R-state haemoglobin with low oxygen affinity: crystal structures of deoxy human and carbonmonoxy horse haemoglobin bound to the effector molecule L35

Although detailed crystal structures of haemoglobin (Hb) provide a clear understanding of the basic allosteric mechanism of the protein, and how this in turn controls oxygen affinity, recent experiments with artificial effector molecules have shown a far greater control of oxygen binding than with natural heterotropic effectors. Contrary to the established text-book view, these non-physiological compounds are able to reduce oxygen affinity very strongly without switching the protein to the T (tense) state. In an earlier paper we showed that bezafibrate (BZF) binds to a surface pocket on the alpha subunits of R state Hb, strongly reducing the oxygen affinity of this protein conformation. Here we report the crystallisation of Hb with L35, a related compound, and show that this binds to the central cavity of both R and T state Hb. The mechanism by which L35 reduces oxygen affinity is discussed, in relation to spectroscopic studies of effector binding.     

Novel allosteric conformation of human HB revealed by the hydration and anion effects on O(2) binding.

The effect of anions on the stability of different functional conformations of Hb is examined through the determination of the dependence of O(2) affinity on water activity (a(w)). The control of a(w) is effected by varying the sucrose osmolal concentration in the bathing solution according to the "osmotic stress" method. Thus, the hydration change following Hb oxygenation is determined as a function of Cl(-) and of DPG concentration. We find that only approximately 25 additional water molecules bind to human Hb during the deoxy-to-oxy conformation transition in the absence of anions, in contrast with approximately 72 that bind in the presence of more than 50 mM Cl(-) or more than 15 microM DPG. We demonstrate that the increase in the hydration change linked with oxygenation is coupled with anion binding to the deoxy-Hb. Hence, we propose that the deoxy-Hb coexists in two allosteric conformations which depend on whether anion is bound or not: the tense T-state, with low oxygen affinity and anion bound, or a new allosteric P-state, with intermediate oxygen affinity and free of bound anions. The intrinsic oxygen affinity of this unforeseen P-state and the differential binding of Cl(-), DPG, and H(2)O between states P and T and P and R are characteristics which are consistent with those expected for a putative intermediate allosteric state of Hb. These findings represent a new opportunity to explore the structure-function relationships of hemoglobin regulation.     

Steric factors moderate conformational fluidity and contribute to the high proton sensitivity of Root effect hemoglobins

The structural basis of the extreme pH dependence of oxygen binding to Root effect Hbs is a long-standing puzzle in the field of protein chemistry. A previously unappreciated role of steric factors in the Root effect was revealed by a comparison of pH effects on oxygenation and oxidation processes in human Hb relative to Spot (Leiostomus xanthurus) and Carp (Cyprinodon carpio) Hbs. The Root effect confers five-fold increased pH sensitivity to oxygenation of Spot and Carp Hbs relative to Hb A(0) in the absence of anionic effectors, and even larger relative elevations of pH sensitivity of oxygenation in the presence of 0.2M phosphate. Remarkably, the Root effect was not evident in the oxidation of the Root effect Hbs. This finding rules out pH-dependent alterations in the thermodynamic properties of the heme iron, measured in the anaerobic oxidation reaction, as the basis of the Root effect. The alternative explanation supported by these results is that the elevated pH sensitivity of oxygenation of Root effect Hbs is attributable to globin-dependent steric effects that alter oxygen affinity by constraining conformational fluidity, but which have little influence on electron exchange via the heme edge. This elegant mode of allosteric control can regulate oxygen affinity within a given quaternary state, in addition to modifying the T-R equilibrium. Evolution of Hb sequences that result in proton-linked steric barriers to heme oxygenation could provide a general mechanism to account for the appearance of the Root effect in the structurally diverse Hbs of many species.     

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.     

