Ligand binding properties and structural studies of recombinant and chemically modified hemoglobins altered at beta 93 cysteine

To investigate the roles of beta93 cysteine in human normal adult hemoglobin (Hb A), we have constructed four recombinant mutant hemoglobins (rHbs), rHb (betaC93G), rHb (betaC93A), rHb (betaC93M), and rHb (betaC93L), and have prepared two chemically modified Hb As, Hb A-IAA and Hb A-NEM, in which the sulfhydryl group at beta93Cys is modified by sulfhydryl reagents, iodoacetamide (IAA) and N-ethylmaleimide (NEM), respectively. These variants at the beta93 position show higher oxygen affinity, lower cooperativity, and reduced Bohr effect relative to Hb A. The response of some of these Hb variants to allosteric effectors, 2,3-bisphosphoglycerate (2,3-BPG) and inositol hexaphosphate (IHP), is decreased relative to that of Hb A. The proton nuclear magnetic resonance (NMR) spectra of these Hb variants show that there is a marked influence on the proximal heme pocket of the beta-chain, whereas the environment of the proximal heme pocket of the alpha-chain remains unchanged as compared to Hb A, suggesting that higher oxygen affinity is likely to be determined by the heme pocket of the beta-chain rather than by that of the alpha-chain. This is further supported by NO titration of these Hbs in the deoxy form. For Hb A, NO binds preferentially to the heme of the alpha-chain relative to that of the beta-chain. In contrast, the feature of preferential binding to the heme of the alpha-chain becomes weaker and even disappears for Hb variants with modifications at beta93Cys. The effects of IHP on these Hbs in the NO form are different from those on HbNO A, as characterized by (1)H NMR spectra of the T-state markers, the exchangeable resonances at 14 and 11 ppm, reflecting that these Hb variants have more stability in the R-state relative to Hb A, especially rHb (betaC93L) and Hb A-NEM in the NO form. The changes of the C2 proton resonances of the surface histidyl residues in these Hb variants in both the deoxy and CO forms, compared with those of Hb A, indicate that a mutation or chemical modification at beta93Cys can result in conformational changes involving several surface histidyl residues, e.g., beta146His and beta2His. The results obtained here offer strong evidence to show that the salt bridge between beta146His and beta94Asp and the binding pocket of allosteric effectors can be affected as the result of modifications at beta93Cys, which result in the destabilization of the T-state and a reduced response of these Hbs to allosteric effectors. We further propose that the impaired alkaline Bohr effect can be attributed to the effect on the contributions of several surface histidyl residues which are altered because of the environmental changes caused by mutations and chemical modifications at beta93Cys.     

Site mutations disrupt inter-helical H-bonds (alpha14W-alpha67T and beta15W-beta72S) involved in kinetic steps in the hemoglobin R-->T transition without altering the free energies of oxygenation

Three recombinant mutant hemoglobins (rHbs) of human normal adult hemoglobin (Hb A), rHb (alphaT67V), rHb (betaS72A), and rHb (alphaT67V, betaS72A), have been constructed to test the role of the tertiary intra-subunit H-bonds between alpha67T and alpha14W and between beta72S and beta15W in the cooperative oxygenation of Hb A. Oxygen-binding studies in 0.1 M sodium phosphate buffer at 29 degrees C show that rHb (alphaT67V), rHb (betaS72A), and rHb (alphaT67V, betaS72A) exhibit oxygen-binding properties similar to those of Hb A. The binding of oxygen to these rHbs is highly cooperative, with a Hill coefficient of approximately 2.8, compared to approximately 3.1 for Hb A. Proton nuclear magnetic resonance (NMR) studies show that rHb (alphaT67V), rHb (betaS72A), rHb (alphaT67V, betaS72A), and Hb A have similar quaternary structures in the alpha(1)beta(2) subunit interfaces. In particular, the inter-subunit H-bonds between alpha42Tyr and beta99Asp and between beta37Trp and alpha94Asp are maintained in the mutants in the deoxy form. There are slight perturbations in the distal heme pocket region of the alpha- and beta-chains in the mutants. A comparison of the exchangeable 1H resonances of Hb A with those of these three rHbs suggests that alpha67T and beta72S are H-bonded to alpha14W and beta15W, respectively, in the CO and deoxy forms of Hb A. The absence of significant free energy changes for the oxygenation process of these three rHbs compared to those of Hb A, even though the inter-helical H-bonds are abolished, indicates that these two sets of H-bonds are of comparable strength in the ligated and unligated forms of Hb A. Thus, the mutations at alphaT67V and betaS72A do not affect the overall energetics of the oxygenation process. The preserved cooperativity in the binding of oxygen to these three mutants also implies that there are multiple interactions involved in the oxygenation process of Hb A.     

