Interface sliding as illustrated by the multiple quaternary structures of liganded hemoglobin

Initial crystallographic studies suggested that fully liganded mammalian hemoglobin can adopt only a single quaternary structure, the quaternary R structure. However, more recent crystallographic studies revealed the existence of a second quaternary structure for liganded hemoglobin, the quaternary R2 structure. Since these quaternary structures can be crystallized, both must be energetically accessible structures that coexist in solution. Unanswered questions include (i) the relative abundance of the R and R2 structures under various solution conditions and (ii) whether other quaternary structures are energetically accessible for the liganded alpha(2)beta(2) hemoglobin tetramer. Although crystallographic methods cannot directly answer the first question, they represent the most direct and most accurate approach to answering the second question. We now have determined and refined three different crystal structures of bovine carbonmonoxyhemoglobin. These structures provide clear evidence that the dimer-dimer interface of liganded hemoglobin has a wide range of energetically accessible structures that are related to each other by a simple sliding motion. The dimer-dimer interface acts as a "molecular slide bearing" that allows the two alpha beta dimers to slide back and forth without greatly altering the number or the nature of the intersubunit contacts. Since the general stereochemical features of this interface are not unusual, it is likely that interface sliding of the kind displayed by fully liganded hemoglobin plays important structural and functional roles in many other protein assemblies.     

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.     

The mutation beta 99 Asp-Tyr stabilizes Y--a new, composite quaternary state of human hemoglobin

Carbonmonoxy hemoglobin Ypsilanti (beta 99 Asp-Tyr) exhibits a quaternary form distinctly different from any structures previously observed for human hemoglobins. The relative orientation of alpha beta dimers in the new quaternary form lies well outside the range of values observed for normal unliganded and liganded tetramers (Baldwin, J., Chothia, C., J. Mol. Biol. 129:175-220, 1979). Despite this large quaternary structural difference between carbonmonoxy hemoglobin Ypsilanti and the two canonical structures, the new quaternary structure's hydrogen bonding interactions in the "switch" region, and packing interactions in the "flexible joint" region, show noncovalent interactions characteristic of the alpha 1 beta 2 contacts of both unliganded and liganded normal hemoglobins. In contrast to both canonical structures, the beta 97 histidine residue in carbonmonoxy hemoglobin Ypsilanti is disengaged from quaternary packing interactions that are generally believed to enforce two-state behavior in ligand binding. These features of the new quaternary structure, denoted Y, may therefore be representative of quaternary states that occur transiently along pathways between the normal unliganded, T, and liganded, R, hemoglobin structures.     

A third quaternary structure of human hemoglobin A at 1.7-A resolution.

Previous crystallographic studies have shown that human hemoglobin A can adopt two stable quaternary structures, one for deoxyhemoglobin (the T-state) and one for liganded hemoglobin (the R-state). In this paper we report our finding of a second quaternary structure (the R2-state) for liganded hemoglobin A. The magnitudes of the spatial differences between the R- and R2-states are as large as those between the R- and T-states. Of particular interest are the structural changes that occur as a result of R-T and R-R2 transitions at the so-called "switch" region of the critical alpha 1 beta 2 interface. In the R-state, His-97 beta 2 is positioned between Thr-38 alpha 1 and Thr-41 alpha 1, whereas in transition to the T-state His 97 beta 2 must "jump" a turn in the alpha 1 C helix to form nonpolar contacts with Thr-41 alpha 1 and Pro-44 alpha 1. This facet of the R-T transition presents a major steric barrier to the quaternary structure change. In the R2-state, His-97 beta 2 simply rotates away from threonines 38 alpha 1 and 41 alpha 1, breaking contact with these residues and allowing water access to the center of the alpha 1 beta 2 interface. With the switch region in an open position in the R2-state, His-97 beta 2 should be able to move by Thr-41 alpha 1 and make the transition to the T-state with a steric barrier that is less than that for the R-T transition. Thus the R2-state may function as a stable intermediate along a R-R2-T pathway. The T-, R-, and R2-states must coexist in solution. That is, the fact that these states can be crystallized implies that they are all energetically accessible structures. What remains to be determined are the T-to-R, T-to-R2, and R-to-R2 equilibrium constants for hemoglobin under various solution conditions and ligation states. Although this may prove to be difficult, we discuss previously published results which indicate that low concentrations of inorganic anions or low pH may favor the R2-state and at least one alpha 1 beta 2 interface mutation stabilizes a quaternary structure that is very similar to the R2-state.     

