Deoxy Quaternary Structure Research Articles

Recombinant hemoglobins with low oxygen affinity and high cooperativity

By introducing an additional H-bond in the α 1β 2 subunit interface or altering the charge properties of the amino acid residues in the α 1β 1 subunit interface of the hemoglobin molecule, we have designed and expressed recombinant hemoglobins (rHbs) with low oxygen affinity and high cooperativity. Oxygen-binding measurements of these rHbs under various experimental conditions show interesting properties in response to pH (Bohr effect) and allosteric effectors. Proton nuclear magnetic resonance studies show that these rHbs can switch from the oxy (or CO) quaternary structure (R) to the deoxy quaternary structure (T) without changing their ligation states upon addition of an allosteric effector, inositol hexaphosphate, and/or reduction of the ambient temperature. These results indicate that if we can provide extra stability to the T state of the hemoglobin molecule without perturbing its R state, we can produce hemoglobins with low oxygen affinity and high cooperativity. Some of these rHbs are also quite stable against autoxidation compared to many of the known abnormal hemoglobins with altered oxygen affinity and cooperativity. These results have provided new insights into the structure–function relationship in hemoglobin.

An additional H-bond in the alpha 1 beta 2 interface as the structural basis for the low oxygen affinity and high cooperativity of a novel recombinant hemoglobin (beta L105W).

Site-directed mutagenesis has been used to construct three recombinant mutant hemoglobins (rHbs), rHb(beta L105W), rHb(alpha D94A/betaL105W), and rHb(alpha D94A). rHb(beta L105W) is designed to form a new hydrogen bond from beta 105Trp to alpha 94Asp in the alpha(1)beta(2) subunit interface to lower the oxygen binding affinity by stabilizing the deoxy quaternary structure. We have found that rHb(beta L105W) does indeed possess a very low oxygen affinity and maintains normal cooperativity (P(50) = 28.2 mmHg, n(max) = 2.6 in 0.1 M sodium phosphate at pH 7.4) compared to those of Hb A (P(50) = 9.9 mmHg, n(max) = 3.2 at pH 7.4). rHb(alpha D94A/beta L105W) and rHb(alpha D94A) are expressed to provide evidence that rHb(betaL 105W) does form a new H-bond from beta 105Trp to alpha 94Asp in the alpha(1)beta(2) subunit interface of the deoxy quaternary structure. Our multinuclear, multidimensional nuclear magnetic resonance (NMR) studies on (15)N-labeled rHb(beta L105W) have identified the indole nitrogen-attached (1)H resonance of beta 105Trp for rHb(beta L105W). (1)H NMR studies on Hb A and mutant rHbs have been used to investigate the structural basis for the low O(2) affinity of rHb(beta L105W). Our NMR results provide evidence that rHb(beta L105W) forms a new H-bond from beta 105Trp to alpha 94Asp in the alpha(1)beta(2) subunit interface of the deoxy quaternary structure. The NMR results also show that these three rHbs can switch from the R quaternary structure to the T quaternary structure in their ligated state upon addition of an allosteric effector, inositol hexaphosphate. We propose that the low O(2) affinity of rHb(beta L105W) is due to the formation of a new H-bond between alpha 105Trp and alpha 94Asp in the deoxy quaternary structure.

Effects of substitutions of lysine and aspartic acid for asparagine at beta 108 and of tryptophan for valine at alpha 96 on the structural and functional properties of human normal adult hemoglobin: roles of alpha 1 beta 1 and alpha 1 beta 2 subunit interfaces in the cooperative oxygenation process.