Quaternary structures of intermediately ligated human hemoglobin a and influences from strong allosteric effectors: resonance Raman investigation

The Fe-histidine stretching (nu(Fe-His)) frequency was determined for deoxy subunits of intermediately ligated human hemoglobin A in equilibrium and CO-photodissociated picosecond transient species in the presence and absence of strong allosteric effectors like inositol(hexakis)phosphate, bezafibrate, and 2,3-bisphosphoglycerate. The nu(Fe-His) frequency of deoxyHb A was unaltered by the effectors. The T-to-R transition occurred around m = 2-3 in the absence of effectors but m > 3.5 in their presence, where m is the average number of ligands bound to Hb and was determined from the intensity of the nu(4) band measured in the same experiment. The alpha1-beta2 subunit contacts revealed by ultraviolet resonance Raman spectra, which were distinctly different between the T and R states, remained unchanged by the effectors. This observation would solve the recent discrepancy that the strong effectors remove the cooperativity of oxygen binding in the low-affinity limit, whereas the (1)H NMR spectrum of fully ligated form exhibits the pattern of the R state.     

Quaternary structure of carbonmonoxyhemoglobins in solution: structural changes induced by the allosteric effector inositol hexaphosphate

We have applied the residual dipolar coupling (RDC) method to investigate the solution quaternary structures of (2)H- and (15)N-labeled human normal adult recombinant hemoglobin (rHb A) and a low-oxygen-affinity mutant recombinant hemoglobin, rHb(alpha96Val-->Trp), both in the carbonmonoxy form, in the absence and presence of an allosteric effector, inositol hexaphosphate (IHP), using a stretched polyacrylamide gel as the alignment medium. Our recent RDC results [Lukin, J. A., Kontaxis, G., Simplaceanu, V., Yuan, Y., Bax, A., and Ho, C. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 517-520] indicate that the quaternary structure of HbCO A in solution is a dynamic ensemble between two previously determined crystal structures, R (crystals grown under high-salt conditions) and R2 (crystals grown under low-salt conditions). On the basis of a comparison of the geometric coordinates of the T, R, and R2 structures, it has been suggested that the oxygenation of Hb A follows the transition pathway from T to R and then to R2, with R being the intermediate structure [Srinivasan, R., and Rose, G. D. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11113-11117]. The results presented here suggest that IHP can shift the solution quaternary structure of HbCO A slightly toward the R structure. The solution quaternary structure of rHbCO(alpha96Val-->Trp) in the absence of IHP is similar to that of HbCO A in the presence of IHP, consistent with rHbCO(alpha96Val-->Trp) having an affinity for oxygen lower than that of Hb A. Moreover, IHP has a much stronger effect in shifting the solution quaternary structure of rHbCO(alpha96Val-->Trp) toward the R structure and toward the T structure, consistent with IHP causing a more pronounced decrease in its oxygen affinity. The results presented in this work, as well as other results recently reported in the literature, clearly indicate that there are multiple quaternary structures for the ligated form of hemoglobin. These results also provide new insights regarding the roles of allosteric effectors in regulating the structure and function of hemoglobin. The classical two-state/two-structure allosteric mechanism for the cooperative oxygenation of hemoglobin cannot account for the structural and functional properties of this protein and needs to be revised.     

Allosteric water and phosphate effects in Hoplosternum littorale hemoglobins.

This paper reports the results obtained using the osmotic stress method applied to the purified cathodic and anodic hemoglobins (Hbs) from the catfish Hoplosternum littorale, a species that displays facultative accessorial air oxygenation. We demonstrate that water potential affects the oxygen affinity of H. littorale Hbs in the presence of an inert solute (sucrose). Oxygen affinity increases when water activity increases, indicating that water molecules stabilize the high-affinity state of the Hb. This effect is the same as that observed in tetrameric vertebrate Hbs. We show that both anodic and cathodic Hbs show conformational substrates similar to other vertebrate Hbs. For both Hbs, addition of anionic effectors, especially chloride, strongly increases the number of water molecules bound, although anodic Hb did not exhibit sensitivity to saturating levels of ATP. Accordingly, for both Hbs, we propose that the deoxy conformations coexist in at least two anion-dependent allosteric states, T(o) and T(x), as occurs for human Hb. We found a single phosphate binding site for the cathodic Hb.     

