The effects of E7 and E11 mutations on the kinetics of ligand binding to R state human hemoglobin

Association and dissociation rate constants for O2, CO, and methyl isocyanide binding to native and distal pocket mutants of R state human hemoglobin were measured using ligand displacement and partial photolysis techniques. Individual rate constants for the alpha and beta subunits were resolved by comparisons between the kinetic behavior of the native and mutant proteins. His-E7 was replaced with Gly and Gln in both alpha and beta subunits and with Phe in beta subunits alone. In separate experiments Val-E11 was replaced with Ala, Leu, and Ile in each globin chain. The parameters describing ligand binding to R state alpha subunits are sensitive to the size and polarity of the amino acids at positions E7 and E11. The distal histidine in this subunit inhibits the bimolecular rate of binding of both O2 and CO, sterically hinders bound CO and methyl isocyanide, and stabilizes bound O2 by hydrogen bonding. The Val-E11 side chain in alpha chains also appears to be part of the kinetic barrier to O2 and CO binding since substitution with Ala causes approximately 10-fold increases in the association rate constants for the binding of these diatomic ligands. However, substitution of Val-E11 by Ile produces only small decreases in the rates of ligand binding to alpha subunits. For R state beta subunits, the bimolecular rates of O2 and CO binding are intrinsically large, approximately 2-5-fold greater than those for alpha subunits, and with the exception of Val-E11----Ile mutation, little affected by substitutions at either the E7 or E11 positions. For the beta Val-E11----Ile mutant the association rate and equilibrium constants for all three ligands decreased 10-50-fold. All of these results agree with Shaanan's conclusions that the distal pocket in liganded beta subunits is more open whereas in alpha subunits bound ligands are more sterically hindered by adjacent distal residues (Shaanan, B. (1983) J. Mol. Biol. 171, 31-59). In the case of O2 binding to alpha subunits, the unfavorable steric effects are compensated by the formation of a hydrogen bond between the nitrogen atom of His-E7 and bound dioxygen.     

X-ray diffraction studies of a partially liganded hemoglobin, [alpha(FeII-CO)beta(MnII)]2

We have applied single-crystal X-ray diffraction methods to analyze the structure of [alpha(FeII-CO)beta(MnII)]2, a mixed-metal hybrid hemoglobin that crystallizes in the deoxyhemoglobin quaternary structure (the T-state) even though it is half liganded. This study, carried out at a resolution of 3.0 A, shows that (1) the Mn(II)-substituted beta subunits are structurally isomorphous with normal deoxy beta subunits, and (2) CO binding to the alpha subunits induces small, localized changes in the T-state that lack the main directional component of the corresponding larger structural changes in subunit tertiary structure that accompany complete ligand binding to all four subunits and the deoxy to oxy quaternary structure change. Specifically, in the T-state, CO binding to the alpha heme group draws the iron atom toward the heme plane, and this in turn pulls the last turn of the F helix (residues 85 through 89) closer to the heme group. The direction of these small movements is almost perpendicular to the axis of the F helix. In contrast, when the structures of fully liganded and deoxyhemoglobin are compared, extensive structural changes occur throughout the F helix and FG corner, and the main component of the atomic movements in the F helix (in addition to the smaller component toward the heme) is in a direction parallel to the heme plane and toward the alpha 1 beta 2 interface. These findings are discussed in terms of the current stereochemical theories of co-operative ligand binding and the Bohr effect.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

Transient kinetics of oxygen dissociation from ferrous subunits of iron-cobalt hybrid hemoglobins. The principal reaction controlling the co-operativity

