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.     

Studies on cobalt myoglobins and hemoglobins, XIII. A consequence of the occurrence of glutamine at the E7 (58) site of alpha subunits in opossum hemoglobin

To investigate the mode of interactions between heme metal, bound oxygen and the distal residue at the E7 site, we have measured accurate oxygen equilibrium curves, oxygen binding relaxations following temperature-jump, and electron paramagnetic resonance spectra of natural and cobalt-substituted opossum hemoglobin, which has glutamine and histidine at the E7 site of the α chain and the β chain, respectively, and compared them with those of natural and cobalt-substituted human hemoglobin, which has histidine at the E7 site of both the α and β chains.Natural opossum hemoglobin has a lower oxygen affinity, slightly smaller and pH-dependent co-operativity, a somewhat greater Bohr effect, and a smaller effect of organic phosphates such as 2,3-diphosphoglycerate and inositol hexaphosphate on oxygen affinity as compared to natural human hemoglobin. Upon substitution of cobalt for iron, these oxygenation characteristics of opossum hemoglobin relative to those of human hemoglobin were preserved well. The behavior of the intrinsic oxygen association constants pertaining to the four oxygenation steps (i.e. the Adair constants) upon addition of the organic phosphates or pH changes indicates that the allosteric equilibrium in opossum hemoglobin is biased towards the T state as compared with that in human hemoglobin, and that the oxygen affinity of the R structure is lower for opossum hemoglobin than for human hemoglobin. The temperature-jump kinetic data indicate that the lower oxygen affinity of opossum cobalt-hemoglobin in comparison with that of human cobalt-hemoglobin can be ascribed to a decreased oxygen association rate constant. The electron paramagnetic resonance experiments on oxy and deoxy opossum and human cobalt-hemoglobins in buffered H₂O and ²H₂O, including their photolysed products at a low temperature, provided the following information. The cobaltous ion of the α subunits of deoxy opossum cobalt-hemoglobin is in an environment that is similar to that for cobaltous ions of deoxy human cobalt-hemoglobin in the T state. The hydrogen bond between the bound oxygen and the residue at E7, which has been shown to exist in oxy human cobalt-hemoglobin and oxy sperm whale cobalt-myoglobin, is absent or, at least, significantly altered in the α subunits of oxy opossum cobalt-hemoglobin, probably resulting in a lower oxygen affinity. Interference by isoleucine at E11α with an oxygen molecule is suggested as an explanation for the lowered affinity of opossum iron-hemoglobin. However, no straightforward structural explanation is available for the lower oxygen affinity of the R structure and the allosteric equilibrium biased towards the T state in opossum iron-hemoglobin.     

The linkage between the four-step binding of oxygen and the binding of heterotropic anionic ligands in hemoglobin

The linkage between the four-step binding of oxygen and the binding of heterotropic anionic ligands in hemoglobin was investigated by accurately measuring and analyzing the oxygen equilibrium curves of human adult hemoglobin in the presence and absence of various concentrations of one or two of the following materials: chloride (Cl-), 2,3-diphosphoglycerate (DPG), and inositol hexaphosphate (IHP). Each equilibrium curve was analyzed according to the Adair equation to evaluate the four-step oxygen equilibrium constants (Adair constants) and the median oxygen pressure. The binding constants of the anions for the molecular species of hemoglobin carrying j oxygen molecules, Hb(O2)j(j=0,1,...,4), were evaluated from the dependences of the Adair constants and the median oxygen pressure on the anion concentration by introducing a model which takes the competitive binding of Cl- and DPG or IHP into account. Assumptions made in the model are: (a) the hemoglobin molecule has two oxygen-linked binding sites for Cl- which are equivalent and independent and (b) no Cl- can be bound to hemoglobin to which DPG or IHP is already bound and vice versa. Thus, we could obtain values for the intrinsic binding constants of Cl- and DPG, i.e., the constants in the absence of other competitive anions. For IHP, only the binding constants and apparent binding constants for Hb and Hb(O2)2 were obtained. Values of the Cl- binding constants and apparent binding constants for DPG and IHP, i.e., the binding constants in the presence of Cl- for Hb and Hb(O2)4, were in reasonable agreement with literature values. From the binding constants we calculated anion binding curves for Hb(O2)j(J=0,1,...,4), the number of anions bound to Hb(O2)J, And the relationship between fractional anion saturation of hemoglobin and fractional oxygen saturation. The numbers of released anions are not uniform with respect to oxygenation step. This non-uniformity is the reason for the changes in the shape of the oxygen equilibrium curve with anion concentration changes and for the non-uniform dependences of the Adair constants on anion concentration, and also results in non-linear relations between anion saturation and oxygen saturation. The anion binding constants and various binding properties of the anions derived from those constants are consistent with those observed by other investigators using different techniques, indicating that the present model describes the oxygen-linked competitive anion binding well.     