High-resolution X-ray study of deoxy recombinant human hemoglobins synthesized from beta-globins having mutated amino termini.

The crystal structures of three mutant hemoglobins reconstituted from recombinant beta chains and authentic human alpha chains have been determined in the deoxy state at 1.8-A resolution. The primary structures of the mutant hemoglobins differ at the beta-chain amino terminus. One mutant, beta Met, is characterized by the addition of a methionine at the amino terminus. The other two hemoglobins are characterized by substitution of Val 1 beta with either a methionine, beta V1M, or an alanine, beta V1A. All the mutation-induced structural perturbations are small intrasubunit changes that are localized to the immediate vicinity of the beta-chain amino terminus. In the beta Met and beta V1A mutants, the mobility of the beta-chain amino terminus increases and the electron density of an associated inorganic anion is decreased. In contrast, the beta-chain amino terminus of the beta V1M mutant becomes less mobile, and the inorganic anion binds with increased affinity. These structural differences can be correlated with functional data for the mutant hemoglobins [Doyle, M. L., Lew, G., DeYoung, A., Kwiatkowski, L., Noble, R. W., & Ackers, G. K. (1992) Biochemistry preceding paper is this issue] as well as with the properties of ruminant hemoglobins and a mechanism [Perutz, M., & Imai, K. (1980) J. Mol. Biol. 136, 183-191] that relates the intrasubunit interactions of the beta-chain amino terminus to changes in oxygen affinity. Since the structures of the mutant deoxyhemoglobins show only subtle differences from the structure of deoxyhemoglobin A, it is concluded that any of the three hemoglobins could probably function as a surrogate for hemoglobin A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recombinant hemoglobin(alpha 29leucine --> phenylalanine, alpha 96valine --> tryptophan, beta 108asparagine --> lysine) exhibits low oxygen affinity and high cooperativity combined with resistance to autoxidation.

Using our hemoglobin expression system in Escherichia coli, we have constructed three recombinant hemoglobins (rHbs) with amino acid substitutions located in the alpha(1)beta(1) and alpha(1)beta(2) subunit interfaces and in the distal heme pocket of the alpha-chain: rHb(alphaV96W, betaN108K), rHb(alphaL29F, alphaV96W, betaN108K), and rHb(alphaL29F). rHb(alphaV96W, betaN108K) exhibits low oxygen affinity and high cooperativity and also ease of autoxidation of the heme iron atoms from the Fe2+ state to the Fe3+ state. It has been reported by Olson and co-workers [Carver et al., (1992) J. Biol. Chem. 267, 14443-14450; Brantley et al. (1993) J. Biol. Chem. 268, 6995-7010] that a mutation at position 29 (B10, helix notation), e.g. , Leu --> Phe, can inhibit the autoxidation of the heme iron of myoglobin. We have introduced such a mutation into our rHb having low oxygen affinity and high cooperativity. This triply mutated rHb(alphaL29F, alphaV96W, betaN108K) is stabilized against autoxidation and azide-induced oxidation compared to the double mutant, rHb(alphaV96W, betaN108K), but still exhibits low oxygen affinity and good cooperativity. According to electron paramagnetic resonance results, the oxidized form of the triple mutant shows a high ratio of an anionic form of bishistidine hemichrome. Previous reports have suggested that this form does not have water present at the distal heme pocket. (1)H nuclear magnetic resonance spectra of the triple mutant in the ferric state also exhibit spectral features characteristic of hemichrome-type signals. We have carried out a series of biochemical measurements to characterize these three interesting rHbs and to compare them to human normal adult hemoglobin. These results provide new insights into the structure-function relationship of hemoglobin with amino acid substitutions in the alpha(1)beta(1) and alpha(1)beta(2) interfaces and in the heme pockets.     