Semihemoglobins, high oxygen affinity dimeric forms of human hemoglobin respond efficiently to allosteric effectors without forming tetramers

Significant reduction in oxygen affinity resulting from interactions between heterotropic allosteric effectors and hemoglobin in not only the unligated derivative but also the fully ligated form has been reported (Tsuneshige, A., Park, S. I., and Yonetani, T. (2002) Biophys. Chem. 98, 49-63; Yonetani, T., Park, S. I., Tsuneshige, A., Imai, K., and Kanaori, K. (2002) J. Biol. Chem. 277, 34508-34520). To further investigate this effect in more detail, alpha- and beta-semihemoglobins, namely, alpha(heme)beta(apo) and alpha(apo)beta(heme), respectively, were prepared and characterized with respect to the impact of allosteric effectors on both conformation and ligand binding properties. Semihemoglobins are dimers characterized by a high affinity for oxygen and lack of cooperativity. We found that, compared with stripped conditions, semihemoglobins responded to effectors (inositol hexaphosphate and L35) by decreasing the affinity for oxygen by 60- and 130-fold for alpha- and beta-semihemoglobins, respectively. 1H NMR and sedimentation velocity experiments carried out with their ligated and unligated forms in the absence and presence of effectors revealed that semihemoglobins always remain as single-heme-carrying dimers. Recombination kinetics of their photolyzed CO derivatives showed that effectors did indeed interact with their ligated forms. Measurements of the Fe-His stretching mode show that the semihemoglobins undergo a large ligand binding-induced conformational shift and that both ligand-free and ligand derivatives respond to the presence of effectors. Contradictions to the Monod-Wyman-Changeaux/Perutz allosteric model arise since 1) the modulation of ligand affinity is not achieved in semihemoglobins by the formation of a low affinity T conformation (quaternary effect) but by direct interaction with effectors, 2) effectors do interact significantly with ligated forms of high affinity semihemoglobins, and 3) modulation of the ligand affinity and the cooperativity are not necessarily linked but instead can be separated into two distinct phenomena that can be isolated.     

Coupling of ferric iron spin and allosteric equilibrium in hemoglobin.

The allosteric transition in triply ferric hemoglobin has been studied with different ferric ligands. This valency hybrid permits observation of oxygen or CO binding properties to the single ferrous subunit, whereas the liganded state of the other three ferric subunits can be varied. The ferric hemoglobin (Hb) tetramer in the absence of effectors is generally in the high oxygen affinity (R) state; addition of inositol hexaphosphate induces a transition towards the deoxy (T) conformation. The fraction of T-state formed depends on the ferric ligand and is correlated with the spin state of the ferric iron complexes. High-spin ferric ligands such as water or fluoride show the most T-state, whereas low-spin ligands such as cyanide show the least. The oxygen equilibrium data and kinetics of CO recombination indicate that the allosteric equilibrium can be treated in a fashion analogous to the two-state model. The binding of a low-spin ferric ligand induces a change in the allosteric equilibrium towards the R-state by about a factor of 150 (at pH 6.5), similar to that of the ferrous ligands oxygen or CO; however, each high-spin ferric ligand induces a T to R shift by a factor of 40.     

T-state hemoglobin with four ligands bound

Flash photolysis kinetics have been measured for ligand recombination to hemoglobin (Hb) in the presence of two effectors: bezafibrate (Bzf) and inositol hexakisphosphate (IHP). The combined influence of the two independent effectors leads to predominantly T-state behavior. Samples equilibrated with 0.1 atm of CO are fully saturated, yet after photodissociation they show only T-state bimolecular recombination rates at all photolysis levels; this indicates that the allosteric transition from R to T occurs before CO rebinding and that the allosteric equilibrium favors the T-state tetramer with up to three ligands bound. Since all four ligands bind at the rate characteristic for the T-state, the return transition from T to R must occur after the fourth ligand was bound. At 1 atm of CO, rebinding to the initial R state competes with the allosteric transition resulting in a certain fraction of CO bound at the rate characteristic for the R state; this fraction is greater the smaller the percentage dissociation. Under 1 atm of oxygen, samples are not more than 93% saturated and show mainly T-state kinetics. The results show that all four hemes can bind oxygen or CO ligands in the T structure. The fraction of the kinetics occurring as geminate is less for partially liganded (T-state) samples than for fully liganded (R-state) Hb.     

Allosteric kinetics and equilibria of triligated, cross-linked hemoglobin.