The oxygen dissociation constants from Fe subunits in the half-ligated intermediate states of Fe-Co hybrid hemoglobins, alpha(Fe-O2)2 beta(Co)2 and alpha(Co)2 beta(Fe-O2)2, have been determined as functions of pH, temperature and inositol hexaphosphate. The oxygen dissociation rates from alpha(Fe-O2)2 beta(Co)2 are estimated to be more than 1300 s-1 for the deoxy quaternary state (T-state) and less than 3 s-1 for the oxy quaternary state (R-state) at 15 degrees C in 50 mM-Tris or Bis-Tris buffer containing 0.1 M-Cl-, while those of alpha(Co)2 beta(Fe-O2)2 are more than 180 s-1 and less than 5 s-1 for the T and R-states, respectively. The pH dependence of the oxygen dissociation rate from Fe subunits is large enough to be accounted for by the R-T transition, and implies that those half-ligated intermediate hybrids mainly exist in the R-state at pH 8.8, and in the T-state at pH 6.6, while other studies indicated that the half-ligated hybrids are essentially in the R-state at pH 7. Large activation energies of the oxygen dissociation process of 19 to 31 kcal/mol determined from the temperature dependence suggest that the process is entropy-driven.     

Effect of the distal residues on the vibrational modes of the Fe-CO bond in hemoglobin studied by protein engineering

Using an Escherichia coli gene expression system, we have engineered human hemoglobin (Hb) mutants having the distal histidine (E7) and valine (E11) residues replaced by other amino acids. The interaction between the mutated distal residues and bound carbon monoxide has been studied by Soret-excited resonance Raman spectroscopy. The replacement of Val-E11 by Ala, Leu, Ile, and Met has no effect on the v(C-O), v(Fe-CO) stretching or delta(Fe-C-O) bending frequencies in both the alpha and beta subunits of Hb, although some of these mutations affect the CO affinity as much as 40-fold. The strain imposed on the protein by the binding of CO is not localized in the Fe-CO bond and is probably distributed among many bonds in the globin. The replacement of His-E7 by Val or Gly brings the stretching frequencies v(Fe-CO) and v(C-O) close to those of free heme complexes. In contrast, the substitution of His-E7 by Gln, which is flexible and polar, produces no effects on the resonance Raman spectrum of either alpha- or beta-globin. The replacement of His-E7 of beta-globin by Phe shows the same effect as replacement by Gly or Val. Therefore, the steric bulk of the distal residues is not the primary determinant of the Fe-CO ligand vibrational frequencies. The ability of both histidine and glutamine to alter the v(C-O), v(Fe-CO), or delta(Fe-C-O) frequencies may be attributed to the polar nature of their side chains which can interact with bound CO in a similar manner.     

R-state AMP complex reveals initial steps of the quaternary transition of fructose-1,6-bisphosphatase

AMP transforms fructose-1,6-bisphosphatase from its active R-state to its inactive T-state; however, the mechanism of that transformation is poorly understood. The mutation of Ala(54) to leucine destabilizes the T-state of fructose-1,6-bisphosphatase. The mutant enzyme retains wild-type levels of activity, but the concentration of AMP that causes 50% inhibition increases 50-fold. In the absence of AMP, the Leu(54) enzyme adopts an R-state conformation nearly identical to that of the wild-type enzyme. The mutant enzyme, however, grows in two crystal forms in the presence of saturating AMP. In one form, the AMP-bound tetramer is in a T-like conformation, whereas in the other form, the AMP-bound tetramer is in a R-like conformation. The latter reveals conformational changes in two helices due to the binding of AMP. Helix H1 moves toward the center of the tetramer and displaces Ile(10) from a hydrophobic pocket. The displacement of Ile(10) exposes a hydrophobic surface critical to interactions that stabilize the T-state. Helix H2 moves away from the center of the tetramer, breaking hydrogen bonds with a buried loop (residues 187-195) in an adjacent subunit. The same hydrogen bonds reform but only after the quaternary transition to the T-state. Proposed here is a model that accounts for the quaternary transition and cooperativity in the inhibition of catalysis by AMP.     