Conformational changes in hemoglobin S (betaE6V) imposed by mutation of the beta Glu7-beta Lys132 salt bridge and detected by UV resonance Raman spectroscopy

The impact upon molecular structure of an additional point mutation adjacent to the existing E6V mutation in sickle cell hemoglobin was probed spectroscopically. The UV resonance Raman results show that the conformational consequences of mutating the salt bridge pair, betaGlu(7)-betaLys(132), are dependent on which residue of the pair is modified. The betaK132A mutants exhibit the spectroscopic signatures of the R --> T state transition in both the "hinge" and "switch" regions of the alpha(1)beta(2) interface. Both singly and doubly mutated hemoglobin (Hb) betaepsilon7Alpha exhibit the switch region signature for the R --> T quaternary state transition but not the hinge signature. The absence of this hinge region-associated quaternary change is the likely origin of the observed increased oxygen binding affinity for the Hb betaepsilon7Alpha mutants. The observed large decrease in the W3 alpha14beta15 band intensity for doubly mutated Hb betaepsilon7Alpha is attributed to an enhanced separation in the A helix-E helix tertiary contact of the beta subunits. The results for the Hb A betaGlu(7)-betaLys(132) salt bridge mutants demonstrate that attaining the T state conformation at the hinge region of the alpha(1)beta(2) dimer interface can be achieved through different intraglobin pathways; these pathways are subject to subtle mutagenic manipulation at sites well removed from the dimer interface.     

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.     

Coarse‐grained and all‐atom modeling of structural states and transitions in hemoglobin

Hemoglobin (Hb), an oxygen‐binding protein composed of four subunits (α1, α2, β1, and β2), is a well‐known example of allosteric proteins that are capable of cooperative ligand binding. Despite decades of studies, the structural basis of its cooperativity remains controversial. In this study, we have integrated coarse‐grained (CG) modeling, all‐atom simulation, and structural data from X‐ray crystallography and wide‐angle X‐ray scattering (WAXS), aiming to probe dynamic properties of the two structural states of Hb (T and R state) and the transitions between them. First, by analyzing the WAXS data of unliganded and liganded Hb, we have found that the structural ensemble of T or R state is dominated by one crystal structure of Hb with small contributions from other crystal structures of Hb. Second, we have used normal mode analysis to identify two distinct quaternary rotations between the α1β1 and α2β2 dimer, which drive the transitions between T and R state. We have also identified the hot‐spot residues whose mutations are predicted to greatly change these quaternary motions. Third, we have generated a CG transition pathway between T and R state, which predicts a clear order of quaternary and tertiary changes involving α and β subunits in Hb. Fourth, we have used the accelerated molecular dynamics to perform an all‐atom simulation starting from the T state of Hb, and we have observed a transition toward the R state of Hb. Further analysis of crystal structural data and the all‐atom simulation trajectory has corroborated the order of quaternary and tertiary changes predicted by CG modeling. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Proton nuclear magnetic resonance studies of hemoglobin Malm?: implications of mutations at homologous positions of the alpha and beta chains.

The abnormal human hemoglobin Malm? (beta97FG4 His leads to Gln) has been studied and its properties are compared with those of normal adult hemoglobin A. The data presented here show that the ring-current shifted proton resonances of both HbCO and HbO2 Malm? are very different from the corresponding forms of Hb A. The hyperfine shifted proton resonances of deoxy-Hb Malm? do not differ drastically from those of deoxy-Hb A. This result, together with the finding that the exchangeable proton resonances of the deoxy form of the two hemoglobins are similar, suggests that unliganded Hb Malm? can assume a deoxy-like quaternary structure both in the absence and presence of organic phosphates We have also compared the properties of Hb Malm? with those of Hb Chesapeake (alpha92FG4 Arg leads to Leu). This allows us to study the properties of two abnormal human hemoglobins with mutations at homologous positions of the alpha and beta chains in the three-dimenstional structure of the hemoglobin molecule. Our present results suggest that the mutaion at betaFG4 has its greatest effect on the teritiary structure of the heme pocket of the liganded forms of the hemoglobin while the mutation at alphaFG4 alters the deoxy structure of the hemoglogin molecule but does not alter the teriary structure of the heme pockets of the liganded form of the hemoglobin molecule. Both hemoglobins undergo a transition from the deoxy (T) to the oxy (R) quaternary structure upon ligation. The abnormally high oxygen affinities and low cooperativities of these two hemoglobins must therefore be due to either the structural differences which we have observed and/or to an altered transition between the T and R structures.     