Binding of diphosphoglycerate and ATP to oxyhemoglobin dimers

The relative affinity of diphosphoglycerate and ATP for hemoglobin dimers and tetramers can be measured under conditions where the protein is in large molar excess over the polyphosphate. Binding of both compounds to dimers was about 25 times stronger than to tetramers in the case of the three low-spin hemoglobins, oxyhemoglobin, carboxyhemoglobin and cyanomethemoglobin. The mutation in hemoglobin Kansas leads to an increased dissociation into alpha beta dimers. The increase in diphosphoglycerate binding by this hemoglobin was in good agreement with that expected from the dimer-tetramer dissociation constant over a wide range of hemoglobin concentrations. In contrast to the liganded hemoglobins, both deoxyhemoglobin and aquomethemoglobin bind the two polyanions as tetramers.     

Carbamoylated hemoglobins A and S: physical properties.

Dimer-tetramer association constants (K2,4) of derivatives of CO-hemoglobins A and S specifically carbamoylated at the NH2-terminal valine residues were measured. Reactivites of the beta-93 sulfhydryls of the hemoglobin A derivatives were also investigated. As compared with the association constants of the parent molecules, the values of K2,4 of both hemoglobin types are raised by carbamoylation of the alpha-chain NH2 terminus, lowered by carbamoylation of the beta-chain NH2 terminus, and raised by carbamoylation of both termini. The apparent second-order rate constant for reaction of p-mercuribenzoate (PMB) with the beta-93 sulfhydryls is, however, unchanged by carbamoylation. These two observations are interpreted to indicate that in the liganded molecule structural changes are produced at the interface between dimers but not in the region of the beta-93 sulfhydryls. From the combination of the K2,4 measurements with ligand-binding data for the same derivatives (Kilmartin, J. V., et al. (1973), J. Biol. Chem. 248, 4039; Nigen, A. M., et al. (1974), J. Biol. Chem. 249, 6611) the carbamoylation-induced changes in the dimer-tetramer association constants of the unliganded derivatives were estimated to be of magnitude equal to or smaller than those in K2,4. It is concluded that much of the change in oxygen affinity that occurs upon carbamoylation of hemoglobins A and S can be accounted for without invoking extensive structural changes in the unliganded molecule.     

Functional properties of human hemoglobins synthesized from recombinant mutant beta-globins.

The previous and following articles in this issue describe the recombinant synthesis of three mutant beta-globins (beta 1 Val----Ala, beta 1 Val----Met, and the addition mutation beta 1 + Met), their assembly with heme and natural alpha chains into alpha 2 beta 2 tetramers, and their X-ray crystallographic structures. Here we have measured the equilibrium and kinetic allosteric properties of these hemoglobins. Our objective has been to evaluate their utility as surrogates of normal hemoglobin from which further mutants can be made for structure-function studies. The thermodynamic linkages between cooperative oxygenation and dimer-tetramer assembly were determined from global regression analysis of multiple oxygenation isotherms measured over a range of hemoglobin concentration. Oxygen binding to the tetramers was found to be highly cooperative (maximum Hill slopes from 3.1 to 3.2), and similar patterns of O2-linked subunit assembly free energies indicated a common mode of cooperative switching at the alpha 1 beta 2 interface. The dimers were found to exhibit the same noncooperative O2 equilibrium binding properties as normal hemoglobin. The most obvious difference in oxygen equilibria between the mutant recombinant and normal hemoglobins was a slightly lowered O2 affinity. The kinetics of CO binding and O2 dissociation were measured by stopped-flow and flash photolysis techniques. Parallel studies were carried out with the mutant and normal hemoglobins in the presence and absence of organic phosphates to assess their allosteric response to phosphates. In the absence of organic phosphates, the CO-binding and O2 dissociation kinetic properties of the mutant dimers and tetramers were found to be nearly identical to those of normal hemoglobin. However, the effects of organic phosphates on CO-binding kinetic properties of the mutants were not uniform: the beta 1 + Met mutant was found to deviate somewhat from normalcy, while the beta 1 Val----Met mutant reproduced the native allosteric response. Further characterization of the allosteric properties of the beta 1 Val----Met mutant was made by measuring the pH dependence of its overall oxygen affinity by tonometry. Regulation of oxygen affinity by protons was found to be nearly identical to normal hemoglobin from pH 5.8 to 9.3 (0.52 +/- 0.07 protons released per oxygen bound at pH 7.4). The present study demonstrates that the equilibrium and kinetic functional properties of the recombinant beta 1 Val----Met mutant mimic reasonably well those of normal hemoglobin. We conclude that this mutant is well-suited to serve as a surrogate system of normal hemoglobin in the production of mutants for structure-function studies.     