Kinetic studies on partially liganded species of carboxyhemoglobin: alpha 2 CO beta 2 and alpha 2 beta 2CO

The valency hybrids of Hb A, alpha 2CO beta 2+, and alpha 2+ beta 2CO have been prepared by a new high pressure liquid chromatography method, and the kinetics of their CO-combination and dissociation reactions have been studied by double mixing and microperoxidase methods. Both reactions are biphasic. The slow phase in CO-combination and the fast phase in CO-dissociation are due to the reactions of alpha CO2 beta T2 or alpha 2 beta 2CO,T. The fast phase in CO-combination reaction has two components, one due to the dimers of the hybrid and the other due to the R-state tetramer. Immediately after the reduction of the valency hybrids, the overall system is represented by the equation: 2 alpha CO beta in equilibrium alpha 2CO beta 2R in equilibrium alpha 2CO beta 2T or (formula: see text) If the solutions are aged for 3-11 s, the R-state population is reduced gradually to a very small size, and the main species after 11 s of aging are dimers and T-state tetramers. Analysis of the kinetic data indicates slow R in equilibrium T equilibria in the absence of phosphates and significant dissociation of the T-state tetramer. It is concluded that the subunit contacts alpha 1-beta 2 (or alpha 2-beta 1) are impaired seriously in the hybrids. Very slow R in equilibrium T relaxation makes these hybrids unlikely intermediates in the sequential binding of CO to Hb tetramer.     

Importance of stalk segment S5 for intramolecular communication in the sarcoplasmic reticulum Ca2+-ATPase

Sixteen residues in stalk segment S5 of the Ca(2+)-ATPase of sarcoplasmic reticulum were studied by site-directed mutagenesis. The rate of the Ca(2+) binding transition, determined at 0 degrees C, was enhanced relative to wild type in mutants Ile(743) --> Ala, Val(747) --> Ala, Glu(748) --> Ala, Glu(749) --> Ala, Met(757) --> Gly, and Gln(759) --> Ala and reduced in mutants Asp(737) --> Ala, Asp(738) --> Ala, Ala(752) --> Leu, and Tyr(754) --> Ala. In mutant Arg(762) --> Ile, the rate of the Ca(2+) binding transition was wild type like at 0 degrees C, whereas it was 3.5-fold reduced relative to wild type at 25 degrees C. The rate of dephosphorylation of the ADP-insensitive phosphoenzyme was increased conspicuously in mutants Ile(743) --> Ala and Tyr(754) --> Ala (close to 20-fold in the absence of K(+)) and increased to a lesser extent in Asn(739) --> Ala, Glu(749) --> Ala, Gly(750) --> Ala, Ala(752) --> Gly, Met(757) --> Gly, and Arg(762) --> Ile, whereas it was reduced in mutants Asp(737) --> Ala, Val(744) --> Gly, Val(744) --> Ala, Val(747) --> Ala, and Ala(752) --> Leu. In mutants Ile(743) --> Ala, Tyr(754) --> Ala, and Arg(762) --> Ile, the apparent affinities for vanadate were enhanced 23-, 30-, and 18-fold, respectively, relative to wild type. The rate of Ca(2+) dissociation was 11-fold increased in Gly(750) --> Ala and 2-fold reduced in Val(747) --> Ala. Mutants with alterations to Arg(751) either were not expressed at a significant level or were completely nonfunctional. The findings show that S5 plays a crucial role in mediating communication between the Ca(2+) binding pocket and the catalytic domain and that Arg(751) is important for both structural and functional integrity of the enzyme.     

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.     

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

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

Analysis of the kinetic barriers for ligand binding to sperm whale myoglobin using site-directed mutagenesis and laser photolysis techniques