Using our Escherichia coli expression system, we have produced five mutant recombinant (r) hemoglobins (Hbs): r Hb (alpha V96 W), r Hb Presbyterian (beta N108K), r Hb Yoshizuka (beta N108D), r Hb (alpha V96W, beta N108K), and r Hb (alpha V96W, beta N108D). These r Hbs allow us to investigate the effect on the structure-function relationship of Hb of replacing beta 108Asn by either a positively charged Lys or a negatively charged Asp as well as the effect of replacing alpha 96Val by a bulky, nonpolar Trp. We have conducted oxygen-binding studies to investigate the effect of several allosteric effectors on the oxygenation properties and the Bohr effects of these r Hbs. The oxygen affinity of these mutants is lower than that of human normal adult hemoglobin (Hb A) under various experimental conditions. The oxygen affinity of r Hb Yoshizuka is insensitive to changes in chloride concentration, whereas the oxygen affinity of r Hb Presbyterian exhibits a pronounced chloride effect. r Hb Presbyterian has the largest Bohr effect, followed by Hb A, r Hb (alpha V96W), and r Hb Yoshizuka. Thus, the amino acid substitution in the central cavity that increases the net positive charge enhances the Bohr effect. Proton nuclear magnetic resonance studies demonstrate that these r Hbs can switch from the R quaternary structure to the T quaternary structure without changing their ligation states upon the addition of an allosteric effector, inositol hexaphosphate, and/or by reducing the temperature. r Hb (alpha V96W, beta N108K), which has the lowest oxygen affinity among the hemoglobins studied, has the greatest tendency to switch to the T quaternary structure. The following conclusions can be derived from our results: First, if we can stabilize the deoxy (T) quaternary structure of a hemoglobin molecule without perturbing its oxy (R) quaternary structure, we will have a hemoglobin with low oxygen affinity and high cooperativity. Second, an alteration of the charge distribution by amino acid substitutions in the alpha 1 beta 1 subunit interface and in the central cavity of the hemoglobin molecule can influence the Bohr effect. Third, an amino acid substitution in the alpha 1 beta 1 subunit interface can affect both the oxygen affinity and cooperativity of the oxygenation process. There is communication between the alpha 1 beta 1 and alpha 1 beta 2 subunit interfaces during the oxygenation process. Fourth, there is considerable cooperativity in the oxygenation process in the T-state of the hemoglobin molecule.

Asymmetric cyanomet valency hybrid hemoglobin, (alpha+CN-beta+CN-)(alpha beta): the issue of valency exchange.

A new framework for hemoglobin cooperativity was proposed by Ackers and colleagues on the basis of the hyper thermodynamic stability and deoxy (T) quaternary structure of one of diliganded deoxy-cyanomet hybrid hemoglobins, (alpha+CN-beta+CN-)(alpha beta), studied by hybridization of the equimolar mixture of deoxyhemoglobin and cyanomethemoglobin through a long (70-100 h) dimer exchange reaction [Daugherty et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1110-1114]. Recently, we reported that the published hyperstability of (alpha+CN-beta+CN-)(alpha beta) is incorrect due to the occurrence of valency exchange between the heme sites of both parental hemoglobins during the long deoxy incubation [Shibayama et al. (1997) Biochemistry 36, 4375-4381]. We also noted a difficulty in maintaining both anaerobicity and excess free cyanide of the sample during the long incubation, which led to formation of cyanide-unbound aqometheme in the original deoxyhemoglobin resulting from the electron transfer to cyanometheme. This paper is a response to a recent argument against our work [Ackers et al. (1997) Biochemistry 36, 10822-10829]. Ackers et al. have claimed that no appreciable formation of aqomethemoglobin with their methods ensures their sample integrity, based on a supposition that our observed valency exchange may have occurred via aqometheme. In this paper, however, we demonstrate that appreciable (>27%) valency exchange really occurs between deoxy and cyanometheme sites during 72 h incubation under conditions where both anaerobicity and excess free cyanide of the sample solution are maintained by a continuous flow of humidified N2 with HCN. This confirms our view that previous experimental data on (alpha+CN-beta+CN-)(alpha beta) obtained by the long incubations should be subject to reexamination while our earlier estimation of a lower limit of free energy of (alpha+CN-beta+CN-)(alpha beta) (i.e., >/= -10.1 kcal/mol) by a rapid method (35 min) is still valid. We also suggest a possibility that the T quaternary structure of (alpha+CN-beta+CN-)(alpha beta) assigned by Ackers and colleagues using the long incubations is an artifact arising from the valency exchange. These results suggest that the putative mechanistic picture for hemoglobin cooperativity inferred from studies on deoxy-cyanomet hybrids is without foundation.

The polymerization of sickle hemoglobin in solutions and cells.