Implication of the alpha 1 beta 1 interface in the hemoglobin affinity changes. A comparative study between normal and San Diego fully ligated hemoglobins

The alpha 1 beta 1 interface of normal and mutated San Diego hemoglobins in their fully liganded form was investigated, through the SH vibrational absorption of beta-112 cysteine, by Fourier-transform infrared spectroscopy. The center frequency of this thiol group was significantly shifted in San Diego hemoglobin compared with normal human hemoglobin. Different dimer organization between the two proteins was also revealed by circular dichroism of the heme. These findings agree well with assessment that the alpha 1 beta 1 interface, far from being inert, is involved in the affinity changes of the hemoglobin molecule.     

Residue F4 plays a key role in modulating oxygen affinity and cooperativity in Scapharca dimeric hemoglobin

Residue F4 (Phe 97) undergoes the most dramatic ligand-linked transition in Scapharca dimeric hemoglobin, with its packing in the heme pocket in the unliganded (T) state suggested to be a primary determinant of its low affinity. Mutation of Phe 97 to Leu (previously reported), Val, and Tyr increases oxygen affinity from 8- to 100-fold over that of the wild type. The crystal structures of F97L and F97V show side chain packing in the heme pocket for both R and T state structures. In contrast, in the highest-affinity mutation, F97Y, the tyrosine side chain remains in the interface (high-affinity conformation) even in the unliganded state. Comparison of these mutations reveals a correlation between side chain packing in the heme pocket and oxygen affinity, indicating that greater mass in the heme pocket lowers oxygen affinity due to impaired movement of the heme iron into the heme plane. The results indicate that a key hydrogen bond, previously hypothesized to have a central role in regulation of oxygen affinity, plays at most only a small role in dictating ligand affinity. Equivalent mutations in sperm whale myoglobin alter ligand affinity by only 5-fold. The dramatically different responses to mutations at the F4 position result from subtle, but functionally critical, stereochemical differences. In myoglobin, an eclipsed orientation of the proximal His relative to the A and C pyrrole nitrogen atoms provides a significant barrier for high-affinity ligand binding. In contrast, the staggered orientation of the proximal histidine found in liganded HbI renders its ligand affinity much more susceptible to packing contacts between F4 and the heme group. These results highlight very different strategies used by cooperative hemoglobins in molluscs and mammals to control ligand affinity by modulation of the stereochemistry on the proximal side of the heme.     