Coupling of tertiary and quaternary changes in human hemoglobin: a 1D and 2D NMR study of hemoglobin Saint Mandé (beta N102Y)

Hemoglobin Saint Mandé (beta N102Y) is a low-affinity mutant with the substitution site situated in the quaternary-sensitive alpha 1 beta 2 interface. In adult hemoglobin the Asn102 beta contributes to the stability of the liganded (R) state, forming a hydrogen bond with Asp94 alpha. The quaternary and tertiary perturbations subsequent to the Tyr for Asn substitution in monocarboxylated hemoglobin Saint Mandé have been investigated by one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy. Analysis of the one-dimensional NMR spectra of the liganded and unliganded samples in 1H2O provides evidence that both R and T quaternary structures of Hb Saint Mandé are different from the corresponding ones in HbA. In the monocarboxylated form of the mutant hemoglobin, at acid pH, we have observed the disappearance of an R-type hydrogen bond and the appearance of a new one whose proton resonates like a deoxy T marker. Using two-dimensional NMR methods and on the basis of previous results on the monocarboxylated HbA, we have obtained a significant number of resonance assignments in the spectra of monocarboxylated Hb Saint Mandé at pH 5.6 in the presence or absence of a strong allosteric effector, inositol hexaphosphate. This enabled us to characterize the tertiary conformational changes (relative to the liganded normal hemoglobin) triggered by the quaternary-state modification. The observed structural variations are confined within the heme pocket regions but concern both the alpha and beta subunits. Most of them, localized in the C, F, G, and FG segments, could result directly from the side-chain substitution, while others, such as Leu141 beta, could be explained only by long-range interactions.     

Evolution of allosteric models for hemoglobin

We compare various allosteric models that have been proposed to explain cooperative oxygen binding to hemoglobin, including the two-state allosteric model of Monod, Wyman, and Changeux (MWC), the Cooperon model of Brunori, the model of Szabo and Karplus (SK) based on the stereochemical mechanism of Perutz, the generalization of the SK model by Lee and Karplus (SKL), and the Tertiary Two-State (TTS) model of Henry, Bettati, Hofrichter and Eaton. The preponderance of experimental evidence favors the TTS model which postulates an equilibrium between high (r)- and low (t)-affinity tertiary conformations that are present in both the T and R quaternary structures. Cooperative oxygenation in this model arises from the shift of T to R, as in MWC, but with a significant population of both r and t conformations in the liganded T and in the unliganded R quaternary structures. The TTS model may be considered a combination of the SK and SKL models, and these models provide a framework for a structural interpretation of the TTS parameters. The most compelling evidence in favor of the TTS model is the nanosecond - millisecond carbon monoxide (CO) rebinding kinetics in photodissociation experiments on hemoglobin encapsulated in silica gels. The polymeric network of the gel prevents any tertiary or quaternary conformational changes on the sub-second time scale, thereby permitting the subunit conformations prior to CO photodissociation to be determined from their ligand rebinding kinetics. These experiments show that a large fraction of liganded subunits in the T quaternary structure have the same functional conformation as liganded subunits in the R quaternary structure, an experimental finding inconsistent with the MWC, Cooperon, SK, and SKL models, but readily explained by the TTS model as rebinding to r subunits in T. We propose an additional experiment to test another key prediction of the TTS model, namely that a fraction of subunits in the unliganded R quaternary structure has the same functional conformation (t) as unliganded subunits in the T quaternary structure.     