The polymerization of sickle hemoglobin occurs by the same mechanisms in solutions and in cells, and involves the formation of 14 stranded fibers from hemoglobin molecules which have assumed a deoxy quaternary structure. The fibers form via two types of highly concentration-dependent nucleation processes: homogeneous nucleation in solutions with hemoglobin activity above a critical activity, and heterogeneous nucleation in similarly supersaturated solutions which also contain hemoglobin polymers. The latter pathway is dominant, and creates polymer arrays called domains. The individual polymers bend, but also cross-link, and the resulting mass behaves as a solid. The concentration of polymerized hemoglobin increases exponentially unless clamped by rate limiting effects such as oxygen delivery.

Identification of the intermediate allosteric species in human hemoglobin reveals a molecular code for cooperative switching.

The 10 ligation species of human cyanomethemoglobin were previously found to distribute into three discrete cooperative free energy levels according to a combinatorial code (i.e., dependent on both the number and configuration of ligated subunits). Analysis of this distribution showed that the hemoglobin tetramer occupies a third allosteric state in addition to those of the unligated (T) and fully ligated (R) species. To determine the nature of the intermediate allosteric state, we have studied the effects of pH, temperature, and single-site mutations on its free energy of quaternary assembly, in parallel with corresponding data on the deoxy (T) and fully ligated (R) species. Results indicate that the intermediate allosteric tetramer has the deoxy (T) quaternary structure. This finding, together with the resolved energetic distribution of the 10 microstates reveals a symmetry rule for quaternary switching--i.e., switching from T to R occurs whenever a binding step creates a tetramer with one or more ligated subunits on each side of the alpha 1 beta 2 intersubunit contact. These studies also reveal significant cooperativity within each alpha 1 beta 1 dimer of the T-state tetramer. The ligand-induced tertiary free energy alters binding affinity within the T structure by 170-fold prior to quaternary switching.

Analysis of proton release in oxygen binding by hemoglobin: implications for the cooperative mechanism.

The relationship in hemoglobin between cooperativity (dependence of the Hill constant on pH0 and the Bohr effect (dependence of the mean oxygen affinity on pH) can be described by a statistical thermodynamic model [Szabo, A., & Karplus, M. (1972) J. Mol. Biol. 72, 163-197; Lee, A., & Karplus, M. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 7055-759]. In this model, salt bridges and other interactions serve to couple tertiary and quaternary structural changes. To test and refine the model, it is applied to the analysis of the pH dependence of the tetramer Adair constants corrected for statistical factors (K4i', i = 1-4). Attention is focused on the proton release of the first (delta H1+ = alpha log K41'/alpha pH) and last (delta H4+ = alpha log K44'/alpha pH) oxygenation steps, where K4i' are the Adair constants corrected for statistical factors. Measurements of delta H1+ and delta H4+ under carefully controlled conditions are reported, and good agreement between the model calculation and these experimental results is obtained. The salt bridges are found to be partially coupled to the ligation state in the deoxy quaternary structure; it is shown that a Monod-Wyman-Changeux-type model, in which the salt bridges are coupled only to quaternary structural change, is inconsistent with the data for delta H1. The significance of the present analysis for an evaluation of the Perutz mechanism [Perutz, M.F. (1970) Nature (London) 228, 726-734, 734-739] and other models for hemoglobin cooperativity is discussed.

Hemoglobin tertiary structural change on ligand binding its role in the co-operative mechanism

