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.     

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.     

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.     

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.     

Structure and function of hemoglobin variants at an internal hydrophobic site: consequences of mutations at the beta 27 (B9) position

We have studied the structure-function relationships in newly discovered hemoglobin (Hb) mutants with substitutions occurring at the tight and highly hydrophobic cluster between the B and G helices in the beta chains, namely, Hb Knossos or beta A27S and Hb Grange-Blanche or beta A27V. The beta A27S mutant has a 50% decrease in oxygen affinity relative to native human Hb A, while the beta A27V mutant has an increased oxygen affinity. We have also engineered the artificial beta A27T mutation through site-directed mutagenesis. This new mutant exhibits functional properties similar to those of Hb A. None of these mutants is unstable. X-ray analyses show that the substitution of Val for Ala may reduce the relative stability of the T structure of the molecule through packing effects in the beta chains; for the beta A27S mutant a new hydrogen bond between serine and the carbonyl O at beta 23 (B5) Val is observed and is likely to increase the relative stability of the T structure in the mutant hemoglobin. However, no significant changes in the crystals were observed for these mutants between the quaternary R and T structures relative to native Hb A. We conclude that small tertiary structural changes in the tight hydrophobic B-G helix interface are sufficient to induce functional abnormalities resulting in either low or high intrinsic oxygen affinities.     

Near-UV Circular Dichroism and UV Resonance Raman Spectra of Individual Tryptophan Residues in Human Hemoglobin and Their Changes upon the Quaternary Structure Transition

The aromatic residues such as tryptophan (Trp) and tyrosine (Tyr) in human adult hemoglobin (Hb A) are known to contribute to near-UV circular dichroism (CD) and UV resonance Raman (RR) spectral changes upon the R → T quaternary structure transition. In Hb A, there are three Trp residues per αβ dimer: at α14, β15, and β37. To evaluate their individual contributions to the R → T spectral changes, we produced three mutant hemoglobins in E. coli; rHb (α14Trp→Leu), rHb (β15Trp→Leu), and rHb (β37Trp→His). Near-UV CD and UVRR spectra of these mutant Hbs were compared with those of Hb A under solvent conditions where mutant rHbs exhibited significant cooperativity in oxygen binding. Near-UV CD and UVRR spectra for individual Trp residues were extracted by the difference calculations between Hb A and the mutants. α14 and β15Trp exhibited negative CD bands in both oxy- and deoxy-Hb A, whereas β37Trp showed positive CD bands in oxy-Hb A but decreased intensity in deoxy-form. These differences in CD spectra among the three Trp residues in Hb A were ascribed to surrounding hydrophobicity by examining the spectral changes of a model compound of Trp, N-acetyl-l-Trp ethyl ester, in various solvents. Intensity enhancement of Trp UVRR bands upon the R → T transition was ascribed mostly to the hydrogen-bond formation of β37Trp in deoxy-Hb A because similar UVRR spectral changes were detected with N-acetyl-l-Trp ethyl ester upon addition of a hydrogen-bond acceptor.     

Proton nuclear magnetic resonance investigation of the allosteric transition in ligated and unligated carp hemoglobin. Evidence for structural heterogeneity in the heme pocket

The proton nuclear magnetic resonance spectra of carp hemoglobin (Hb) in the unligated deoxy and ligated met-cyano and met-azido forms have been recorded as a function of pH and upon addition of inositol hexaphosphate. All protein derivatives yield spectra that are consistent with appreciable molecular heterogeneity in the heme cavity. The pattern of heme methyl hyperfine shifts in carp met-cyano Hb indicates that this heterogeneity arises from the presence of heme rotational disorder, as found in native myoglobin. In carp deoxy Hb, the T----R transition manifests itself in nuclear magnetic resonance spectral changes similar to those found in modified human Hb species; namely, a decrease in heme methyl and an increase in proximal histidyl imidazole ring NH hyperfine shifts indicative of a strengthening of the iron-histidine bond. The met-cyano complex exhibits heme methyl hyperfine shifts similar to the analogous R state complex of Hb A; addition of inositol hexaphosphate did not give evidence for a quaternary structural change. Carp met-azido Hb in the R state also closely resembles the electronic structure of the HbA complex. Addition of inositol hexaphosphate appeared to effect at least a partial conversion to a T state with larger high-spin content than that observed for T state human metHbN3.     