Time courses for NO, O2, CO, methyl and ethyl isocyanide rebinding to native and mutant sperm whale myoglobins were measured at 20 degrees C following 17-ns and 35-ps laser excitation pulses. His64 (E7) was replaced with Gly, Val, Leu, Phe, and Gln, and Val68 (E11) was replaced with Ala, Ile, and Phe. For both NO and O2, the effective picosecond quantum yield of unliganded geminate intermediates was roughly 0.2 and independent of the amino acids at positions 64 and 68. Geminate recombination of NO was very rapid; 90% rebinding occurred within 0.5-1.0 ns for all of the myoglobins examined; and except for the Gly64 and Ile68 mutants, the fitted recombination rate parameters were little influenced by the size and polarity of the amino acid at position 64 and the size of the residue at position 68. The rates of NO recombination and ligand movement away from the iron atom in the Gly64 mutant increased 3-4-fold relative to native myoglobin. For Ile68 myoglobin, the first geminate rate constant for NO rebinding decreased approximately 6-fold, from 2.3 x 10(10) s-1 for native myoglobin to 3.8 x 10(9) s-1 for the mutant. No picosecond rebinding processes were observed for O2, CO, and isocyanide rebinding to native and mutant myoglobins; all of the observed geminate rate constants were less than or equal to 3 x 10(8) s-1. The rebinding time courses for these ligands were analyzed in terms of a two-step consecutive reaction scheme, with an outer kinetic barrier representing ligand movement into and out of the protein and an inner barrier representing binding to the heme iron atom by ligand occupying the distal portion of the heme pocket. Substitution of apolar amino acids for His64 decreased the absolute free energies of the outer and inner kinetic barriers and the well for non-covalently bound O2 and CO by 1 to 1.5 kcal/mol, regardless of size. In contrast, the His64 to Gln mutation caused little change in the barrier heights for all ligands, showing that the polar nature of His64 inhibits both the bimolecular rate of ligand entry into myoglobin and the unimolecular rate of binding to the iron atom from within the protein. Increasing the size of the position 68(E11) residue in the series Ala to Val (native) to Ile caused little change in the rate of O2 migration into myoglobin or the equilibrium constant for noncovalent binding but did decrease the unimolecular rate for iron-O2 bond formation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Crystal structure of horse carbonmonoxyhemoglobin-bezafibrate complex at 1.55-A resolution. A novel allosteric binding site in R-state hemoglobin

Bezafibrate, an antilipidemic drug, is known as a potent allosteric effector of hemoglobin. The previously proposed mechanism for the allosteric potency of this drug was that it stabilizes and constrains the T-state of hemoglobin by specifically binding to the large central cavity of the T-state. Here we report a new allosteric binding site of fully liganded R-state hemoglobin for this drug. The high resolution crystal structure of horse carbonmonoxyhemoglobin in complex with bezafibrate reveals that the bezafibrate molecule lies near the surface of the E-helix of each alpha subunit and the complex maintains the quaternary structure of the R-state. Binding is caused by the close fit of bezafibrate into the binding pocket, which is composed of some hydrophobic residues and the heme edge, suggesting the importance of hydrophobic interactions. Upon binding of bezafibrate, the distance between Fe and the N epsilon(2) of distal His E7(alpha 58) is shortened by 0.22 A in the alpha subunit, whereas no significant structural changes are transmitted to the beta subunit. Oxygen equilibrium studies of R-state-locked hemoglobin with bezafibrate in a wet porous sol-gel indicate that bezafibrate selectively lowers the oxygen affinity of one type of subunit within the R-state, consistent with the structural data. These results disclose a new allosteric mechanism of bezafibrate and offer the first demonstration of how the allosteric effector interacts with R-state hemoglobin.     

The primary structure of sperm whale hemoglobin (Physeter catodon, cetacea)

The complete primary structure of the two major hemoglobin components of sperm whale (Physeter catodon) is presented. The major components A and B account for 55% and 40% respectively whereas the minor component constitutes for 5% of the total hemoglobin. The globin chains were separated on CM-Cellulose in 8M urea buffer. The sequence was determined by automatic Edman degradation of tryptic and hydrolytic peptides in a liquid phase sequencer. Alignment of the sequence with human hemoglobin shows 22 exchanges each for the alpha I and alpha II and 21 exchanges for the beta I and beta II chains. Within the two beta-chains three differences have been located, beta NA2 His/Gln, beta A2 Gly/Ala and beta A8 Leu/Val. The two alpha-chains are characterized by heterogeneities at position alpha A8 Val/Ile or Ala/Ile (ratio of the phenylthiohydantoin derivatives of the amino acids 1:1) and alpha AB1 Asn/Ser (ratio of the phenylthiohydantoin derivatives of the amino acids 6:4). The role of these exchanges in modulating oxygen affinity is discussed.     