Analysis of the tertiary structural alterations in hemoglobin induced by ligand binding demonstrates that an allosteric core composed of the heme, histidine F8, the FG corner and part of the F-helix plays an essential role in co-operativity. This conclusion is based on structural and spectroscopic data and theoretical studies of hemoglobin chains. The methodology employed in the calculations is presented with details of the empirical energy function. Energy minimized structures of the unliganded hemoglobin chains, which serve as reference systems for the analysis, are described. To determine the structural changes induced by ligand binding, the effects of FeN bond shortening and of heme translation and tilting perturbations are examined. Energy minimization in the presence of the perturbations serves to provide information concerning the globin structural modifications produced by them. The validity of the results is supported by comparisons with the X-ray data of Anderson, Pulsinelli, Baldwin and Chothia on tertiary changes in the hemoglobin subunits. Internal to the allosteric core, there appear to be two stable positions for its elements: one of these corresponds to the liganded and the other to the unliganded species. The unliganded geometry fits without strain into the deoxy tetramer, while the liganded one fits without strain into the oxy tetramer. On ligation of a subunit in the deoxy tetramer, the structural changes within the allosteric core are in the direction of those found in going from the unliganded deoxy to the liganded oxy system, although they are reduced by the presence of constraints due to the other subunits in the deoxy tetramer. In addition, the quaternary constraints in the deoxy tetramer prevent the large overall displacement of the allosteric core that occurs in the transition to the liganded oxy tetramer. The coupling between the changes internal to the allosteric core, produced on ligation and the overall displacement of the core that accompanies the quaternary transition, is an essential element of the co-operative mechanism. As shown in previous work (Gelin & Karplus, 1977), the proximal histidine serves as the link between the position of the heme and the F-helix; the asymmetric orientation of the histidine in the deoxy structure, coupled with contributions from other heme-protein interactions, appears to initiate the tertiary structural changes induced by ligand binding. The reduced oxygen affinity of hemoglobin results not from tension on the heme in the unliganded structure (there is none) but instead from strain in the liganded subunit of the tetramer within the deoxy quaternary structure. Further, the changes in the allosteric core provide a relatively localized reaction path for transmitting information concerning ligand binding from the heme group to the surface of the subunit; particularly in the α-chain, the residue Val FG5 appears to play an important role in the reaction path. The present analysis has important implications for realistic statistical thermodynamic models of hemoglobin co-operativity. It suggests that the previously formulated model (Szabo & Karplus, 1972) should be generalized by the introduction of two different subunit tertiary structures in the deoxy and in the oxy tetramer; they would be associated with the unliganded and the liganded allosteric core, respectively, and would take account of steric constraints that reduce the ligand affinity of the deoxy tetramer.

Oxygen equilibrium studies on hemoglobin from the bluefin tuna ( Thunnus thynnus)

Oxygen equilibrium curves of the purified hemoglobin component I from the Atlantic bluefin tuna (Thunnus thynnus) have been determined between pH 6.5 and 8.75 at 25 degrees C, and for five temperatures between 10 and 30 degrees C at pH 7.0 and 7.5. From the equilibrium data oxygen equilibrium constants for four oxygenation steps, Ki (i = 1 to 4) were estimated. The number of the Bohr protons released on the ith oxygenation (delta Hi+), and the enthalpy and entropy changes at each oxygenation step (delta Hi and delta Si) were calculated. The Hill plot for oxygenation below neutral pH is biphasic; the top asymptote lies to the right of the bottom one and the linking limb between them exhibits a slope less than unity, exhibiting apparent negative co-operativity. The values of K1 and K2 exhibit little pH dependence, while those of K3 and K4 increase by two orders of magnitudes as the pH is changed from 6.5 to 8.75. In consequence, oxygen equilibrium above neutral pH exhibits a normal positive co-operativity. The oxygen equilibrium at lower temperature is biphasic as is that below neutral pH. The shape of the Hill plot is temperature-dependent. The affinity at low saturation decreases, and that at high saturation increases upon raising the temperature from 10 to 30 degrees C, resulting in crossing of the middle portion of the equilibrium curves at different temperatures. The delta H1 and delta H2 values are negative as are those of most other hemoglobins, but the delta H3 and delta H4 values are positive. Consideration of these results in a framework of the allosteric model extended to take account of differences between subunits has indicated that the deoxy quaternary structure is stabilized at low pH or low temperature, and that subunit heterogeneity gives rise to the biphasic oxygen equilibrium curve. An analysis of delta Hi+ suggests that the large number of the Bohr groups is responsible for the biased allosteric equilibrium towards the deoxy quaternary structure. The positive delta H3 and delta H4 values are also considered to arise from the large endothermic contribution of the Bohr protons released at the third and fourth steps of oxygenation.

Thermodynamic analysis of human hemoglobins in terms of the Perutz mechanism: extensions of the Szabo--Karplus model to include subunit assembly.