A study on the quaternary structure change of hemoglobin in the ligation process

In order to inquire into the molecular mechanism underlying the cooperative ligand binding to hemoglobin (Hb), conformational interaction at the interfaces between subunits are investigated on the basis of the atomic coordinates of human deoxy and human carbonmonoxy Hbs. Hypothetical intermediate structures are used, each of which is obtained from the procedure where one or more subunits in deoxy Hb are replaced by the corresponding CO-liganded subunits in carbonmonoxy Hb using the method of superimposition of two sets of atomic coordinates. When either alpha or beta subunit is substituted with the corresponding subunit in carbonmonoxy Hb, serious steric hindrances are produced between alpha 1FG4(92)Arg and beta 2C3(37)Trp or between alpha 1C6(41)Thr and beta 2FG4(97)His, all of which belong to the allosteric core affected directly by ligand binding. These steric hindrances become more serious when both alpha 1(alpha 2) and beta 2(beta 1) subunits are substituted. Therefore the change in the relative distance between iron atom and porphyrin by ligation results in strain in the C-terminal residues as an effect of the steric hindrance between the FG and C segments. However, no steric hindrance can be seen between subunits when the subunits in carbonmonoxy Hb are substituted with the corresponding subunits in deoxy Hb. The nature of the quaternary structural change from liganded to deoxy Hb seems to be different from that from deoxy to liganded Hb.     

Effect of ligands of ferric hemes on interaction between ferric and ferrous chains in partially oxidized hemoglobin A.

Normal values of Bohr effect of oxygenation of partially oxidized hemoglobin A with ferrihemes liganded either with H2O and OH or with CN have been found in the range of pH values from 6.8 to 7.6 in 45 micrometer (Fe)-hemoglobin containing 36--38% of ferrihemes. As the changes of oxygen affinity of Hb A induced by changes of pH are due to the modifications of R state, this quaternary conformation is thought to be unchanged in the studied of R state, this quaternary conformation is thought to be unchanged in the studied forms of partially oxidized hemoglobin. It is suggested that interactions between ferric and ferrous hemes leading to the increased affinity of ferrous hemes to oxygen occur in deoxygenated form of partially oxidized hemoglobin. In partially oxidized hemoglobin with ferric hemes liganded with H2O asymmetry of oxygen binding curves has been noted, which is not observed in forms with ferric hemes liganded with OH ot CN. This shows the effect of ligands of ferric hemes on interactions between chains containing ferric and ferrous hemes.     

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.     

Enhanced inhibition of polymerization of sickle cell hemoglobin in the presence of recombinant mutants of human fetal hemoglobin with substitutions at position 43 in the gamma-chain

Four recombinant mutants of human fetal hemoglobin [Hb F (alpha2gamma2)] with amino acid substitutions at the position 43 of the gamma-chain, rHb (gammaD43L), rHb (gammaD43E), rHb (gammaD43W), and rHb (gammaD43R), have been expressed in our Escherichia coli expression system and used to investigate their inhibitory effect on the polymerization of deoxygenated sickle cell hemoglobin (Hb S). Oxygen-binding studies show that rHb (gammaD43E), rHb (gammaD43W), and rHb (gammaD43R) exhibit higher oxygen affinity than human normal adult hemoglobin (Hb A), Hb F, or rHb (gammaD43L), and all four rHbs are cooperative in binding O2. Proton nuclear magnetic resonance (NMR) studies of these four rHbs indicate that the quaternary and tertiary structures around the heme pockets are similar to those of Hb F in both deoxy (T) and liganded (R) states. Solution light-scattering experiments indicate that these mutants remain mostly tetrameric in the liganded (R) state. In equimolar mixtures of Hb S and each of the four rHb mutants (gammaD43L, gammaD43E, gammaD43R, and gammaD43W), the solubility (Csat) of each of the pairs of Hbs is higher than that of a similar mixture of Hb S and Hb A, as measured by dextran-Csat experiments. Furthermore, the Csat values for Hb S/rHb (gammaD43L), Hb S/rHb (gammaD43E), and Hb S/rHb (gammaD43R) mixtures are substantially higher than that for Hb S/Hb F. The results suggest that these three mutants of Hb F are more effective than Hb F in inhibiting the polymerization of deoxy-Hb S in equimolar mixtures.     