Analysis of the interaction of organic phosphates with hemoglobin.

The interaction of organic phosphates with hemoglobin is studied by use of a simple thermodynamic approach. A model-independent analysis is employed to evaluate the accuracy of Adair constants determined in the presence of 2,3-diphosphoglycerate (DPG). The change of oxygen affinity in the presence of phosphates is related to the macroscopic phosphate binding constants of oxy- and deoxyhemoglobin and used to extract such binding constants from oxygen equilibrium measurements. The change of the Bohr effect in the presence of phosphates and the competitive binding of carbon dioxide and DPG are treated quantitatively. The binding of organic phosphates is incorporated into an allosteric model, in which the effect of phosphate on both tertiary and quaternary structure changes is included. By use of this model, the factors which can be responsible for the increased functional heterogeneity of alpha and beta chains in the presence of phosphates are clarified.     

On the validity of the spectrophotometric determination of oxygen saturation of hemoglobin. The wavelength dependence of observed oxygen equilibrium parameter values

Spectral changes of oxyhemoglobin induced by such anions as 2,3-diphosphoglycerate, inositol hexaphosphate, and Cl- may affect the validity of the spectrophotometric determination of oxygen saturation of hemoglobin. Therefore, the anion-induced difference spectra were extensively measured under a variety of conditions and accurate oxygen equilibrium curves were determined under representative conditions with detection at different wavelengths selected from peaks, troughs, and zero difference points of the difference spectra in the visible and Soret regions. Oxygen equilibrium parameters including the four Adair constants (i.e., equilibrium constants for four steps of oxygenation) estimated from the equilibrium curves did not show any dependence on wavelength within the limits of experimental error. These results indicate that anion-induced spectral changes do not invalidate the spectrophotometric determination of oxygen saturation and confirm the validity of the previous conclusions drawn in our series of studies on the effects of anions, pH and temperature on oxygen equilibrium parameters.     

Precision determination and Adair scheme analysis of oxygen equilibrium curves of concentrated hemoglobin solution. A strict examination of Adair constant evaluation methods

To examine the validity of the recent finding by Gill et al. (S.J. Gill, E. Di Cera, M.L. Doyle, G.A. Bishop and C.H. Robert, Biochemistry 26 (1987) 3995) that the third overall Adair constant (A3) for human hemoglobin tetramers (Hb A) is too small to be determined and therefore that the contribution of the triply ligated species in the oxygenation process is negligibly small, highly accurate oxygen equilibrium curves for concentrated pure Hb A solutions were determined with an automatic oxygenation apparatus and analyzed by a least-squares curve-fitting method with various options. The present results indicate that an appropriate choice of weighting for data points is the key to the correct evaluation of the Adair constants and the present experimental data cannot accommodate the Adair scheme with A3 = 0, giving distinctly positive values for A3. Several criteria for correct determination of the Adair constants are presented.     

Statistical analysis of data pertaining to complex state systems by stepwise regression with reformulated parameters; Application to spectroscopically monitored hemoglobin oxygen binding data

