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.     

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.     

Control of the allosteric equilibrium of hemoglobin by cross-linking agents

The kinetics of ligand rebinding have been studied for modified or cross-linked hemoglobins (Hbs). Several compounds were tested that interact with alpha Val 1 or involve a cross-link between alpha Val 1 and alpha Lys 99 of the opposite dimer. By varying the length of certain cross-linking molecules, a wide range in the allosteric equilibrium could be obtained. Several of the mono-aldehyde modified Hbs show a shift toward the high affinity conformation of Hb. At the other extreme, for certain di-aldehyde cross-linked Hbs, the CO kinetics are typical of binding to deoxy Hb, even at low photodissociation levels, with which the dominant photoproduct is the triply liganded species; in these cases the hemoglobin does not switch from the low to high affinity state until after the fourth ligand is bound. Although each modified Hb shows only two distinct rates, the kinetic data as a function of dissociation level cannot be simulated with a simple two-state model. A critical length is observed for the maximum shift toward the low affinity T-state. Longer or shorter lengths of the cross-linker yielded more high affinity R-state. Unlike native Hb, which is in equilibrium with free dimers, the cross-linked Hbs maintain the fraction slow kinetics, which is unique to Hb tetramers, even at 0.5 microM (total heme). Addition of HbCN to unmodified HbCO solutions results in dimer exchange, which decreases the relative fraction of slow bimolecular kinetics; the cross-linked Hbs did not show such an effect, indicating that they do not participate in dimer exchange.     

Functional consequences of mutations at the allosteric interface in hetero- and homo-hemoglobin tetramers.

A seminal difference exists between the two types of chains that constitute the tetrameric hemoglobin in vertebrates. While alpha chains associate weakly into dimers, beta chains self-associate into tightly assembled tetramers. While heterotetramers bind ligands cooperatively with moderate affinity, homotetramers bind ligands with high affinity and without cooperativity. These characteristics lead to the conclusion that the beta 4 tetramer is frozen in a quaternary R-state resembling that of liganded HbA. X-ray diffraction studies of the liganded beta 4 tetramers and molecular modeling calculations revealed several differences relative to the native heterotetramer at the "allosteric" interface (alpha 1 beta 2 in HbA) and possibly at the origin of a large instability of the hypothetical deoxy T-state of the beta 4 tetramer. We have studied natural and artificial Hb mutants at different sites in the beta chains responsible for the T-state conformation in deoxy HbA with the view of restoring a low ligand affinity with heme-heme interaction in homotetramers. Functional studies have been performed for oxygen equilibrium binding and kinetics after flash photolysis of CO for both hetero- and homotetramers. Our conclusion is that the "allosteric" interface is so precisely tailored for maintaining the assembly between alpha beta dimers that any change in the side chains of beta 40 (C6), beta 99 (G1), and beta 101 (G3) involved in the interface results in increased R-state behavior. In the homotetramer, the mutations at these sites lead to the destabilization of the beta 4 hemoglobin and the formation of lower affinity noncooperative monomers.     

Reduced sensitivity of O2 transport to allosteric effectors and temperature in loggerhead sea turtle hemoglobin: functional and spectroscopic study.

The functional and spectroscopic (EPR and absorbance) properties of the adult loggerhead sea turtle (Caretta caretta) hemoglobin have been studied with special reference to the action of allosteric effectors and temperature. Present results indicate that turtle Hb displays a very low O2 affinity and a very small sensitivity to allosteric effectors and temperature. Furthermore, the amplitude of the Bohr effect for O2 binding is strongly reduced. In parallel, EPR and absorbance spectroscopic properties of the nitrosylated derivative of turtle Hb suggest that the hemoprotein is in a low-affinity conformation, even in the absence of allosteric effectors. These findings suggest the existence of unusual molecular mechanisms modulating the basic reaction of Hb with O2, which may be linked to specific physiological needs related to the diving behavior of the turtle.     

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.     

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.     

The X-ray structure determination of bovine carbonmonoxy hemoglobin at 2.1 A resoultion and its relationship to the quaternary structures of other hemoglobin crystal froms

Crystallographic studies of the intermediate states between unliganded and fully liganded hemoglobin (Hb) have revealed a large range of subtle but functionally important structural differences. Only one T state has been reported, whereas three other quaternary states (the R state, B state, and R2 or Y state) for liganded Hb have been characterized; other studies have defined liganded Hbs that are intermediate between the T and R states. The high-salt crystal structure of bovine carbonmonoxy (CO bovine) Hb has been determined at a resolution of 2.1 A and is described here. A detailed comparison with other crystallographically solved Hb forms (T, R, R2 or Y) shows that the quaternary structure of CO bovine Hb closely resembles R state Hb. However, our analysis of these structures has identified several important differences between CO bovine Hb and R state Hb. Compared with the R state structures, the beta-subunit N-terminal region has shifted closer to the central water cavity in CO bovine Hb. In addition, both the alpha- and beta-subunits in CO bovine Hb have more constrained heme environments that appear to be intermediate between the T and R states. Moreover, the distal pocket of the beta-subunit heme in CO bovine Hb shows significantly closer interaction between the bound CO ligand and the Hb distal residues Val 63(E11) and His 63(E7). The constrained heme groups and the increased steric contact involving the CO ligand and the distal heme residues relative to human Hb may explain in part the low intrinsic oxygen affinity of bovine Hb.     