Using modulated excitation, we have measured the forward and reverse rates of the allosteric transition between relaxed (R) and tense (T) quaternary structures for triply ligated hemoglobin (Hb), cross-linked between the alpha chains at Lys 99. Oxygen, carbon monoxide, and water were used as ligands and were studied in phosphate and low Cl- bis-Tris buffers at neutral pH. Since the cross-link prohibits disproportionation, triply ligated aquomet Hb species with ferrous beta chains were specifically isolated by isoelectric focusing. Modulated excitation provides rate pairs and therefore gives equilibrium constants between quaternary structures. To coordinate with that information, oxygen binding curves of fully ferrous and tri-aquomet Hb were also measured. L3, the equilibrium constant between three liganded R and T structures, is determined by modulated excitation to be of order unity for O2 or CO (1.1 to 1.5 for 3O2 and 0.7 for 3CO bound), while with three aquomet subunits it is much greater (> or = 23). R-->T conversion rates are similar to those found for HbA, with weak sensitivity to changes in L3. The L3 values from HbXL O2 were used to obtain a unique allosteric decomposition of the ferrous O2 binding curve in terms of KT, KR, and L3. From these values and the O2 binding curve of tri-aquomet HbXL, L3 was calculated to be 2.7 for the tri-aquomet derivative. Consistency in L3 values between equilibrium and modulated excitation data for tri-aquomet-HbXL can be achieved if the equilibrium constant for O2 binding to the alpha chains is six times lower than that for binding to the beta chains in the R state, while the cooperative properties remain homogeneous. The results are in quantitative agreement with other studies, and suggest that the principal effect of the cross-link is to decrease the R state and T state affinity of the alpha subunits with almost no change in the affinity of the beta subunits, leaving the allosteric parameters L and c unchanged.     

High pressure reveals that the stability of interdimeric contacts in the R- and T-state of HbA is influenced by allosteric effectors: Insights from computational simulations

The molecular details of the mechanism of action of allosteric effectors on hemoglobin oxygen affinity are not clearly understood. The global allostery model proposed by Yonetani et al. suggests that the binding of allosteric effectors can take place both in the R and T states and that they influence oxygen affinity through inducing global tertiary changes in the subunits. Recently published high pressure studies yielded dissociation constants at atmospheric pressure that showed a stabilizing effect of heterotropic allosteric effectors on the dimer interface in the R state, and a more pronounced destabilizing effect in a T state model. In the present work, we report on computational modeling used to interpret the high pressure experimental data. We show structural changes in the hemoglobin interdimeric interfaces, indicative of a global tertiary structural change induced by the binding of allosteric effectors. We also show that the number of water molecules bound at the interface is significantly influenced by binding effectors in the T state in accordance with the experimental data. Our results suggest that the binding of effectors at definite sites leads to tertiary changes that propagate to the interfaces and results in overall structural re-organizations.     

Allosteric effectors do not alter the oxygen affinity of hemoglobin crystals.

In solution, the oxygen affinity of hemoglobin in the T quaternary structure is decreased in the presence of allosteric effectors such as protons and organic phosphates. To explain these effects, as well as the absence of the Bohr effect and the lower oxygen affinity of T-state hemoglobin in the crystal compared to solution, Rivetti C et al. (1993a, Biochemistry 32:2888-2906) suggested that there are high- and low-affinity subunit conformations of T, associated with broken and unbroken intersubunit salt bridges. In this model, the crystal of T-state hemoglobin has the lowest possible oxygen affinity because the salt bridges remain intact upon oxygenation. Binding of allosteric effectors in the crystal should therefore not influence the oxygen affinity. To test this hypothesis, we used polarized absorption spectroscopy to measure oxygen binding curves of single crystals of hemoglobin in the T quaternary structure in the presence of the "strong" allosteric effectors, inositol hexaphosphate and bezafibrate. In solution, these effectors reduce the oxygen affinity of the T state by 10-30-fold. We find no change in affinity (< 10%) of the crystal. The crystal binding curve, moreover, is noncooperative, which is consistent with the essential feature of the two-state allosteric model of Monod J, Wyman J, and Changeux JP (1965, J Mol Biol 12:88-118) that cooperative binding requires a change in quaternary structure. Noncooperative binding by the crystal is not caused by cooperative interactions being masked by fortuitous compensation from a difference in the affinity of the alpha and beta subunits. This was shown by calculating the separate alpha and beta subunit binding curves from the two sets of polarized optical spectra using geometric factors from the X-ray structures of deoxygenated and fully oxygenated T-state molecules determined by Paoli M et al. (1996, J Mol Biol 256:775-792).     

