A detailed description of hemoglobin cooperativity requires knowledge of the dimer-dimer interactions responsible for the low ligand affinity of the quaternary-T tetramer, the "quaternary-T constraints", along with stereochemical pathways that specify how ligand binding disrupts these quaternary constraints. The recent mutagenic screen of Noble et al. [Noble, R. W., et al. (2001) Biochemistry 40, 12357-12368] has identified the major region of quaternary constraint to be a cluster of residues at the alpha1beta2 interface that is centered at Trp37beta. In this paper, crystallographic studies are presented for most of the mutant hemoglobins studied by Noble et al. These crystallographic experiments identify structural transitions-referred to as T-to-T(High) transitions-between the quaternary-T structure of wild-type deoxyhemoglobin and an ensemble of related T-like quaternary structures that are induced by some mutations in the Trp37beta cluster and/or by exposing crystals of wild-type or mutant deoxyhemoglobins to oxygen. The T-to-T(High) quaternary transitions consist of a rotation of the alpha1beta1 dimer relative to the alpha2beta2 dimer as well as a coupled alphabeta dimer bending component that consists of a small rotation of the alpha1 subunit relative to the beta1 subunit (and a symmetry related rotation of the alpha2 subunit relative to the beta2 subunit). In addition, differences in subunit tertiary structure associated with the T-to-T(High) transitions suggest two stereochemical pathways (one associated with the alpha subunits and one associated with the betasubunits) by which ligand binding specifically disrupts quaternary constraints in the Trp37beta cluster. In the alpha subunits, ligand binding induces a shift of the heme iron producing tension in a chain of covalent bonds that extends from the Fe-N(epsilon)(2)His(F8)alpha1 bond to the peptide backbone bonds of residues His87(F8)alpha1 and Ala88(F9)alpha1. This tension induces an alpha-to-pi transition in the COOH-terminal end of the F-helix that shifts the beta-carbon of Ala88alpha1 by approximately 1.5 A directly into the side chain of Tyr140alpha1 (a key residue in the Trp37beta2 cluster). Collectively these structural changes constitute a relatively short pathway by which ligand binding forces Tyr140alpha1 into the alpha1beta2 interface disrupting quaternary constraints associated with the Trp37beta2 cluster. In the beta subunits, our analysis suggests a more extended energy transduction pathway in which ligand-induced beta1-heme movement triggers tertiary changes in the beta1 subunit that promote alpha1beta1 dimer bending that disrupts quaternary constraints in the Trp37beta2 cluster at the alpha1beta2 interface.

Previous mutational studies on Tyr42 alpha variants as well as the current Studies on the mutant hemoglobin alpha Y42A show that the intersubunit interactions associated with Tyr42 alpha significantly stabilize the alpha 1 beta 2 interface of the quaternary-T deoxyhemoglobin tetramer. However, crystallographic studies, UV and visible resonance Raman spectroscopy, CO combination kinetic measurements, and oxygen binding measurements on alpha Y42A show that the intersubunit interactions formed by Tyr42 alpha have only a modest influence on the structural properties and ligand affinity of the deoxyhemoglobin tetramer. Therefore, the alpha 1 beta 2 interface interactions associated with Tyr42 alpha do not contribute significantly to the quaternary constraints that are responsible for the low oxygen affinity of deoxyhemoglobin. The slight increase in the ligand affinity of deoxy alpha Y42A correlates with small, mutation-induced structural changes that perturb the environment of Trp37 beta, a critical region of the quaternary-T alpha 1 beta 2 interface that has been shown to be the major source of quaternary constraint in deoxyhemoglobin.

In addition to interacting with hemoglobin as a heme ligand to form nitrosylhemoglobin, NO can react with cysteine sulfhydryl groups to form S-nitrosocysteine or cysteine oxides such as cysteinesulfenic acid. Both modes of interaction are very sensitive to the quaternary structure of hemoglobin. To directly view the interaction of NO with quaternary-T deoxyhemoglobin, crystallographic studies were carried out on crystals of deoxyhemoglobin that were exposed to gaseous NO under a variety of conditions. Consistent with previous spectroscopic studies in solution, these crystallographic studies show that the binding of NO to the heme groups of crystalline wild-type deoxyhemoglobin ruptures the Fe-proximal histidine bonds of the alpha-subunits but not the beta-subunits. This finding supports Perutz's theory that ligand binding induces tension in the alpha Fe-proximal histidine bond. To test Perutz's theory, deoxy crystals of the mutant hemoglobin betaW37E were exposed to NO. This experiment was carried out because previous studies have shown that this mutation greatly reduces the quaternary constraints that oppose the ligand-induced movement of the alpha-heme Fe atom into the plane of the porphyrin ring. As hypothesized, the Fe-proximal histidine bonds in both the beta- and the alpha-subunits remain intact in crystalline betaW37E after exposure to NO. With regard to S-nitrosocysteine or cysteine oxide formation, no evidence for the reaction of NO with any cysteine residues was detected under anaerobic conditions. However, when deoxyhemoglobin crystals are first exposed to air and then to NO, the appearance of additional electron density indicates that Cys93(F9)beta has been modified, most likely to cysteinesulfenic acid. This modification of Cys93(F9)beta disrupts the intrasubunit salt bridge between His146(HC3)beta and Asp94(FG1)beta, a key feature of the quaternary-T hemoglobin structure. Also presented is a reanalysis of our previous crystallographic studies [Chan, N.-L., et al. (1998) Biochemistry 37, 16459-16464] of the interaction of NO with liganded hemoglobin in the quaternary-R2 structure. These studies showed additional electron density at Cys93(F9)beta that was consistent with an NO adduct. However, for reasons discussed in this paper, we now believe that this adduct may be the Hb-S-N.-O-H radical intermediate and not Hb-S-N=O as previously suggested.