Nanosecond optical spectra of iron-cobalt hybrid hemoglobins: geminate recombination, conformational changes, and intersubunit communication

Hybrid hemoglobins were prepared in which cobalt was substituted for the heme iron in either the alpha or beta subunits. Transient optical absorption spectra were measured at room temperature for these hybrids at time intervals between 0 and 50 ms following photodissociation of the carbon monoxide complex with 10-ns laser pulses. The cobalt porphyrins do not bind carbon monoxide, making it possible to investigate the time-resolved response of the cobalt-containing subunits to photodissociation of carbon monoxide in the iron-containing subunits. At the same time the response of the iron-containing subunits to the photolysis event can be studied, permitting an independent determination of the kinetics of ligand rebinding and conformational changes in the alpha and beta subunits of an intact tetramer. The data were analyzed by using singular-value decomposition to obtain the kinetic progress curve for ligand rebinding, the deoxyheme and cobalt porphyrin spectral changes, and the time course of these spectral changes. The geminate rebinding kinetics following photodissociation of alpha(Co)2 beta(Fe-CO)2 were very similar to those found unsubstituted hemoglobin, alpha(Fe-CO)2 beta(Fe-CO)2, indicating equivalence of the geminate kinetics for alpha and beta subunits within the R-state tetramer. The results for alpha(Fe-CO)2 beta(Co)2 were consistent with this conclusion, even though the analysis was complicated by the presence of comparable populations of R- and T-state species. Comparison of the deoxyheme spectral changes and relaxation times among the three molecules indicated that both alpha and beta subunits contribute to the deoxyheme spectral changes that signal tertiary and quaternary conformational changes in the unsubstituted tetramer. The response of the cobalt porphyrins to photodissociation was similar in the two hybrids. No structural changes were detected in the cobalt-containing subunits until the second tertiary conformational change in the iron-containing subunits observed at 1-2 microseconds. Much larger structural changes, as judged by the amplitude of the spectral changes, occurred in the cobalt-containing subunits concomitant with the R----T quaternary change at about 20 microseconds.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

Involvement of the distal histidine in the low affinity exhibited by Hb Chico (Lys beta 66----Thr) and its isolated beta chains.

Hemoglobin (Hb) Chico (Lys beta 66----Thr at E10) has a diminished oxygen affinity (Shih, D. T.-b., Jones, R. T., Shih, M. F.-C., Jones, M. B., Koler, R. D., and Howard, J. (1987) Hemoglobin 11, 453-464). Our studies show that its P50 is about twice that of Hb A and that its cooperativity, anion, and Bohr effects between pH 7 and 8 are normal. The Bohr effect above pH 8 is somewhat reduced, indicating a small but previously undocumented involvement of the ionic bond formed by Lys beta 66 in the alkaline Bohr effect. Since the oxygen affinity of the alpha-hemes is likely to be normal, that of the beta-hemes in the tetramer is likely to be reduced by the equivalent of 1.2 kcal/mol beta-heme in binding energy. Remarkably, both initial and final stages of oxygen binding to Hb Chico are of lowered affinity relative to Hb A under all conditions examined. The isolated beta chains also show diminished oxygen affinity. In T-state Hb A, Lys(E10 beta) forms a salt bridge with one of the heme propionates, but comparison with other hemoglobin variants shows that rupture of this bridge cannot be the cause of the low oxygen affinity. X-ray analysis of the deoxy structure has now shown that Thr beta 66 either donates a hydrogen bond to or accepts one from His beta 63 via a bridging water molecule. This introduces additional steric hindrance to ligand binding to the T-state that results in slower rates of ligand binding. We measured the O2/CO partition coefficient and the kinetics of oxygen dissociation and carbon monoxide binding and found that lowered O2 and CO affinity is also exhibited by the R-state tetramers and the isolated beta chains of Hb Chico.     

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.     