A biochemical--biophysical study of hemoglobins from woolly mammoth, Asian elephant, and humans

This study is aimed at investigating the molecular basis of environmental adaptation of woolly mammoth hemoglobin (Hb) to the harsh thermal conditions of the Pleistocene ice ages. To this end, we have carried out a comparative biochemical-biophysical characterization of the structural and functional properties of recombinant hemoglobins (rHb) from woolly mammoth (rHb WM) and Asian elephant (rHb AE) in relation to human hemoglobins Hb A and Hb A(2) (a minor component of human blood). We have obtained oxygen equilibrium curves and calculated O(2) affinities, Bohr effects, and the apparent heat of oxygenation (ΔH) in the presence and absence of allosteric effectors [inorganic phosphate and inositol hexaphosphate (IHP)]. Here, we show that the four Hbs exhibit distinct structural properties and respond differently to allosteric effectors. In addition, the apparent heat of oxygenation (ΔH) for rHb WM is less negative than that of rHb AE, especially in phosphate buffer and the presence of IHP, suggesting that the oxygen affinity of mammoth blood was also less sensitive to temperature change. Finally, (1)H NMR spectroscopy data indicates that both α(1)(β/δ)(1) and α(1)(β/δ)(2) interfaces in rHb WM and rHb AE are perturbed, whereas only the α(1)δ(1) interface in Hb A(2) is perturbed compared to that in Hb A. The distinct structural and functional features of rHb WM presumably facilitated woolly mammoth survival in the Arctic environment.     

Inositol tripyrophosphate: a new membrane permeant allosteric effector of haemoglobin

Nine inositol tripyrophosphate (ITPP) salts have been synthesized. Their ability to act as allosteric effectors of haemoglobin (Hb) has been measured in vitro with free Hb and whole blood. All the synthesized compounds bound to free Hb and were also able to cross, to a certain extent, the plasma membrane of the red blood cells (RBCs) in whole blood samples, lowering the affinity of Hb for oxygen. The oxy-haemoglobin dissociation curves were significantly shifted towards higher values of oxygen partial pressures, both for free Hb and for intracellular Hb in whole blood.     

Allosteric interpretation of the oxygen-binding reaction of human hemoglobin tetramers

An allosteric model is presented that provides a simple explanation for the low population of triply ligated species, relative to the other species, in the oxygenation of human hemoglobin tetramers as found in high-concentration studies [Gill, S. J., Di Cera, E., Doyle, M. L., Bishop, G. A., & Robert, C. H. (1987) Biochemistry (preceding paper in this issue)]. The model is a quantitative interpretation of the Perutz mechanism [Perutz, M. F. (1970) Nature (London) 228, 726-739] and is based on a number of structural and thermodynamic findings so far reported in the analysis of hemoglobin properties. Human hemoglobin is assumed to exist in two quaternary states: the T or low-affinity state and the R or high-affinity state. An extreme chain heterogeneity in the T state is postulated so that oxygen binds only to the alpha chains. Nearest-neighbor interactions between the alpha chains may lead to cooperativity within the T state. The R state is noncooperative, and both the alpha and beta chains have equal oxygen affinity.     

Effectors of hemoglobin. Separation of allosteric and affinity factors.