Dependence of the quantum efficiency for photolysis of carboxyhemoglobin on the degree of ligation.

A combined stopped flow-laser photolysis apparatus was used to measure the quantum efficiency for removal of carbon monoxide bound to human hemoglobin as a function of fractional CO saturation. This flow-flash technique allows the properties of partially liganded hemoglobin molecules, which are sparsely populated under equilibrium conditions, to be conveniently studied. Experiments performed at pH 7 and 20 degrees C both in the presence and absence of phosphates gave a similar dependence of quantum efficiency on fractional saturation. The observed quantum efficiency was 0.90 +/- 0.06 at 10% saturation and decreased to 0.47 +/- 0.02 as full saturation was approached. An allosteric model in which Hb(CO)1 has a quantum efficiency of 0.99 while other liganded species have quantum efficiencies of 0.47 was used to produce a good simulation of the results.     

Antagonistic interaction between oxygenation-linked lactate and CO2 binding to human hemoglobin

Oxygen binding to hemoglobin (Hb) depends on allosteric effectors (CO(2), lactate and protons) that may increase drastically in concentration during exercise. The effectors share common binding sites on the Hb molecules, predicting mutual interaction in their effects on Hb (de)oxygenation. We analysed the effects of lactate and CO(2), separately and in combination, on O(2) binding of purified human Hb at 37 degrees C and physiological pH and chloride values. We demonstrate pH-dependent, inhibitory interactions between lactate binding and CO(2) binding (carbamate formation); at pH 7.4, physiological CO(2) tension ( approximately 43 mm Hg) reduced lactate binding more markedly ( approximately 75%), than lactate (50 mM) inhibited carbamate formation ( approximately 25%). In contrast to previous studies on blood and Hb solutions, we moreover find that added lactate neither 'reverses' oxylabile carbamate formation (resulting in lower carbamate levels in deoxyHb than in oxyHb) nor exerts greater allosteric effects on Hb-O(2) affinity than equal increases in chloride ion concentrations.     

Ligand binding in the ferric and ferrous states of Paramecium hemoglobin

The unicellular protozoan Paramecium caudatum contains a monomeric hemoglobin (Hb) that has only 116 amino acid residues. This Hb shares the simultaneous presence of a distal E7 glutamine and a B10 tyrosine with several invertebrate Hbs. In the study presented here, we have used ligand binding kinetics and resonance Raman spectroscopy to characterize the effect of the distal pocket residues of Paramecium Hb in stabilizing the heme-bound ligands. In the ferric state, the high-spin to low-spin (aquo-hydroxy) transition takes place with a pK(a) of approximately 9.0. The oxygen affinity (P(50) = 0.45 Torr) is similar to that of myoglobin. The oxygen on- and off-rates are also similar to those of sperm whale myoglobin. Resonance Raman data suggest hydrogen bonding stabilization of bound oxygen, evidenced by a relatively low frequency of Fe-OO stretching (563 cm(-1)). We propose that the oxy complex is an equilibrium mixture of a hydrogen-bonded closed structure and an open structure. Oxygen will dissociate preferentially from the open structure, and therefore, the fraction of open structure population controls the rate of oxygen dissociation. In the CO complex, the Fe-CO stretching frequency at 493 cm(-1) suggests an open heme pocket, which is consistent with the higher on- and off-rates for CO relative to those in myoglobin. A high rate of ligand binding is also consistent with the observation of an Fe-histidine stretching frequency at 220 cm(-1), indicating the absence of significant proximal strain. We postulate that the function of Paramecium Hb is to supply oxygen for cellular oxidative processes.     