Quaternary relaxations in sol-gel encapsulated hemoglobin studied via NIR and UV spectroscopy

In this work, we study the kinetics of the R --> T transition in hemoglobin using a combination of near-infrared and near-ultraviolet spectroscopy. We use a sol-gel encapsulation protocol to decelerate the conformational transitions and to avoid spectral perturbations arising from ligand migration and recombination. We monitor two spectroscopic markers: band III in the near-IR, which is a fine probe of the heme pocket conformation, and the tryptophan band in the near-UV, which probes the formation of the Trpbeta37-Aspalpha94 hydrogen bond, characteristic of the T structure, at the critical alpha1beta2 subunit interface. The time evolution of these two bands is monitored after deoxygenation of encapsulated oxyhemoglobin, obtained by diffusion of a reducing agent into the porous silica matrix. Characteristic spectral shifts are observed: comparison with myoglobin enables us to assign them to quaternary structure relaxations. Band III spectral relaxation is clearly nonexponential, and analysis with the Maximum Entropy Method enables us to identify three processes. On the other hand, near-UV spectral relaxation follows an exponential decay with a time constant closely corresponding to the second process observed in the near IR. Very interestingly, the rates of all processes markedly depend on the viscosity of the co-encapsulated solvent, following a power law. Our results reveal correlations between heme pocket relaxations, induced by the R --> T transition, and structural event(s) occurring at the alpha1beta2 interface and highlight their solvent dependence. The power law viscosity dependence of relaxation rates suggests that the observed protein relaxations are "slaved" to the co-encapsulated solvent. The stepwise character of the quaternary transition is also evidenced.     

Site-directed mutations of human hemoglobin at residue 35beta: a residue at the intersection of the alpha1beta1, alpha1beta2, and alpha1alpha2 interfaces

Because Tyr35beta is located at the convergence of the alpha1beta1, alpha1beta2, and alpha1alpha2 interfaces in deoxyhemoglobin, it can be argued that mutations at this position may result in large changes in the functional properties of hemoglobin. However, only small mutation-induced changes in functional and structural properties are found for the recombinant hemoglobins betaY35F and betaY35A. Oxygen equilibrium-binding studies in solution, which measure the overall oxygen affinity (the p50) and the overall cooperativity (the Hill coefficient) of a hemoglobin solution, show that removing the phenolic hydroxyl group of Tyr35beta results in small decreases in oxygen affinity and cooperativity. In contrast, removing the entire phenolic ring results in a fourfold increase in oxygen affinity and no significant change in cooperativity. The kinetics of carbon monoxide (CO) combination in solution and the oxygen-binding properties of these variants in deoxy crystals, which measure the oxygen affinity and cooperativity of just the T quaternary structure, show that the ligand affinity of the T quaternary structure decreases in betaY35F and increases in betaY35A. The kinetics of CO rebinding following flash photolysis, which provides a measure of the dissociation of the liganded hemoglobin tetramer, indicates that the stability of the liganded hemoglobin tetramer is not altered in betaY35F or betaY35A. X-ray crystal structures of deoxy betaY35F and betaY35A are highly isomorphous with the structure of wild-type deoxyhemoglobin. The betaY35F mutation repositions the carboxyl group of Asp126alpha1 so that it may form a more favorable interaction with the guanidinium group of Arg141alpha2. The betaY35A mutation results in increased mobility of the Arg141alpha side chain, implying that the interactions between Asp126alpha1 and Arg141alpha2 are weakened. Therefore, the changes in the functional properties of these 35beta mutants appear to correlate with subtle structural differences at the C terminus of the alpha-subunit.     

Structure of deoxy hemoglobin C (beta six Glu replaced by Lys) in two crystal forms

Five crystal forms of the abnormal human hemoglobin Hb³ C (beta six Glu → Lys) have been grown. Two of them are grown with liganded Hb C, three with deoxy Hb C. The structures of two of the deoxy crystal forms were determined by the method of molecular replacement, using deoxy Hb A as the model structure. Fourier maps were calculated for each Hb C structure, using data to a resolution of 5 Å in one case and 4 Å in the second case. The structural differences between each deoxy Hb C structure and the deoxy Hb A model are found mostly at the molecular surface. Energetically favorable interactions involving the variant residue, beta six lysine, occur in both Hb C crystal forms, and could explain the lowered solubility and enhanced tendency of deoxy Hb C to crystallize in vivo.     

