essential functional properties and correlate them to the tertiary and quaternary structures available in the protein databank to infer on the molecular basis of the evolution of oxygen transporters

Studies on the Relations between Molecular and Functional Properties of Hemoglobin: VII. KINETIC EFFECTS OF THE REVERSIBLE DISSOCIATION OF HEMOGLOBIN INTO SINGLE CHAIN MOLECULES

Abstract The kinetics of the reactions of human hemoglobin with carbon monoxide and oxygen has been studied in photochemical and rapid mixing experiments over a large range of hemoglobin concentration. When the reaction is initiated by rapid removal of the ligand from ligand-bound hemoglobin, the kinetics of the combination of hemoglobin with CO shows a marked concentration dependence in both the photochemical and the rapid mixing experiments. In dilute hemoglobin solutions (below 10-5 m in heme), dissociation of the ligand from oxyhemoglobin or carbonmonoxyhemoglobin is followed by slow changes (half-time of the order of seconds) in the properties of the system. These results lead to the following picture, which is also consistent with other as yet unexplained aspects of hemoglobin kinetics. (a) Ligand-bound hemoglobin dissociates reversibly into single chain molecules at concentrations below 10-5 m. (b) Deoxygenated hemoglobin has a much lower tendency to dissociate into single chain molecules, and there is no appreciable dissociation even at concentrations of the order of 10-6 to 10-7 m. (c) The association of deoxygenated α and β chains is a relatively slow process. Therefore, after sudden dissociation of the ligand from dilute hemoglobin solutions, the properties of the system, for a brief time, are those of a mixture of deoxygenated hemoglobin and deoxygenated α and β chains. (d) The properties of the single chain molecules obtained by dilution of ligand-bound hemoglobin are the same as those of isolated α and β hemoglobin chains as obtained by preparative procedures

Properties of modified cytochromes. I. Equilibrium and kinetics of the pH-dependent transition in carboxymethylated horse heart cytochrome c.

Reduced (Fe+2) carboxymethylated cytochrome c, Cm-cyt. c, undergoes a reversible pH-dependent transition with a pK of 7.16 at 20°. This pK is found to be nearly temperature-independent indicating that the over-all enthalpy for the transition is close to zero. The kinetics of this transition have been investigated by the temperature jump technique. A single well resolved relaxation process (in the millisecond time range) is observed over the pH region of the static titrations. The amplitude of this relaxation at different wave lengths fits the statically derived difference spectrum between the alkaline and acid forms of the protein. Both the amplitude and the relaxation time τ are pH dependent; the over-all enthalpy of the process is estimated to be ∼ +1 Cals per mole. The observed behavior may be accounted for by a model in which a proton-linked conformational change in the protein is responsible for the spectral changes. It is suggested that the deprotonation of an e-amino group of lysine (possibly that of lysine 79) is followed by the binding of this group to the ferrous iron to fill the vacant sixth coordination position. The observed spectral changes are attributed to the binding of a nitrogen atom to the iron. The thermodynamic parameters governing the conformational part of the reaction are calculated on the basis of the above model and values of ΔH = -10 Cals per mole and ΔS = -20 e.u. are found. These values are discussed in the context of the binding of protein residue to the iron and the consequent changes in the crevice structure

Properties of the Product of Partial Photodissociation of Carbon Monoxide Hemoglobin

Abstract Some properties of the quickly reacting hemoglobin generated by partial photodissociation of human CO hemoglobin have been studied using flash photolysis methods. The absorption spectrum of the rapidly reacting photoproduct is different from that of slowly reacting "normal" hemoglobin and, similarly to other quickly reacting forms, corresponds to that of isolated deoxyhemoglobin chains. The fraction of the fast reacting material increases linearly with the fraction of nonphotodissociated hemoglobin. On the basis of these results and of other information on hemoglobin kinetics it is suggested that this fast reacting species represents partially saturated intermediate which appears transiently when the ligand is suddenly removed from ligand-bound hemoglobin

Nitric oxide, cytochrome c oxidase and myoglobin: Competition and reaction pathways

AbstractIt is relevant to cell physiology that nitric oxide (NO) reacts with both cytochrome oxidase (CcOX) and oxygenated myoglobin (MbO2). In this respect, it has been proposed [Pearce, L.L., et al. (2002) J. Biol. Chem. 277, 13556–13562] that (i) CcOX in turnover out-competes MbO2 for NO, and (ii) NO bound to reduced CcOX is “metabolized” in the active site to nitrite by reacting with O2. In contrast, rapid kinetics experiments reported in this study show that (i) upon mixing NO with MbO2 and CcOX in turnover, MbO2 out-competes the oxidase for NO and (ii) after mixing nitrosylated CcOX with O2 in the presence of MbO2, NO (and not nitrite) dissociates from the enzyme causing myoglobin oxidation

Folding and Misfolding in a Naturally Occurring Circularly Permuted PDZ Domain

One of the most extreme and fascinating examples of naturally occurring mutagenesis is represented by circular permutation. Circular permutations involve the linking of two chain ends and cleavage at another site. Here we report the first description of the folding mechanism of a naturally occurring circularly permuted protein, a PDZ domain from the green alga Scenedesmus obliquus. Data reveal that the folding of the permuted protein is characterized by the presence of a low energy off-pathway kinetic trap. This finding contrasts with what was previously observed for canonical PDZ domains that, although displaying a similar primary structure when structurally re-aligned, fold via an on-pathway productive intermediate. Although circular permutation of PDZ domains may be necessary for a correct orientation of their functional sites in multi-domain protein scaffolds, such structural rearrangement may compromise their folding pathway. This study provides a straightforward example of the divergent demands of folding and function