Familial secondary erythrocytosis due to increased oxygen affinity is caused by destabilization of the T state of hemoglobin Brigham (α(2) β(2) (Pro100Leu) )

Hemoglobin Brigham (β Pro100 to Leu) was first reported in a patient with familial erythrocytosis. Erythrocytes of an affected individual from the same family contain both HbA and Hb Brigham and exhibit elevated O(2) affinity compared with normal cells (P(50) = 23 mm Hg vs. 31 mmHg at pH 7.4 at 37°C). O(2) affinities measured for hemolysates were sensitive to changes in pH or chloride concentrations, indicating little change in the Bohr and Chloride effects. Hb Brigham was separated from normal HbA by nondenaturing cation exchange liquid chromatography, and the amino acid substitution was verified by mass spectrometry. The properties of Hb Brigham isolated from the patient's blood were then compared with those of recombinant Hb Brigham expressed in Escherichia coli. Kinetic experiments suggest that the rate constants for ligand binding and release in the high (R) and low (T) affinity quaternary states of Hb Brigham are similar to those of native hemoglobin. However, the Brigham mutation decreases the T to R equilibrium constant (L) which accelerates the switch to the R state during ligand binding to deoxy-Hb, increasing the rate of association by approximately twofold, and decelerates the switch during ligand dissociation from HbO(2) , decreasing the rate approximately twofold. These kinetic data help explain the high O(2) affinity characteristics of Hb Brigham and provide further evidence for the importance of the contribution of Pro100 to intersubunit contacts and stabilization of the T quaternary structure.     

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.     

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.     

Tryptophan fluorescence of human hemoglobin. I. Significant change of fluorescence intensity and lifetimes in the T − R transition

The fluorescence spectra and fluorescence lifetimes due to tryptophan residues in HbA, Hb Chesapeake, NES-des-Arg Hb and Hb Kempsey were determined at room temperature. The fluorescence intensity and apparent fluorescence lifetimes decrease when the deoxy or T structure in HbA changes to the oxy or R structure, while no significant difference was observed in Hb Kempsey. The difference of fluorescence behavior was ascribed to the quaternary conformational transition of T- and R-states.     

The quaternary structure of tetrameric hemoglobin regulates the oxygen affinity of polymerized hemoglobin

This study focuses on the effect of the initial quaternary structure of bovine hemoglobin (Hb) on the physical properties of glutaraldehyde polymerized Hb (PolyHb) solutions. Tense (T) state PolyHb was synthesized by maintaining the pO₂ of Hb before and after polymerization at 0 mm Hg. In contrast, relaxed (R) state PolyHb was generated by maintaining the pO₂ of Hb before and after polymerization to >749 mm Hg. PolyHb solutions were characterized by measuring the pO₂, methemoglobin (metHb) level, molecular weight distribution, O₂ affinity and cooperativity coefficient. The metHb level of all PolyHb solutions was low (<2%). Analysis of the molecular weight distribution of PolyHb solutions indicates that in general, the molecular weight of PolyHb solutions increased with increasing cross‐link density. T‐state PolyHb solutions exhibited lower O₂ affinity compared to unmodified Hb, whereas R‐state PolyHb solutions exhibited higher O₂ affinity compared to unmodified Hb. In addition, the polymerization reaction resulted in a significant decrease in cooperativity that was more pronounced at higher cross‐link densities. All of these results were explained in terms of the quaternary structure of Hb. Taken together, our results yield more insight into the importance Hb's quaternary structure plays in defining the physical properties of glutaraldehyde PolyHb solutions. This information will be useful in designing optimized glutaraldehyde PolyHb oxygen carriers for various applications in transfusion medicine. © 2009 American Institute of Chemical Engineers Biotechnol. Prog. 2009     

Changes in the abnormal alpha-subunit upon CO-binding to the normal beta-subunit of Hb M Boston: resonance Raman,EPR and CD study

Heme-heme interaction in Hb M Boston (His alpha 58-->Tyr) was investigated with visible and UV resonance Raman (RR), EPR, and CD spectroscopies. Although Hb M Boston has been believed to be frozen in the T quaternary state, oxygen binding exhibited appreciable co-operativity (n=1.4) and the near-UV CD spectrum indicated weakening of the T marker at pH 9.0. Binding of CO to the normal beta-subunit gave no change in the EPR and visible Raman spectra of the abnormal alpha-subunit at pH 7.5, but it caused an increase of EPR rhombicity and significant changes in the Raman coordination markers as well as the Fe(III)-tyrosine related bands of the alpha-subunit at pH 9.0. The UVRR spectra indicated appreciable changes of Trp but not of Tyr upon CO binding to the alpha-subunit at pH 9.0. Therefore, we conclude that the ligand binding to the beta heme induces quaternary structure change at pH 9.0 and is communicated to the alpha heme, presumably through His beta 92-->Trp beta 37-->His alpha 87.     