Inversion of axial coordination in myoglobin to create a "proximal" ligand binding pocket

A ligand binding pocket has been created on the proximal side of the heme in porcine myoglobin by site-directed mutagenesis. Our starting point was the H64V/V68H double mutant which has been shown to have bis-histidine (His68 and His93) heme coordination [Dou, Y., Admiraal, S. J., Ikeda-Saito, M., Krzywda, S., Wilkinson, A. J., Li, T., Olson, J. S., Prince, R. C., Pickering, I. J., George, G. N. (1995) J. Biol. Chem. 270, 15993-16001]. The replacement of the proximal His93 ligand by noncoordinating Ala (H64V/V68H/H93A) or Gly (H64V/V68H/H93G) residues resulted unexpectedly in a six-coordinate low-spin species in both ferric and ferrous states. To test the hypothesis that the sixth coordinating ligand in the triple mutants was the imidazole of His97, this residue was mutated to Phe, in the quadruple mutants, H64V/V68H/H93A/H97F and H64V/V68H/H93G/H97F. The ferric quadruple mutants show a clear water/hydroxide alkaline transition and high cyanide and CO affinities, characteristics similar to those of wild-type myoglobin. The nu(Fe-CO) and nu(C-O) stretching frequencies in the ferrous-CO state of the quadruple mutants indicate that the "proximal" ligand binding heme pocket is less polar than the distal pocket in the wild-type protein. Thus, we conclude that the proximal heme pocket in the quadruple mutants has a similar affinity for exogenous ligands to the distal pocket of wild-type myoglobin but that the two pockets have different polarities. The quadruple mutants open up new approaches for developing heme chemistry on the myoglobin scaffold.     

Dynamic properties of some β-chain mutant hemoglobins

The thermal behavior of the Soret band relative to the carbonmonoxy derivatives of some β-chain mutant hemoglobins is studied in the temperature range 300–10 K and compared to that of wild-type carbonmonoxy hemoglobin. The band profile at various temperatures is modeled as a Voigt function that accounts for homogeneous broadening and for the coupling with high- and low-frequency vibrational modes, while inhomogeneous broadening is taken into account with a gaussian distribution of purely electronic transition frequencies. The various contributions to the overall bandwidth are singled out With this analysis and their temperature dependence, in turn, gives information on structural and dynamic properties of the system studied. In the wildtype and mutant hemoglobins, the values of homogeneous bandwidth and of the coupling constants to high-frequency vibrational modes are not modified with respect to natural human hemoglobin, thus indicating that the local electronic and vibrational properties of the heme–CO complex are not altered by the recombinant procedures. On the contrary, differences in the protein dynamic behavior are observed. The most relevant are those relative to the “polar isosteric” βVal-67(Ell) →Thr substitution, localized in the heme pocket, which results in decreased coupling with low-frequency modes and increased anharmonic motions. Mutations involving residue βLys-144(HC1) at the C-terminal and residue βCys-112(G14) at the α₁β₁ interface have a smaller effect consisting in an increased coupling with low-frequency modes. Mutations at the β-N-terminal and at the α₁β₂ interface have no effect on the dynamic properties of the heme pocket. © 1995 Wiley-Liss, Inc.     

Proton nuclear magnetic resonance investigation of the spin-state equilibrium of the alpha and beta subunits in intact azidomethemoglobin

The hyperfine-shifted proton NMR spectra of human azidomethemoglobin were examined at 300 MHz in the 2-60 degree C range. From analysis of the temperature-dependent heme methyl shifts, the thermal spin-state equilibria of the alpha and beta subunits were independently analyzed in the intact tetramer. The thermodynamic values of the spin equilibrium of the alpha and beta subunits were comparable, suggesting that the spin equilibrium properties of the constituent subunits are similar to each other. Examination of the azidomethemoglobins reconstituted with deutero- or mesohemin further shows that the alpha and beta subunit difference is still small in these hemoglobins probably due to the smallness of the steric and electronic difference of the heme 2,4-substituents of the examined porphyrins. The similarity of the spin equilibrium profiles of the subunits indicates that the strain imposed from the globin to the heme iron is of comparable magnitude for the alpha and beta subunits within the azidomethemoglobins.     

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.     