The relative contributions of the allosteric and affinity factors toward the change in p50 have been calculated for a series of effectors of hemoglobin (Hb). Shifts in the ligand affinity of deoxy Hb and the values for 50% ligand saturation (p50) were obtained from oxygen equilibrium data. Because the high-affinity parameters (liganded conformation) are poorly determined from the equilibrium curves, they were determined from kinetic measurements of the association and dissociation rates with CO as ligand. The CO on-rates were obtained by flash photolysis measurements. The off-rates were determined from the rate of oxidation of HbCO by ferricyanide, or by replacement of CO with NO. The partition function of fully liganded hemoglobin for oxygen and CO is only slightly changed by the effectors. Measurements were made in the presence of the effectors 2,3-diphosphoglycerate (DPG), inositol hexakisphosphate (IHP), bezafibrate (Bzf), and two recently synthesized derivatives of Bzf (LR16 and L35). Values of p50 change by over a factor of 60; the on-rates decrease by nearly a factor of 8, with little change in the off-rates for the liganded conformation. The data indicate that both allosteric and affinity parameters are changed by the effectors; the changes in ligand affinity represent the larger contribution toward shifts in p50.     

T-quaternary structure of oxy human adult hemoglobin in the presence of two allosteric effectors, L35 and IHP

The cooperative O(2)-binding of hemoglobin (Hb) have been assumed to correlate to change in the quaternary structures of Hb: T(deoxy)- and R(oxy)-quaternary structures, having low and high O(2)-affinities, respectively. Heterotropic allosteric effectors have been shown to interact not only with deoxy- but also oxy-Hbs causing significant reduction in their O(2)-affinities and the modulation of cooperativity. In the presence of two potent effectors, L35 and inositol hexaphosphate (IHP) at pH 6.6, Hb exhibits extremely low O(2)-affinities (K(T)=0.0085mmHg(-1) and K(R)=0.011mmHg(-1)) and thus a very low cooperativity (K(R)/K(T)=1.3 and L(0)=2.4). (1)H-NMR spectra of human adult Hb with these two effectors were examined in order to determine the quaternary state of Hb in solution and to clarify the correlation between the O(2)-affinities and the structural change of Hb caused by the heterotropic effectors. At pH 6.9, (1)H-NMR spectrum of deoxy-Hb in the presence of L35 and IHP showed a marker of the T-quaternary structure (the T-marker) at 14ppm, originated from inter- dimeric α(1)β(2)- (or α(2)β(1)-) hydrogen-bonds, and hyperfine-shifted (hfs) signals around 15-25ppm, caused by high-spin heme-Fe(II)s. Upon addition of O(2), the hfs signals disappeared, reflecting that the heme-Fe(II)s are ligated with O(2), but the T-marker signals still remained, although slightly shifted and broadened, under the partial pressure of O(2) (P(O2)) of 760mmHg. These NMR results accompanying with visible absorption spectroscopy and visible resonance Raman spectroscopy reveal that oxy-Hb in the presence of L35 and IHP below pH 7 takes the ligated T-quaternary structure under the P(O2) of 760mmHg. The L35-concentration dependence of the T-marker in the presence of IHP indicates that there are more than one kind of L35-binding sites in the ligated T-quaternary structure. The stronger binding sites are probably intra-dimeric binding sites between α(1)G- and β(1)G-helices, and the other weaker binding site causes the R→T transition without release of O(2). The fluctuation of the tertiary structure of Hb seems to be caused by both the structural perturbation of α(1)β(1) (or α(2)β(2)) intra-dimeric interface, where the stronger L35-binding sites exist, and by the IHP-binding to the α(1)α(2)- (or β(1)β(2)-) cavity. The tertiary structural fluctuation induced by the allosteric effectors may contribute to the significant reduction of the O(2)-affinity of oxy-Hb, which little depends on the quaternary structures. Therefore, the widely held assumptions of the structure-function correlation of Hb - [the deoxy-state]=[the T-quaternary structure]=[the low O(2)-affinity state] and [the oxy-state]=[the R-quaternary structure]=[the high O(2)-affinity state] and the O(2)-affiny of Hb being regulated by the T/R-quaternary structural transition - are no longer sustainable. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.     

On the pH-dependence of the reaction of hemoglobin with carbon monoxide.

Flash-photolysis experiments were performed on solutions of carbonmonoxy hemoglobin (human Hb A) as function as pH. The fraction of fast reaction and the amount of photodissociation as produced by a given amount of light quanta has been analyzed in terms of the allosteric model of ligand binding by Monod, Wyman and Changeux. It is shown how the switch-over point of the allosteric transition from the T or R state is controlled by the protons, which act as allosteric effectors.