A method is described for the statistical analysis of data pertaining to complex state systems, based on the concept of reformulating the parameters describing the system as a hierarchy of interactions, and this method demonstrated on the analysis of spectroscopically monitored hemoglobin oxygen binding data [K. Imai, Biophys. Chem. 37 (1990) 197-210]. The concept of reformulation was first extended to state parameters other than ΔG°s, such as the extinction coefficients (εs) associated with different ligation states during hemoglobin oxygen binding. The reformulated parameters are incrementally allowed to vary in the data fitting procedure, and the statistical significance of the added parameters tested by F and Kolmogorov-Smirnov tests. The result of this method is the minimal set of statistically significant parameters required to describe the data. The hierarchical nature of reformulated parameters allows the physical significance of the subset of statistically significant parameters to be discussed even when all reformulated terms may not be statistically significant. Applying this method to hemoglobin oxygen binding data with the reformulated Adair model demonstrated that at least two, and at most three, of the four reformulated Adair constants are statistically significant. A reformulated square model was found to give a statistically indistinguishable fit from the Adair model, with the statistically significant thermodynamic terms essentially those proposed by Linus Pauling in 1935. A change in Δ ε with subsequent oxygen binding events was found to be significant in both models. These results are consistent with a model for hemoglobin oxygen binding where a subunit changes its conformation upon oxygen binding, and affects the conformation of adjacent subunits.     

Effects of polyvalent anion binding to hemoglobin on oxygen and oxidation-reduction equilibria and their relevance to allosteric transition.

The reaction of human hemoglobin and some of its derivatives with aliphatic and aromatic compounds carrying from two to six carboxylate groups has been studied. The effect of the polycarboxylates as well as of three co-ordinate anions (respectively tri-, tetra- and pentavalent) on the oxygenation and oxidation-reduction equilibria and optical spectra have been compared to those of 2,3-diphosphoglycerate and inositol hexaphosphate.All the polyvalent anions raise the P_0.5³ and the E_m values of human hemoglobin and are thus bound more strongly to deoxyhemoglobin than to oxy- or methemoglobin. Binding of benzene hexacarboxylate and benzene pentacarboxylate to oxyhemoglobin is demonstrated through a study of oxygenation curves, that of these reagents, and ferrocyanide, to methemoglobin through their effect on redox potential as well as on optical spectra. Methemoglobin and oxyhemoglobin are shown to bind more than one molecule of the carboxylates at high anion concentrations. Results bearing on the anion binding site for deoxy- as well as for methemoglobin are reported.Two appropriate human hemoglobin derivatives, namely Hb_BME and Hb_{NES desArg} have been examined in search of relations between the effect of anions on oxygen equilibria and that on quaternary structure: in both of these derivatives the chemical modifications inhibit quaternary conformational change that would result from oxygen binding, the deoxy structure being strongly destabilized. Several of the polyanions significantly raise the P_0.5 values of these derivatives but do not modify the quaternary structure, as judged from the absence of characteristic spectral changes. The results imply that anion binding by these proteins somehow inhibits the change in tertiary structure produced by oxygen binding; similar considerations may also apply in the case of the normal hemoglobin-diphosphoglycerate complex.     

Ruthenium-iron hybrid hemoglobins as a model for partially liganded hemoglobin: oxygen equilibrium curves and resonance Raman spectra

The structure and function of iron(II)-ruthenium(II) hybrid hemoglobins alpha(Ru-CO)2 beta(Fe)2 and alpha(Fe)2 beta(Ru-CO)2, which can serve as models for the intermediate species of the oxygenation step in native human adult hemoglobin, were investigated by measuring oxygen equilibrium curves and the Fe(II)-N epsilon (His F8) stretching resonance Raman lines. The oxygen equilibrium properties indicated that these iron-ruthenium hybrid hemoglobins are good models for the half-liganded hemoglobin. The pH dependence of the oxygen binding properties and the resonance Raman line revealed that the quaternary and tertiary structural transition was induced by pH changes. When the pH was lowered, both the iron-ruthenium hybrid hemoglobins exhibited relatively higher cooperativity and a Raman line typical of normal deoxy structure, suggesting that their structure is stabilized at a "T-like" state. However, the oxygen affinity of alpha(Fe)2 beta(Ru-CO)2 was lower than that of alpha(Ru-CO)2 beta(Fe)2, and the transition to the "deoxy-type" Fe-N epsilon stretching Raman line of alpha(Fe2)beta(Ru-CO)2 was completed at pH 7.4, while that of the complementary counterpart still remained in an "oxy-like" state under the same condition. These observations clearly indicate that the beta-liganded hybrid has more "T"-state character than the alpha-liganded hybrid. In other words, the ligation to the alpha subunit induces more pronounced changes in the structure and function in Hb than the ligation to the beta subunit. This feature agrees with our previous observations by NMR and sulfhydryl reactivity experiments. The present results are discussed in relation to the molecular mechanism of the cooperative stepwise oxygenation in native human adult hemoglobin.     