Phosphines as a new structural probe of hemoglobin. 1H-NMR evidence for perturbations in the beta heme pocket induced by a thiol reagent

Binding of trimethylphosphine to myoglobins and hemoglobins from a variety of sources has been examined by 1H-nuclear magnetic resonance. The hemoglobins exhibit two resonances at high field (approx. -3.5 ppm) which have been assigned to PMe3 bound to alpha or to beta subunits. Perturbations in the beta heme pocket induced by a thiol reagent have been detected both in 1H and 31P spectra.     

Site-directed mutagenesis in hemoglobin: test of functional homology of the F9 amino acid residues of hemoglobin alpha and beta chains

The cysteine residue at F9(93) of the human hemoglobin (Hb A) beta chain, conserved in mammalian and avian hemoglobins, is located near the functionally important alpha1-beta2 interface and C-terminal region of the beta chain and is reactive to sulfhydryl reagents. The functional roles of this residue are still unclear, although regulation of local blood flow through allosteric S-nitrosylation of this residue is proposed. To clarify the role of this residue and its functional homology to F9(88) of the alpha chain, we measured oxygen equilibrium curves, UV-region derivative spectra, Soret-band absorption spectra, the number of titratable -SH groups with p-mercuribenzoate and the rate of reaction of these groups with 4, 4'-dipyridine disulfide for three recombinant mutant Hbs with single amino acid substitutions: Ala-->Cys at 88alpha (rHb A88alphaC), Cys-->Ala at 93beta (rHb C93betaA) and Cys-->Thr at 93beta (rHb C93betaT). These Hbs showed increased oxygen affinities and impaired allosteric effects. The spectral data indicated that the R to T transition upon deoxygenation was partially restricted in these Hbs. The number of titratable -SH groups of liganded form was 3.2-3.5 for rHb A88alphaC compared with 2.2 for Hb A, whereas those for rHb C93betaA and rHb C93betaT were negligibly small. The reduction of rate of reaction with 4,4'-dipyridine disulfide upon deoxygenation in rHb A88alphaC was smaller than that in Hb A. Our experimental data have shown that the residues at 88alpha and 93beta have definite roles but they have no functional homology. Structure-function relationships in our mutant Hbs are discussed.     

Molecular engineering of a polymer of tetrameric hemoglobins.

We have engineered a recombinant mutant human hemoglobin, Hb Prisca beta(S9C+C93A+C112G), which assembles in a polymeric form. The polymerization is obtained through the formation of intermolecular S-S bonds between cysteine residues introduced at position beta9, on the model of Hb Porto Alegre (beta9Ser --> Cys) (Bonaventura and Riggs, Science 1967;155:800-802). Cbeta93 and Cbeta112 were replaced in order to prevent formation of spurious S&bond;S bonds during the expression, assembly, and polymerization events. Dynamic light scattering measurements indicate that the final polymerization product is mainly formed by 6 to 8 tetrameric hemoglobin molecules. The sample polydispersity Q = 0.07 +/- 0.02, is similar to that of purified human hemoglobin (Q = 0.02 +/- 0.02), consistent with a good degree of homogeneity. In the presence of strong reducing agents, the polymer reverts to its tetrameric form. During the depolymerization process, a direct correlation is observed between the hydrodynamic radius and the light scattering of the system, which, in turn, is proportional to the mass of the protein. We interpret this to indicate that the hemoglobin molecules are tightly packed in the polymer with no empty spaces. The tight packing of the hemoglobin molecules suggests that the polymer has a globular shape and, thus, allows estimation of its radius. An illustration of an arrangement of a finite number of tetrameric hemoglobin molecules is presented. The conformational and functional characteristics of this polymer, such as heme pocket conformation, stability to denaturation, autoxidation rate, oxygen affinity, and cooperativity, remain similar to those of tetrameric human hemoglobin.     