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.     

Oligomerization and ligand binding in a homotetrameric hemoglobin: two high-resolution crystal structures of hemoglobin Bart's (gamma(4)), a marker for alpha-thalassemia

Hemoglobin (Hb) Bart's is present in the red blood cells of millions of people worldwide who suffer from alpha-thalassemia. alpha-Thalassemia is a disease in which there is a deletion of one or more of the four alpha-chain genes, and excess gamma and beta chains spontaneously form homotetramers. The gamma(4) homotetrameric protein known as Hb Bart's is a stable species that exhibits neither a Bohr effect nor heme-heme cooperativity. Although Hb Bart's has a higher O(2) affinity than either adult (alpha(2)beta(2)) or fetal (alpha(2)gamma(2)) Hbs, it has a lower affinity for O(2) than HbH (beta(4)). To better understand the association and ligand binding properties of the gamma(4) tetramer, we have solved the structure of Hb Bart's in two different oxidation and ligation states. The crystal structure of ferrous carbonmonoxy (CO) Hb Bart's was determined by molecular replacement and refined at 1.7 A resolution (R = 21.1%, R(free) = 24.4%), and that of ferric azide (N(3)(-)) Hb Bart's was similarly determined at 1.86 A resolution (R = 18.4%, R(free) = 22.0%). In the carbonmonoxy-Hb structure, the CO ligand is bound at an angle of 140 degrees, and with an unusually long Fe-C bond of 2.25 A. This geometry is attributed to repulsion from the distal His63 at the low pH of crystallization (4.5). In contrast, azide is bound to the oxidized heme iron in the methemoglobin crystals at an angle of 112 degrees, in a perfect orientation to accept a hydrogen bond from His63. Compared to the three known quaternary structures of human Hb (T, R, and R2), both structures most closely resemble the R state. Comparisons with the structures of adult Hb and HbH explain the association and dissociation behaviour of Hb homotetramers relative to the heterotetrameric Hbs.     

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.     

Flash photolytic studies of carbon monoxide binding to the ferrous chains of [Mn(II),Fe(II)] hybrid hemoglobins: kinetic mechanism for the early stages of hemoglobin ligation

Flash photolysis is employed to investigate the kinetics of CO recombination to the ferrous chains of [Mn(II),Fe(II)] hemoglobin (Hb) hybrids. At low pH (6.6), Hb remains predominantly in the T quaternary state for the first two CO ligation steps, when binding to either the alpha chains or beta chains. At elevated pH, CO binding to the alpha chains produces a larger degree of T to R conversion than binding to the beta chains, in support of earlier equilibrium measurements. This study provides the full pH dependence of the CO binding rate constants for both alpha- and beta-Fe chains within the T state and at elevated values of pH gives the R-state rate constants for the monoliganded analogues. The data can be analyzed within the context of a two-state model for Hb cooperativity, but they give clear evidence for slow quaternary structure interconversion at the monoliganded level.     

Quaternary conformational changes in human hemoglobin studied by laser photolysis of carboxyhemoglobin.

These experiments indicate that absorbance changes observed at the 425 nm isosbestic point of the Hb and HbCO following laser photolysis of HbCO provide a direct measure of the rates of quaternary conformational changes between rapidly reacting Hb (the immediate product of full photolysis) and slowly reacting normal deoxyhemoglobin. Hb, first observed by Gibson (Gibson, Q.H. (1959) Biochem. J. 71, 293-303), Has been interpreted as deoxyhemoglobin remaining in the liganded quaternary conformation following rapid removal of ligand by a light pulse. In borate buffers between pH 8.4 and 9.6 particularly simple pH-independent results were obtained which allowed the use of a Monod. Wyman, and Changeux model (Monod, J., Wyman, J., and Changeux, J (1965) J. Mol. Biol. 12, 88-118) to fit the data. In this case Hb is taken to be R state deoxyhemoglobin. Partial photolysis experiments at 425 nm show that the rate of the R - T conformational change at 20 degrees decreases by about a factor of 2 for each additional bound ligand. The rate of the ligand-free conformational change is found to be 920 +/- 60s(-1), 6400 +/- 600s(-1), and 15,700 +/- 700(-1) respectively at 3 degrees, 20 degrees, and 30 degrees. The previously uninterpreted effects of flash length and partial photolysis on the CO recombination kinetics can be explained in terms of the present model. Kinetic results obtained below pH 8 are found to be inconsistent with a two-state model. It appears that binding of inositol hexaphosphate produces a new rapidly reacting quaternary conformation of HbCO.     

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.