Functional and spectroscopic characterization of half-liganded iron-zinc hybrid hemoglobin: evidence for conformational plasticity within the T state

Functionally distinct conformations of HbA (human adult hemoglobin) were probed using deoxy and diliganded derivatives of symmetric Fe-Zn hybrids of HbA. To expand the range of accessible structures, different environments were utilized including solution, sol-gel encapsulation, and crystals. Further structural and functional modulation was achieved by the addition of allosteric effectors. Functional characterization included oxygen affinity measurements, CO combination rates, and geminate and bimolecular CO recombination, after photodissociation. The conformational properties were studied using visible resonance Raman spectroscopy as a probe of local tertiary structure at the iron-containing hemes and UV resonance Raman spectroscopy as a probe of elements of the globin known to be sensitive to quaternary structure. The combined results show a pattern in which there is a progression of conformational and functional properties that are consistent with a picture in which the T quaternary structure can accommodate a range of tertiary conformations (plasticity). At one end of the distribution is the equilibrium deoxy T state conformation that has the lowest ligand reactivity. At the other end of the distribution are T state conformations with higher ligand reactivity that exhibit "loosened" T state constraints within the globin including the alpha(1)beta(2) interface and reduced proximal strain at the heme.     

Involvement of Glu G3(101)beta in the function of hemoglobin. Comparative O2 equilibrium studies of human mutant hemoglobins

The glutamyl residue at G3(101)beta of normal hemoglobin (Hb A) is one of the alpha 1 beta 2 subunit contacts which are vital to O2 binding properties of the molecule. The O2 equilibrium properties of the four mutants with different substitutions at this site are studied in order to elucidate the role of this residue. Under stripped conditions with minimum chloride the order of O2 affinity is: Hb A (Glu) much less than Hb Rush (Gln) less than or equal to Hb British Columbia (Lys) less than or equal to Hb Potomac (Asp) less than or equal to Hb Alberta (Gly). The first Adair constants, K1, for the mutant hemoglobins are greater than that for Hb A whereas the fourth, K4, are similar, indicating that the allosteric constants (L) of these mutants are greatly reduced. Therefore, the G3(101)beta residue contributes intrinsically to the strengthening of the structural constraints that are imposed upon the deoxy (T) forms but not the oxy (R) form. On addition of 0.1 M Cl- and further addition of 2,3-diphosphoglycerate or inositol hexaphosphate, their O2 affinities and cooperativities are altered, reflecting different responses to anionic ligands. Hb Rush exhibits a stronger chloride effect than Hb A and the other variants and, as a result, an increased Bohr effect and a smaller heat of oxygenation at pH 6.5. These changes are consistent with an increased positive net charge in the central cavity of Hb Rush and subsequent extra anion binding in the deoxy form. The tetramer to dimer dissociation constants are estimated to be greater than normal for Hb British Columbia and less than normal for Hb Alberta. This comparative study of the G3(101)beta mutants indicates that the size and the charge of this residue may influence the switching of two neighboring interchain hydrogen bonds that occurs during oxygenation of normal hemoglobin.     

Allosteric interactions in sipunculid and brachiopod hemerythrins

Chemical and spectroscopic consequences of allosteric interactions for ligand binding to sipunculid (Phascolopsis gouldii) and brachiopod (Lingula reevii) hemerythrins (Hrs) have been investigated. Possible allosteric effectors for homotropic effects in sipunculid Hrs have been examined, but only reduction in ligand affinity is observed without cooperativity. In contrast to sipunculid Hr, L. reevii Hr binds O2 cooperatively in the pH range 7-8 and exhibits a Bohr effect. Spectroscopic comparisons of the sipunculid and brachiopod Hrs show no significant differences in the active site structures; therefore, modulation of oxygen affinity is attributable to effects linking the site to quaternary structural changes in the octamer. Oxygen equilibria can be fit with a conformational model incorporating a minimum of three states, tensed (T), relaxed (R), and an R-T hybrid. Resonance Raman spectra of L. reevii oxyHr show a shift in the peroxo stretching frequency when the pH is lowered from pH 7.7 (predominantly R oxyHr) to pH 6.3 (a mixture of R, T, and R-T hybrid), but P. gouldii Hr does not have a frequency shift under the same conditions. In contrast to hemoglobins, ligand binding to the deoxy and met forms is noncooperative for brachiopod (and sipunculid) Hrs. It is thus suggested that conformational changes in the protein are linked to the oxidation state change that accompanies oxygenation of the coupled binuclear iron site (deoxy [FeIIFeII]----oxy [FeIIIFeIII]). The total allosteric energy expended in oxygenation is about 1.4 kcal/mol, and such a shift is possible in the relaxed-tense conversion with relatively limited constraints of the iron coordination environment via the protein quaternary structure. The mechanism of cooperativity in the binuclear copper oxygen carrier hemocyanin is discussed in light of these results.     