The molecular code for hemoglobin allostery revealed by linking the thermodynamics and kinetics of quaternary structural change. 2. Cooperative free energies of (alphaFeCObetaFe)2 and (alphaFebetaFeCO)2 T-state tetramers

Ligand photodissociation experiments are used to measure the prephotolysis equilibria between doubly liganded R and T quaternary conformers of the symmetric Fe-Co HbCO hybrids, (alpha(FeCO)beta(Co))(2) and (alpha(Co)beta(FeCO))(2). The free energies obtained from these data are used to calculate the cooperative free energies of the (alpha(FeCO)beta(Fe))(2) and (alpha(Fe)beta(FeCO))(2) intermediate CO-ligation states of normal hemoglobin in the T conformation, quantities important to the evaluation of current models of cooperativity. The symmetry rule model, incorporating sequential cooperativity of T-state ligand binding within an alphabeta dimer in addition to the traditional two-state cooperativity of the tetramer, predicts a larger free energy penalty for disturbing both dimers in a doubly liganded T tetramer than would be expected in the two-state model as currently formulated. (Cooperative energy penalties are simply proportional to the number of tetramer-bound ligands in the traditional two-state model.) The value found here for the energies of doubly liganded T microstates in which both dimers are perturbed, 7.9 +/- 0.3 kcal/mol, is consistent with the symmetry rule model but significantly higher than that expected (5-6 kcal/mol) in the two-state model of cooperativity.     

Structure-function relationships in hemoglobin Kariya, Lys-40(C5) alpha----Glu, with high oxygen affinity. Functional role of the salt bridge between Lys-40 alpha and the beta chain COOH terminus

The epsilon-amino group of Lys-40 alpha forms a salt bridge with the alpha-carboxyl group of beta chain in deoxyhemoglobin and is considered to impose a constraint upon hemoglobin tetramer, stabilizing the T quaternary structure. Hb Kariya, in which Lys-40 alpha is replaced by Glu, provides a unique opportunity to investigate the functional role of this salt bridge. Hb Kariya showed oxygen binding properties characterized by a high affinity, diminished cooperativity, a reduced alkaline Bohr effect, and a decreased effect of phosphates upon oxygen affinity. In deoxyHb Kariya the reactivity of the sulfhydryl groups of cysteins-93 beta with 4,4'-dipyridine disulfide was profoundly enhanced, being comparable to that for normal oxyhemoglobin (oxyHb A). The Soret band spectra, UV derivative spectra, and UV oxyminus-deoxy difference spectra indicated that oxyHb Kariya assumes a quaternary structure similar to that of oxyHb A whereas the T structure of deoxyHb Kariya is destabilized, and Hb Kariya remains predominantly in the R state upon deoxygenation. Resonance Raman scattering by deoxyHb Kariya showed that the Fe-N epsilon(proximal His) bond is less stretched than that of deoxyHb A. These experimental results provide structural basis for explaining the oxygen binding characteristics of Hb Kariya and further give direct evidence that the intersubunit salt bridge between Lys-40 alpha and the beta chain COOH terminus actually contributes to stabilization of the T quaternary structure, thereby playing a key role in cooperative oxygen binding by hemoglobin. The nature of another salt bridge between Asp-94 beta and the COOH-terminal His of beta chain was also discussed in comparison with the salt bridge involving Lys-40 alpha.     

Modulation of reactivity and conformation within the T-quaternary state of human hemoglobin: the combined use of mutagenesis and sol-gel encapsulation

A range of conformationally distinct functional states within the T quaternary state of hemoglobin are accessed and probed using a combination of mutagenesis and sol-gel encapsulation that greatly slow or eliminate the T --> R transition. Visible and UV resonance Raman spectroscopy are used to probe the proximal strain at the heme and the status of the alpha(1)beta(2) interface, respectively, whereas CO geminate and bimolecular recombination traces in conjunction with MEM (maximum entropy method) analysis of kinetic populations are used to identify functionally distinct T-state populations. The mutants used in this study are Hb(Nbeta102A) and the alpha99-alpha99 cross-linked derivative of Hb(Wbeta37E). The former mutant, which binds oxygen noncooperatively with very low affinity, is used to access low-affinity ligated T-state conformations, whereas the latter mutant is used to access the high-affinity end of the distribution of T-state conformations. A pattern emerges within the T state in which ligand reactivity increases as both the proximal strain and the alpha(1)beta(2) interface interactions are progressively lessened after ligand binding to the deoxy T-state species. The ligation and effector-dependent interplay between the heme environment and the stability of the Trp beta37 cluster in the hinge region of the alpha(1)beta(2) interface appears to determine the distribution of the ligated T-state species generated upon ligand binding. A qualitative model is presented, suggesting that different T quaternary structures modulate the stability of different alphabeta dimer conformations within the tetramer.