In 1947, Perutz and co-workers reported that crystalline horse methemoglobin undergoes a large lattice transition as the pH is decreased from 7.1 to 5.4. We have determined the pH 7.1 and 5.4 crystal structures of horse methemoglobin at 1.6 and 2.1 A resolution, respectively, and find that this lattice transition involves a 23 A translation of adjacent hemoglobin tetramers as well as changes in alpha heme ligation and the tertiary structure of the alpha subunits. Specifically, when the pH is lowered from 7.1 to 5.4, the Fe(3+) alpha heme groups (but not the beta heme groups) are converted from the aquomet form, in which the proximal histidine [His87(F8)alpha] and a water molecule are the axial heme ligands, to the hemichrome (bishistidine) form, in which the proximal histidine and the distal histidine [His58(E7)alpha] are the axial heme ligands. Hemichrome formation is coupled to a large tertiary structure transition in the eight-residue segment Pro44(CD2)alpha-Gly51(D7)alpha that converts from an extended loop structure at pH 7.1 to a pi-like helix at pH 5.4. The formation of the pi helix forces Phe46(CD4)alpha out of the alpha heme pocket and into the interface between adjacent hemoglobin tetramers where it participates in crystal lattice contacts unique to the pH 5.4 structure. In addition, the transition from aquomet alpha subunits to bishistidine alpha subunits is accompanied by an approximately 1.2 A movement of the alpha heme groups to a more solvent-exposed position as well as the creation of a solvent channel from the interior of the alpha heme pocket to the outside of the tetramer. These changes and the extensive rearrangement of the crystal lattice structure allow the alpha heme group of one tetramer to make direct contact with an alpha heme group on an adjacent tetramer. These results suggest possible functional roles for hemichrome formation in vivo.

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(β)->Lys mutation–induced low-oxygen affinity of Hb. The nuclear magnetic resonance studies of Tg-HbP revealed that the conformation of the α1β1 and α1β2 interfaces of the protein in the deoxy state are indistinguishable from that of deoxy HbA, whereas the conformation of the microenvironment of His-103(α) of Tg-HbP, a residue of the α1β1 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 α1β2 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(β). In short, Lys-108(β) 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.

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.

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 arp 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.

Human hemoglobin produced in the Escherichia coli coexpression system of Hernan et al. [(1992) Biochemistry 31, 8619-8628] has been transformed into a functionally homogeneous protein whose properties closely approximate those of normal hemoglobin A. Both of the alpha and beta chains of this hemoglobin contain a valine-methionine substitution at position 1 in order to accommodate the difference in specificity of the protein-processing enzymes of procaryotes. Despite extensive purification, functional homogeneity of the E. coli expressed hemoglobin was achieved only by the complete disassembly of the hemoglobin into its component alpha and beta globins and their reassembly in the presence of hemin. The kinetics of CO combination and the thermodynamics of O2 binding and cooperativity of the reassembled alphaV1M-betaV1M hemoglobin closely approximate those of HbA. The alpha globin obtained from the E. coli expressed hemoglobin was also combined with normal human beta chains and hemin to form the alphaV1M variant. The alpha+M variant of HbA, in which the normal N-terminal valine of the alpha chains is preceded by a methionine residue, was prepared by the same procedure. The kinetics of the reactions of CO with the alphaV1M and alpha+M variants are similar to those for HbA. The equilibria of oxygen binding to alphaV1M and HbA are similar whereas alpha+M exhibits a significantly higher oxygen affinity. The three-dimensional structures of alphaV1M and alpha+M offer an explanation for the latter affinity difference. Although the structures of alphaV1M and HbA, which have been determined by X-ray crystallography, are virtually indistinguishable except at the N-terminal residues, that of alpha+M indicates the displacement of a solvent molecule, possibly a chloride ion, from arginine 141alpha. Such an alteration in an anion binding site could result in increased oxygen affinity.