Properties of human hemoglobin immobilized on Sepharose 4B.

This paper reports the properties of human hemoglobin covalently bound to Sepharose 4B both in 'high-affinity' and 'low-affinity' conformations. The results suggest that the coupling reaction is strongly affected by the conformational changes linked to oxygenation of the protein. The rate and the extent of the reaction are different for the oxy and deoxyderivatives, probably due to the change in reactivity of the amino groups in the liganded and unliganded tetramer. The data on the equilibrium which is established between matrix-bound and soluble subunits, measured by the 'subunit-exchange chromatography', indicate that the system displays a minimal heterogeneity when hemoglobin is coupled to the gel in the deoxy state at intermediate protein concentration and pH 8. Maxtrix-bound hemoglobin is characterized by a higher oxygen affinity and by decreased homotropic and heterotropic interactions with respect to hemoglobin in solution, but the changes depend strongly on the conditions used in the coupling procedure.     

The crystal structure of bar-headed goose hemoglobin in deoxy form: the allosteric mechanism of a hemoglobin species with high oxygen affinity

The crystal structure of a high oxygen affinity species of hemoglobin, bar-headed goose hemoglobin in deoxy form, has been determined to a resolution of 2.8 A. The R and R(free) factor of the model are 0.197 and 0.243, respectively. The structure reported here is a special deoxy state of hemoglobin and indicates the differences in allosteric mechanisms between the goose and human hemoglobins. The quaternary structure of the goose deoxy hemoglobin shows obvious differences from that of human deoxy hemoglobin. The rotation angle of one alphabeta dimer relative to its partner in a tetramer molecule from the goose oxy to deoxy hemoglobin is only 4.6 degrees, and the translation is only 0.3 A, which are much smaller than those in human hemoglobin. In the alpha(1)beta(2) switch region of the goose deoxy hemoglobin, the imidazole ring of His beta(2)97 does not span the side-chain of Thr alpha(1)41 relative to the oxy hemoglobin as in human hemoglobin. And the tertiary structure changes of heme pocket and FG corner are also smaller than that in human hemoglobin. A unique mutation among avian and mammalian Hbs of alpha119 from proline to alanine at the alpha(1)beta(1 )interface in bar-headed goose hemoglobin brings a gap between Ala alpha119 and Leu beta55, the minimum distance between the two residues is 4.66 A. At the entrance to the central cavity around the molecular dyad, some residues of two beta chains form a positively charged groove where the inositol pentaphosphate binds to the hemoglobin. The His beta146 is at the inositol pentaphosphate binding site and the salt-bridge between His beta146 and Asp beta94 does not exist in the deoxy hemoglobin, which brings the weak chloride-independent Bohr effect to bar-headed goose hemoglobin.     

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...     

Consequences of neglecting the hemoglobin concentration on the determination of Adair binding constants

It is commonly believed that the tetrameric Adair constants for oxygen binding to human hemoglobin can be evaluated from a single oxygenation experiment at 'high' hemoglobin concentration without considering the consequence of the presence of alpha beta dimers. We present examples which demonstrate that this is a very dangerous assumption. Without a knowledge of the complete oxygenation-linked dimer-tetramer association reaction (alpha beta Xi----(alpha beta)2Xj), it is impossible to predict a priori how high of a hemoglobin concentration would be required to make this assumption. Furthermore, without a knowledge of the complete oxygenation-linked dimer-tetramer association reaction, it is impossible to predict a priori the direction and magnitude of the systematic errors which are induced by making this assumption.     

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.