Enhancing stability and expression of recombinant human hemoglobin in E. coli: Progress in the development of a recombinant HBOC source

The commercial feasibility of recombinant human Hb (rHb) as an O(2) delivery pharmaceutical is limited by the production yield of holoprotein in E. coli. Currently the production of rHb is not cost effective for use as a source in the development of third and fourth generation Hb-based oxygen carriers (HBOCs). The major problems appear to be aggregation and degradation of apoglobin at the nominal expression temperatures, 28-37 degrees C, and the limited amount of free heme that is available for holohemoglobin assembly. One approach to solve the first problem is to inhibit apoglobin precipitation by a comparative mutagenesis strategy to improve apoglobin stability. alpha Gly15 to Ala and beta Gly16 to Ala mutations have been constructed to increase the stability of the alpha helices of both subunits of HbA, based on comparison with the sequences of the more stable sperm whale hemoglobin subunits. Fetal hemoglobin is also known to be more stable than human HbA, and sequence comparisons between human beta and gamma (fetal Hb) chains indicate several substitutions that stabilize the alpha1beta1 interface, one of which, beta His116 to Ile, increases resistance to denaturation and enhances expression in E. coli. These favorable effects of enhanced globin stability can be augmented by co-expression of bacterial membrane heme transport systems to increase the rate and extent of heme uptake through the bacterial cell membranes. The combination of increased apoglobin stability and active heme transport appear to enhance holohemoglobin production to levels that may make rHb a plausible starting material for all extracellular Hb-based oxygen carriers.     

NMR study of human mutant hemoglobins synthesized in Escherichia coli. Consequences of tyrosine alpha 42 substitutions

The hydroxyl group of Tyr alpha 42 in human hemoglobin forms a hydrogen bond with the carboxylate of Asp beta 99 which is considered to be one of the most important hydrogen bonds for stabilizing the "T-state." However, no spontaneous mutation at position 42 of the alpha subunit has been reported, and the role of the tyrosine has not been tested experimentally. Two artificial human mutant hemoglobins in which Tyr alpha 42 was replaced by phenylalanine or histidine were synthesized in Escherichia coli, and their proton NMR spectra were studied with particular attention to the hyperfine-shifted and hydrogen-bonded proton resonances. The site-directed mutagenesis of the Tyr alpha 42----Phe removes the hydrogen bond described above and prevents transition to the T-state so that the mutant Hb is rather similar to the "R-state" even when deoxygenated. On the other hand, the mutation from tyrosine to histidine causes less drastic structural changes, and its quaternary and tertiary structures are almost the same as native deoxy-Hb A. This may be attributed to the formation of a new hydrogen bond between His alpha 1(42) and Asp beta 2(99). These observations indicate that the hydrogen bond formed between Tyr alpha 42 and Asp beta 99 is required to convert unliganded Hb to the T-state.     

Thermal stability of hemoglobin crosslinked in the T-state by bis(3,5-dibromosalicyl) fumarate

Bis(3,5-dibromosalicyl) fumarate was used to crosslink oxyhemoglobin between Lys 82 beta 1 and Lys 82 beta 2 (Walder, J. A., et al. (1979) Biochemistry 18, 4265) and deoxyhemoglobin between Lys 99 alpha 1 and Lys 99 alpha 2 (Chatterjee R.Y., et al. (1986) J. Biol. Chem. 261, 9929). Thermal denaturations demonstrated that alpha crosslinked hemoglobin (alpha 99XLHb A) has the same stability as the beta crosslinked one (beta 82XLHb A). Both alpha and beta crosslinked methemoglobins have a denaturation temperature in 0.9 M guanidine of 57 degrees C compared to 41 degrees C of Hb A. The second product from the T-state crosslinking reaction was found to be crosslinked between the beta chains by chain separation and amino acid analysis. The possible positions for this crosslink are limited to the bisphosphoglycerate binding site in the three-dimensional structure. Its stability is comparable to that of the alpha 99XLHb A or beta 82XLHb A. These modified hemoglobins are potential blood substitutes.