The stereochemical postulates of Perutz for the mechanism of hemoglobin [Perutz, M. F. (1970) Nature (London) 228, 726--739] have been formulated into a statistical thermodynamic model. The model is based on that of Szabo and Karplus [Szabo, A., & Karplus, M. (1972) J. Mol. Biol 72, 163--197] but has been extended to include the properties of dissociated dimers in equilibrium with tetramers. The dissociation eliminates the alpha 1 beta 2 intersubunit contact which is the major site of ligand-linked structure change. The model quantitatively describes the coupling between binding of oxygen and protons in dimers and tetramers, the change in quaternary structure, and the breaking of salt bridges which are assumed to stabilize the deoxy quaternary structure. The extended model has been tested against an extensive series of recent experimental data from our laboratory and elsewhere on the ligand-linked dimer-tetramer assembly in normal human hemoglobin A and in the variant hemoglobin Kansas (beta 102 Asp leads to Asn). Two versions of the model were used which differ in the properties of the dissociated dimers. For both hemoglobins, the models were found capable of simultaneously describing the data on the ligand-linked dimer-tetramer assembly and predicting the tetramer Bohr effect. However, neither model predicted reasonable values for the tetramer Bohr effect without simultaneously predicting unreasonable values for the affinities of individual chains. Both models incorrectly predict preferential binding of oxygen to the alpha or beta chains within the tetramer. These results argue against the Perutz mechanism for the molecular processes of hemoglobin.

Mutual effects of protons, NaCl, and oxygen on the dimer-tetramer assembly of human hemoglobin. The dimer Bohr effect.

The dimer-tetramer equilibrium constants of human oxyhemoglobin (4K2) and deoxyhemoglobin (0K2) have been determined at 21.5 degrees C as a function of pH and chloride concentration. In buffers containing 0.1 M NaCl, 1 mM EDTA, the apparent numbers of protons released upon assembly of dimers into tetramers were determined from the pH dependencies of 4K2 and 0K2. At pH 7.4, the assembly of unliganded tetramers is accompanied by the absorption of 0.9 +/- 0.1 mol of H+. For assenbly of oxyhemoglobin, there are 0.8 +/- 0.1 mol of H+ released per mol of tetramer formed. From these results and the value of 2.1 mol of H+/402 for the tetramer Bohr effect, a Bohr effect for dimers is determined as 0.2 +/- .08 mol of H+ released upon binding 2 mol of 02. Thus, the dimer Bohr effect is approximately 20% as large as the tetramer Bohr effect. At pH 7.4, the value of 0K2 is insensitive to [Cl-], whereas 4K2 varies inversely with [Cl-]. At pH 8.95, both 4K2 and 0K2 decrease with increasing [Cl-]. These and previous results indicate that salt bridges are not the dominant energetic factor in stabilizing the deoxy quaternary structure of hemoglobin. In buffer conditions of 0.1 M Tris-HCl, 0.1 M NaCl, 1 mM EDTA, pH 7.4, we estimate 1.8 mol of Cl- bound upon dissociation of 1 mol of oxy tetramers into oxy dimers, whereas the deoxy molecules dissociate without any change in bound chloride. From the [Cl-] dependence of oxygenation curves, we estimate 1.8 mol of [Cl-] released upon binding 4 mol of O2 to tetramers. Thus, oxygenation of dimers at pH 7.4 apparently involves no change in bound chloride.

Energetics of subunit assembly and ligand binding in human hemoglobin

An extensive and self-consistent set of thermodynamic properties has recently been established for the coupled processes of subunit assembly and ligand binding (oxygen and protons) in human hemoglobin. The resulting thermodynamic values permit a consideration of the possible sources of energetic terms accounting for stability of the tetrameric quaternary structures at different stages of ligation, and of the possible sources of cooperative energy. The analysis indicates that: (a) The change in buried surface ara upon oxygenation (i.e., hydrophobic stabilization) does not play a dominant role in stabilizing the unliganded tetramer relative to the liganded tetramer. (b) The pattern of enthalpic and entropic contributions to the free energies of dimer-tetramer. (c) The thermodynamic results are consistent with a dominant role of increased hydrogen bond formation in the deoxy quaternary structure. (d) Within tetramers the variation in free energy for successive oxygenation steps arises from both enthalpic and entropic contributions and the enthalpic contributions are almost entirely attributable to the heats of Bohr proton release. At pH 7.4 the pattern of thermodynamic values suggests that a large contribution to the free energy of cooperativity may arise from the energetics of Bohr proton release. It is suggested that a combination of proton ionization and hydrogen bonding may account for the main energetic features of cooperativity. Possible contributions from fluctuation behavior cannot presently be evaluated.

Proton nuclear magnetic resonance and biochemical studies of oxygenation of human adult hemoglobin in deuterium oxide.