Waterproofing the heme pocket. Role of proximal amino acid side chains in preventing hemin loss from myoglobin

The ability of myoglobin to bind oxygen reversibly depends critically on retention of the heme prosthetic group. Globin side chains at the Leu(89)(F4), His(97)(FG3), Ile(99)(FG5), and Leu(104)(G5) positions on the proximal side of the heme pocket strongly influence heme affinity. The roles of these amino acids in preventing heme loss have been examined by determining high resolution structures of 14 different mutants at these positions using x-ray crystallography. Leu(89) and His(97) are important surface amino acids that interact either sterically or electrostatically with the edges of the porphyrin ring. Ile(99) and Leu(104) are located in the interior region of the proximal pocket beneath ring C of the heme prosthetic group. The apolar amino acids Leu(89), Ile(99), and Leu(104) "waterproof" the heme pocket by forming a barrier to solvent penetration, minimizing the size of the proximal cavity, and maintaining a hydrophobic environment. Substitutions with smaller or polar side chains at these positions result in exposure of the heme to solvent, the appearance of crystallographically defined water molecules in or near the proximal pocket, and large increases in the rate of hemin loss. Thus, the naturally occurring amino acid side chains at these positions serve to prevent hydration of the His(93)-Fe(III) bond and are highly conserved in all known myoglobins and hemoglobins.     

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.     

Ligand binding and hexacoordination in synechocystis hemoglobin.

A large and phylogenetically diverse group of organisms contain truncated hemoglobins, including the unicellular cyanobacterium Synechocystis (Pesce, A., Couture, M., Dewilde, S., Guertin, M., Yamauchi, K., Ascenzi, P., Moens, L., and Bolognesi, M. (2000) EMBO J. 19, 2424-2434). Synechocystis hemoglobin is also hexacoordinate, with a heme pocket histidine that reversibly coordinates the ligand binding site. Hexacoordinate hemoglobins are ubiquitous in plants and are now being identified in a diverse array of organisms including humans (Arredondo-Peter, R., Hargrove, M. S., Moran, J. F., Sarath, G., and Klucas, R. V. (1998) Plant Physiol. 118, 1121-1125; Trent, J. T., III, Watts, R. A., and Hargrove, M. S. (2001) J. Biol. Chem. 276, 30106-30110). Rate constants for association and dissociation of the hexacoordinating amino acid side chain in Synechocystis hemoglobin have been measured along with bimolecular rate constants for association of oxygen and carbon monoxide following laser flash photolysis. These values were compared with ligand binding initiated by rapid mixing. Site-directed mutagenesis was used to determine the roles of several heme pocket amino acids in facilitating hexacoordination and stabilizing bound oxygen. It is demonstrated that Synechocystis hemoglobin contains a very reactive binding site and that ligand migration through the protein is rapid. Rate constants for hexacoordination by His(46) are also large and facilitated by other heme pocket amino acids including Gln(43).     

Slow ligand binding kinetics dominate ferrous hexacoordinate hemoglobin reactivities and reveal differences between plants and other species

Hexacoordinate hemoglobins are found in many living organisms ranging from prokaryotes to plants and animals. They are named "hexacoordinate" because of reversible coordination of the heme iron by a histidine side chain located in the heme pocket. This endogenous coordination competes with exogenous ligand binding and causes multiphasic relaxation time courses following rapid mixing or flash photolysis experiments. Previous rapid mixing studies have assumed a steady-state relationship between hexacoordination and exogenous ligand binding that does not correlate with observed time courses for binding. Here, we demonstrate that this assumption is not valid for some hexacoordinate hemoglobins, and that multiphasic time courses are due to an appreciable fraction of pentacoordinate heme resulting from relatively small equilibrium constants for hexacoordination (K(H)). CO binding reactions initiated by rapid mixing are measured for four plant hexacoordinate hemoglobins, human neuroglobin and cytoglobin, and Synechocystis hemoglobin. The plant proteins, while showing a surprising degree of variability, differ from the others in having much lower values of K(H). Neuroglobin and cytoglobin display dramatic biphasic time courses for CO binding that have not been observed using other techniques. Finally, an independent spectroscopic quantification of K(H) is presented that complements rapid mixing for the investigation of hexacoordination. These results demonstrate that hexacoordination could play a much larger role in regulating affinity constants for ligand binding in human neuroglobin and cytoglobin than in the plant hexacoordinate hemoglobins.