Geminate ligand recombination as a probe of the R, T equilibrium in hemoglobin

Flash photolysis kinetics of carbon monoxide hemoglobin show a decrease in the fraction of ligand recombination occurring as geminate when the hemoglobin has fewer ligands bound. Fully saturated samples, normally referred to as R state, show approximately 50% geminate phase, while samples at low saturation (T state) show less than 3%. The latter result was obtained by photolysis of samples with a short delay after stopped flow of solutions of deoxy hemoglobin (Hb) and ligand. The decrease in the fraction of geminate phase was also observed using a double flash technique. The transient mixture of R and T states generated by flash photolysis of Hb-CO was probed with a weaker time-delayed photolysis pulse. The kinetics of both the geminate and bimolecular phases following the second pulse were measured. The fraction geminate signal was least at delays where the maximum proportion of liganded T state tetramer is expected. The biphasic bimolecular process is also an indicator of the allosteric state of Hb. The populations of R and T may be determined from the overall ligand recombination kinetics; however, the analysis is model-dependent. The fraction geminate reaction may provide a rapid measure of the amount of liganded hemes in the R and T states.     

Ligand binding properties of hemoglobin 3 of the trout, Salmo gairdneri. The occurrence of an acid Bohr effect in the absence of heme-heme interaction.

The four components of hemoglobin from the rainbow trout (Salmo gairdneri) have been isolated. The oxygen affinities of the first two components eluted from the DEAE-cellulose column have much smaller pH dependencies than the last two components. These components have very low O2 affinities at low pH. The effect of pH on the equilibrium and kinetics of ligand binding to the third fraction, the pH-dependent component present in greatest amounts, has been studied. Measurements of ligand binding equilibria demonstrate the presence of both an alkaline and an acid Bohr effect. In the region of the alkaline Bohr effect the value of n in the Hill equation is a function of ligand affinity. For CO binding n decreases as the pH is decreased until at pH 6, the minimum ligand affinity is reached. At this pH there is also a complete loss of cooperative ligand binding. Decreasing the pH further results in an increase of ligand affinity, but this acid Bohr effect is not associated with a reappearance of cooperativity. This suggests that Fraction 3 of S. gairdneri is frozen in the low affinity, deoxygenated conformation at low pH and that the quaternary structure does not change even when fully liganded. However, the properties of the low affinity conformation of this hemoglobin are pH-dependent.     

Crystal structure of unliganded phosphofructokinase from Escherichia coli

In an attempt to characterize the mechanism of co-operativity in the allosteric enzyme phosphofructokinase from Escherichia coli, crystals were grown in the absence of activating ligands. The crystal structure was determined to a resolution of 2.4 A by the method of molecular replacement, using the known structure of the liganded active state as a starting model, and has been refined to a crystallographic R-factor of 0.168 for all data. Although the crystallization solution would be expected to contain the enzyme in its inactive conformation, with a low affinity for the co-operative substrate fructose 6-phosphate, the structure in these crystals does not show the change in quaternary structure seen in the inactive form of the Bacillus stearothermophilus enzyme (previously determined at low resolution), nor does it show any substantial change in the fructose 6-phosphate site from the structure seen in the liganded form. Compared to the liganded form, there are considerable changes around the allosteric effector site, including the disordering of the last 19 residues of the chain. It seems likely that the observed conformation corresponds an active unliganded form, in which the absence of ligand in the effector site induces structural changes that spread through much of the subunit, but cause only minor changes in the active site. It is not clear why the crystals should contain the enzyme in a high-affinity conformation, which presumably represents only a small fraction of the molecules in the crystallizing solution. However, this structure does identify the conformational changes involved in binding of the allosteric effectors.     

Additive effects of beta chain mutations in low oxygen affinity hemoglobin betaF41Y,K66T.

In order to decrease significantly the oxygen affinity of human hemoglobin, we have associated the mutation betaF41Y with another point mutation also known to decrease the oxygen affinity of Hb. We have synthesized a recombinant Hb (rHb) with two mutations in the beta chains: rHb betaF41Y,K66T. In the absence of 2, 3-diphosphoglycerate, additive effects of the mutations are evident, since the doubly mutated Hb exhibits a larger decrease in oxygen affinity than for the individual single mutations. In the presence of 2,3-diphosphoglycerate, the second mutation did not significantly increase the P(50) value relative to the single mutations. However, the kinetics of CO binding still indicate combined effects on the allosteric equilibrium, as evidenced by more of the slow bimolecular phase characteristic of binding to the deoxy conformation. Dimer-tetramer equilibrium studies indicate an increase in stability of the mutants relative to rHb A; the double mutant rHb betaF41Y, K66T at pH 7.5 showed a K(4,2) value of 0.26 microM. Despite the lower oxygen affinity, the single mutant betaF41Y and double mutant betaF41Y,K66T show only a moderate increase of 20% in the autoxidation rate. These mutations are thus of interest in developing a Hb-based blood substitute.     