The proton nuclear magnetic resonance spectrum of human adult deoxyhemoglobin in D2O in the region from 6 to 20 ppm downfield from the proton resonance of residual water shows a number of hyperfine shifted proton resonances that are due to groups on or near the alpha and beta hemes. The sensitivity of these resonances to the ligation of the heme groups and the assignment of these resonances to the alpha and beta chains provide an opportunity to investigate the cooperative oxygenation of an intact hemoglobin molecule in solution. By use of the nuclear magnetic resonance correlation spectroscopy technique, at least two resonances, one at approximately 18 ppm downfield from HDO due to the beta chain and the other at approximately 12 ppm due to the alpha chain, can be used to study the binding of oxygen to the alpha and beta chains of hemoglobin. The present results using approximately 12% hemoglobin concentration in 0.1 M Bistris buffer at pD 7 and 27 degrees C with and without organic phosphate show that there is no significant line broadening on oxygenation (from 0 to 50% saturation) to affect the determination of the intensities or areas of these resonances. It is found that the ratio of the intensity of the alpha-heme resonance at 12 ppm to that of the beta-heme resonance at 18 ppm is constant on oxygenation in the absence of organic phosphate but decreases in the presence of 2,3-diphosphoglycerate or inositol hexaphosphate, with the effect of the latter being the stronger. On oxygenation, the intensities of the alpha-heme resonance at 12 ppm and of the beta-heme resonance at 18 ppm decreases more than the total number of deoxy chains available as measured by the degree of O2 saturation of hemoglobin. This shows the sensitivity of these resonances to structural changes which are believed to occur in the unligated subunits upon the ligation of their neighbors in an intact tetrameric hemoglobin molecule. A comparison of the nuclear magnetic resonance data with the populations of the partially saturated hemoglobin tetramers (i.e., hemoglobin with one, two, or three oxygen molecules bound) leads to the conclusion that in the presence of organic phosphate the hemoglobin molecule with one oxygen bound maintains the beta-heme resonance at 18 ppm but not the alpha-heme resonance at 12 ppm. These resluts suggest that some cooperativity must exist in the deoxy quaternary structure of the hemoglobin molecule during the oxygenation process. Hence, these results are not consistent with the requirements of two-state concerted models for the oxygenation of hemoglobin. In addition, we have investigated the effect of D2O on the oxygenation of hemoglobin by measuring the oxygen dissociation curves of normal adult hemoglobin as a function of pH in D2O andH2O media. We have found that (1) the pH dependence of the oxygen equilibrium of hemoglobin (the Bohr effect) in higher pH in comparison to that in H2O medium and (2) the Hill coefficients are essentially the same in D2O and H2O media over the pH range from 6.0 to 8.2...

Cobalt-substituted hemoglobin Zürich ( α2β263His→Arg). Oxygen equilibria and EPR spectra

Cobalt hemoglobin Zürich ( α 2 β 2 63His→Arg) has been successfully reconstituted from the apohemoglobin Zürich and cobaltous protoporphyrin IX. The oxygen affinity of cobalt hemoglobin Zurich, as well as that of iron hemoglobin Zürich, were measured in the absence and presence of organic phosphate and Cl −. The overall oxygen affinity of cobalt hemoglobin Zürich was found to be higher and the cooperativity as measured by the n value was smaller than those of cobalt hemoglobin A. Organic phosphate and Cl − affect the oxygen equilibrium properties of cobalt hemoglobin Zürich in a manner similar to that of cobalt hemoglobin A, but to a lesser extant than cobalt hemoglobin A. The EPR spectrum of oxy cobalt hemoglobin Zürich is less sensitive to the replacement of the buffer system from H 2O to 2H 2O, indicating that the hydrogen bond between the distal amino acid residue and the bound oxygen is not formed in the abnormal β subunits. The deoxy EPR spectrum of cobalt hemoglobin Zürich is similar to that of deoxy cobalt hemoglobin A, suggesting that the deoxy cobalt hemoglobin Zürich is predominantly in the deoxy quaternary structure (T state).