Although numerous reports have documented that the S-nitrosylation of cysteine residues by NO alters the activities of a wide variety of proteins, the direct visualization and the structural consequences of this reversible modification have not yet been reported for any protein. Here we describe the crystal structure of S-nitroso-nitrosylhemoglobin determined at a resolution of 1.8 A. The specific reaction of NO with Cys93beta is confirmed in this structure, and a large S-nitrosylation-induced change in the tertiary structure of the COOH-terminal dipeptides of the beta subunits provides additional insight into the stereochemical mechanism by which blood flow is regulated by the interaction of NO with hemoglobin.

The high-resolution X-ray structures of the deoxy forms of four recombinant hemoglobins in which Trp37 (C3)beta is replaced with Tyr (beta W37Y), Ala (beta W37A), Glu (beta W37E), or Gly (beta W37G) have been refined and analyzed with superposition methods that partition mutation-induced perturbations into quaternary structure changes and tertiary structure changes. In addition, a new cross-validation statistic that is sensitive to local changes in structure (a "local R-free" parameter) was used as an objective measure of the significance of the tertiary structure changes. No significant mutation-induced changes in tertiary structure are detected at the mutation site itself for any of the four mutants studied. Instead, disruption of the intersubunit contacts associated with Trp37(C3)beta results in (1) a change in quaternary structure at the alpha 1 beta 2 interface, (2) alpha subunit tertiary structure changes that are centered at Asp94(G1)alpha-Pro95(G2)alpha, (3)beta subunit tertiary structure changes that are located between residues Asp99(G1)beta and Asn102(G4)beta, (4) increased mobility of the alpha subunit COOH-terminal dipeptide, and (5) shortening of the Fe-N(epsilon 2)His(F8) bond in the alpha and beta subunits of the beta W37G and beta W37E mutants. In each case, the magnitude of the change in a particular structural parameter increases in the order beta W37Y < beta W37A < beta W37E approximate to beta W37G, which corresponds closely to the degree of functional disruption documented in the preceding papers.

To correlate directly structure with function, the oxygen affinity and the three-dimensional structure of crystals of the T quaternary state of des-His-146β human hemoglobin have been determined by polarized absorption microspectrophotometry and x-ray diffraction crystallography. In des-His-146β, the COOH-terminal histidine residues of the β chains of hemoglobin A have been removed. Oxygen binding to crystalline des-His hemoglobin is non-cooperative and independent of pH. The oxygen affinity is 1.7-fold greater than that of the crystalline state of hemoglobin A. Removal of His-146β results in a small movement of the truncated COOH-terminal peptide and in a very small change in quaternary structure. Previously, similar studies on T state crystals of des-Arg-141α hemoglobin showed that removal of the COOH termini of the α chains results in much larger effects on oxygen affinity and on quaternary structure. Kinetic studies in solution reveal that at pH 7.0, the rates of CO combination with deoxygenated des-His-146β in the absence and presence of inositol hexaphosphate are 2.5- and 1.3-fold, respectively, more rapid than for hemoglobin A. The values for des-Arg are 7.6- and 3.9-fold. The properties of the T state of hemoglobin both in the crystal and in solution are influenced to a greater degree by the interactions associated with Arg-141α than those associated with His-146β.

The correlation of a protein structure determined crystallographically to its functional properties determined in solution can be an extremely complex problem due to potential differences of protein conformational flexibility in the two physical states. A more direct approach to the correlation of structure with function is to examine both the structure and the function of a protein in the same crystalline environment. In this paper, the structural and functional properties of T state desArg hemoglobin (human hemoglobin modified by removal of the α-chain COOH-terminal residue, Arg141α) have been studied in the same crystal form by high resolution X-ray diffraction methods and by polarized absorption microspectrophotometry. Specifically, the crystal structure of deoxygenated desArg human hemoglobin has been refined at a 2.1 Å resolution using crystals grown at low salt concentration from solutions of polyethylene glycol. The loss of Arg141α and all of the salt bridges in which it participates is associated with subtle structural perturbations of the α-chains which include an increase in the conformational flexibility of both the NH2and COOH-terminal peptides. Although the heme pockets appear unchanged and even the side-chain of Tyr140 is oriented nearly as in HbA, the functional characterization by microspectrophotometric measurements indicates that crystals of desArg hemoglobin bind oxygen with an affinity which is roughly 15-fold greater than that of crystals of human hemoglobin A. There is no alkaline Bohr effect or effect of chloride ions, but an acid Bohr effect is observed. The oxygen affinities measured along two principal axes of the crystals differ by 25% indicating heterogeneity in the affinities of the oxygen binding sites. This finding and the measured Hill coefficient of unity suggest significant cooperativity in the binding of oxygen in these crystals. The origins of the observed heterogeneity and the implied cooperativity are unknown.