Probing the conformation of hemoglobin presbyterian in the R-state

The influence of allosteric effectors on the R-state (liganded) conformation of Tg-HbP (human hemoglobin Presbyterian expressed in transgenic pig) has been probed using a number of biophysical techniques, and the results have been compared with that of liganded of HbA (human normal adult hemoglobin) to gain insight into the molecular basis of Asn-108()->Lys mutation–induced low-oxygen affinity of Hb. The nuclear magnetic resonance studies of Tg-HbP revealed that the conformation of the ₁₁ and ₁₂ interfaces of the protein in the deoxy state are indistinguishable from that of deoxy HbA, whereas the conformation of the microenvironment of His-103() of Tg-HbP, a residue of the ₁₁ interface, is distinct from that of HbA in the R-state. In addition, the Presbyterian mutation also influences the structure of oxy Hb in other regions of the molecule. First, it facilitates the generation of deoxy (T)-state marker at 14.2 ppm (from 2,2-dimethyl-s-silapentane-5-sulfonate) on the interaction of oxy Hb with inositol hexa-phosphate without changing the ligation state. Second, it increases the geminate yield of the 10 ns photoproduct of CO-Hb. Third, it enhances the propensity of phosphate to increase the geminate yield. Fourth, it potentiates the ability of phosphate to induce deoxy-like features at the heme environment in the R-state. Fifth, it induces T-state-like signatures at the switch and hinge regions of the ₁₂ interface. Finally, molecular modeling studies have indicated an increased affinity for the four anion binding sites mapped in the midcentral cavity of Hb caused by the presence of Lys-108(). In short, Lys-108() in HbP induces a propensity for oxy Hb to access T-like conformational features in different regions of the oxy Hb molecule and also enhances the T-like signatures in the oxy state on interaction with allosteric effectors without changing its ligation. Interestingly, the intrinsic T-like conformational features of the R-state of HbP, in addition to those induced by the addition of allosteric effectors to liganded HbP, appear to be reminiscent of features of the B-state conformation of Hb found in rHb 1.1 (recombinant hemoglobin). We propose that the lowered oxygen affinity of Tg-HbP in the presence of allosteric effectors is a consequence of an altered R-state conformation of Hb, which reflects the facilitation of switching the R-state of HbP to the T-state compared with the normal R-state of HbA, thereby reducing HbA's affinity to oxygen.     

Interaction of butene with human hemoglobin A.

The binding of various alkanes by proteins was recognized years ago. We have studied the effect of butene (C4H8), a short-chain aliphatic hydrocarbon, on the functional properties of human adult hemoglobin. Under 1 atm pressure (100 kPa) butene decreased the affinity of hemoglobin (Hb) for oxygen (p50) by 45% without altering the cooperativity of ligand binding. This effect was independent of pH (from 7.0 to 8.0) and of ionic strength. The changes in the affinity of hemoglobin for oxygen were dependent upon the partial pressure of butene and evoked a saturating mechanism of the binding site(s). Mathematical simulation of the curve relating p50 to the concentration of dissolved butene allowed us to calculate the apparent association constants for one single binding site KHb = 10.4 mmol-1 and KHbO2 = 1.53 mmol-1 to Hb and HbO2 respectively. The larger binding of butene by Hb was confirmed by a 25% decrease in K1, the first association constant of oxygen to the tetrameric hemoglobin. It is concluded that butene is an allosteric effector of human Hb which acts most likely through hydrophobic interactions. It is postulated that the oxygen-linked binding site may be located at the alpha 1 beta 2 interface.     

The enigma of the liganded hemoglobin end state: a novel quaternary structure of human carbonmonoxy hemoglobin

The liganded hemoglobin (Hb) high-salt crystallization condition described by Max Perutz has generated three different crystals of human adult carbonmonoxy hemoglobin (COHbA). The first crystal is isomorphous with the "classical" liganded or R Hb structure. The second crystal reveals a new liganded Hb quaternary structure, RR2, that assumes an intermediate conformation between the R form and another liganded Hb quaternary structure, R2, which was discovered more than a decade ago. Like the R2 structure, the diagnostic R state hydrogen bond between beta2His97 and alpha1Thr38 is missing in the RR2 structure. The third crystal adopts a novel liganded Hb conformation, which we have termed R3, and it shows substantial quaternary structural differences from the R, RR2, and R2 structures. The quaternary structure differences between T and R3 are as large as those between T and R2; however, the T --> R3 and T --> R2 transitions are in different directions as defined by rigid-body screw rotation. Moreover, R3 represents an end state. Compared to all known liganded Hb structures, R3 shows remarkably reduced strain at the alpha-heme, reduced steric contact between the beta-heme ligand and the distal residues, smaller alpha- and beta-clefts, and reduced alpha1-alpha2 and beta1-beta2 iron-iron distances. Together, these unique structural features in R3 should make it the most relaxed and/or greatly enhance its affinity for oxygen compared to the other liganded Hbs. The current Hb structure-function relationships that are now based on T --> R, T -->R --> R2, or T --> R2 --> R transitions may have to be reexamined to take into account the RR2 and R3 liganded structures.