Haemoglobin: The structural changes related to ligand binding and its allosteric mechanism

The structural changes that occur on ligand binding to haemoglobin have been studied by comparison of the atomic co-ordinates of human deoxy, horse met and human carbonmonoxy haemoglobin, using computer graphics and least-squares fitting methods. The changes that occur on going from deoxy to either of the liganded forms are very similar. These include tertiary structure changes within the α 1 β 1 dimer and a quaternary structure change in which the packing of α 1 β 1 against α 2 β 2 alters. On going from deoxy to liganded haemoglobin, no significant structural change occurs in the central regions of the α 1 β 1 dimer, including the α 1 β 1 interface and nearby helices B, C, G and H in both subunits. Movements occur in the outer parts of the dimer, where the haems, F helices and FG corners of both subunits move towards the centre of the molecule. The two haems and the two FG corners come ~2 Å closer together. One important effect of the changes in both subunits is to translate the F helix across the face of the haem by ~1 Å. This moves the haem-linked histidine F8 from a position that is asymmetric with respect to the porphyrin nitrogens in deoxy to a more symmetric position in liganded haemoglobin. The motion of the β haem removes the ligand-binding site from the vicinity of ValβE11, which hinders ligand binding in deoxy. The changes in tertiary structure are linked to the quaternary change through the motion of the FG corners. The C helices and FG corners of α 1 β 1 are in contact with the FG corners and C helices of α 2 β 2 in both quaternary structures. In the quaternary change the contacts between α 1FG and β 2C and between α 2FG and β 1C act as “flexible joints” allowing small relative motions. The same side-chains are involved in the contacts in both structures. The contacts between α 1C and β 2FG and between α 2C and β 1FG act as “switch” regions, having two different stable positions with different side-chains in contact. The change between the two positions involves a relative movement of ~6 Å. The quaternary structure change to liganded haemoglobin destroys the contacts made by the C-terminal residues of each subunit in deoxy haemoglobin, and these residues rotate freely in the liganded form. These structural results, together with other work, particularly the calculations of Gelin & Karplus and of Warshel, support a description of the haemoglobin mechanism in which (1) the binding of ligand to the deoxy form is accompanied by steric strain, originating from the particular position of the F helix and of His F8 relative to the haem. (2) The strain leads to decreased stability of the deoxy quaternary structure relative to the liganded quaternary structure, so that the proportion of molecules in the high-affinity form increases as successive ligands bind. (3) The quaternary structure change to the high-affinity form induces tertiary structure changes that reposition the F helix and HisF8 relative to the haem and there is then no strain on ligand binding. In the absence of ligand the deoxy structure is favoured by the greater surface area buried between α 1 β 1 and α 2 β 2 in this quaternary structure. Further implications of the structural results are discussed.

Ligand binding and the gelation of sickle cell hemoglobin

The solubility equilibrium between monomer and polymer which has been shown to exist in deoxyhemoglobin S solutions is examined in solutions partially saturated with carbon monoxide. The total solubility is found to increase monotonically with increasing fractional saturation. At low fractional saturations the increase is nearly linear, amounting roughly to an increase of 0.01 g cm −3 in solubility for each 10% increase in fractional saturation. Linear dichroism measurements on the spontaneously aligned polymer phase are used to examine the composition of the polymer as a function of the fractional saturation of the corresponding solution phase. The dichroism experiments show that the polymer phase contains less than 5% of CO-liganded hemes even at supernatant fractional saturations in excess of 70%. The polymer selects against totally liganded hemoglobin molecules by a minimum factor of 65 and against singly liganded molecules by a factor of at least 2.5. Consequently, polymerized hemoglobin S has a ligand affinity which is significantly lower than that of monomeric hemoglobin S in the deoxy quaternary structure. The kinetics of the polymerization reaction in the presence of CO are similar to those observed in pure deoxyhemoglobin S solutions. The polymerization is preceded by a pronounced delay, the duration of which, t d, is proportional roughly to the 30th power of the solubility. At low fractional saturations, this amounts to a tenfold increase in t d for each 10% increase in the fractional saturation. These results show that the polymerization reaction is nearly specific for deoxyhemoglobin. Models for the dependence of the solubility and the polymer saturation on ligand partial pressure demonstrate the importance of solution phase non-ideality in determining the solubility of mixtures. The results require selection against partially liganded species which is significantly greater than is predicted by the two-state allosteric model. The data are compatible with either sequential or allosteric models in which the major polymerized component is the unliganded hemoglobin molecule.

Kinetics of iron hexacyanide release from the ferric hemes of partially oxidized intermediates of human deoxy hemoglobin

A new experiment is presented in which the kinetics of iron hexacyanide release is measured from partially oxidized intermediates of human deoxy hemoglobin (T state). The intermediates were generated by briefly exposing a solution of hemoglobin and dithionite to various concentrations of ferricyanide at 6° C in 0.1 M phosphate buffer, pH 7. The reaction of the residual dithionite with the partially oxidized intermediates was limited by the release of ferrocyanide and was strongly biphasic with about 75% fast phase and 25% slow and with the two phases differing in rate by a factor of 26. The predominance of rapid over slow phase is the result of a wide (approx. factor of 5) difference in the kinetics of oxidation of the alpha and beta chains by ferricyanide and is not due to heme-heme interaction. This experimental approach appears to offer a clear example of wide kinetic differences between the two types of subunits within the deoxy quaternary structure of human hemoglobin, uncomplicated by strong cooperative effects.

Proton nuclear magnetic resonance studies of hemoglobins Osler (beta145HC2 Tyr replaced by Asp) and McKee Rocks (beta145HC2 Tyr replaced by term): an assignment for an important tertiary structural probe in hemoglobin.

High-resolution proton nuclear magnetic resonance studies of deoxyhemoglobins Osler (beta145HC2 Tyr replaced by Asp) and McKees Rocks (beta 145HC2 Tyr replaced by term) indicate that these hemoglobins are predominately in the oxy quaternary structure in 0.1 M [bis(2-hydroxyethyl)imino]-tris(hydroxymethyl) methane buffer at pH 7. Upon the addition of inositol hexaphosphate, the proton nuclear magnetic resonance spectra of these hemoglobins become similar to those characteristic of a hemoglobin molecule in the deoxy quaternary structure. The exchangeable proton resonance which is found at -6.4 ppm from H2O in the spectrum of normal human adult deoxyhemoglobin is absent in the spectra of these two mutant hemoglobins. Consequently we believe the hydrogen bond between the hydroxyl group of tyrosine-beta145HC2 and the carboxyl oxygen of valine-beta98FG5 gives rise to this resonance. This assignment allows us to use the -6.4ppm resonance as an important tertiary structural probe in the investigation of the cooperative oxygenation of hemoglobin.

A plausible sequence of the conformational changes of hemoglobin induced by oxygenation

Recent experimental data of oxygen equilibrium constants of human adult hemoglobin, which are measured over a wide range of oxygen pressures, are analyzed successfully from the viewpoint that the change in the molecular structure of hemoglobin induced by oxygenation is considered individually at each stage of oxygenation. Then, a simple phenomenological rule, which explains quantitatively the values of the four Adair constants with only three parameters, is found for hemoglobin under normal physiological conditions. The temperature dependence of these parameters suggests a sequence of the conformational changes such that until the third stage of oxygenation the conformational changes occur within the deoxy quaternary structure and at the fourth stage of oxygenation the deoxy quaternary structure is altered to the oxy one. The effects of pH and phosphate compounds on the Adair constants are discussed, and a possible modification and extension of the rule is suggested. The connection between the rule and the molecular structures of deoxy- and oxyhemoglobin is also discussed.

The effect of ferric ligands on the oxygen affinity of the ferrous subunits in valency hybrid haemoglobins

Oxygen equilibrium curves of various valency hybrid haemoglobins (cyanide, azide, fluoride, aquomet hybrids) have been measured in the presence of inorganic phosphate or inositol hexaphosphate. All the hybrids showed asymmetric oxygen equilibrium curves which is inconsistent with the presence of only two oxygen binding sites. This inconsistency was found to be due to a small amount of oxidation of the ferrous subunits. Except for α 2 + β 2 combined with inositol hexaphosphate, the oxygen affinity was always lower in high compared to low spin hybrids implying that the deoxy quaternary structure is more stable for high spin derivatives. This result is consistent with the stereochemical interpretation of the co-operative effects proposed by Perutz (1970) . However, in the presence of inositol hexaphosphate, α 2 +CN β 2 and α 2 +F β 2 have similar oxygen affinity, showing that here the effect of spin state is no longer dominant. The reason for this is not yet clear. The oxygen binding parameters of the valency hybrids have been analysed according to the two-state allosteric model ( Monod et al. , 1965 ). This model adequately describes the oxygen binding properties in the presence of inositol hexaphosphate but not in phosphate buffer.