The effect of quaternary structure on the state of the alpha and beta subunits within nitrosyl haemoglobin. Low temperature photodissociation and the ESR spectra.

Photodissociation of nitrosyl haemoglobin and nitrosyl hybrids, in which either the alpha or beta subunit is in the nitrosyl form has been stidued at liquid helium temperature (4.2 degrees K) by electron spin resonance and optical absorption spectroscopy. In the presence of inositol hexaphosphate, the photodissociated form of nitrosyl haemoglobin showed an anomalous absorption spectrum in the near infrared region. The experiments with nitrosyl hybrids showed that the alphaNO subunit within the T state haemoglobin is predominantly responsible for the anomalous photodissociated form and the ESR spectrum with three distinct hypefines. The ESR spectrum of alphaNO2betadeoxy2 with inositol hexaphosphate appeared to be very similar to that of the 5-coordinated NO-haem complexes but the absorption spectrum of its photodissociated form was similar to none of protoporphyrin Fe(II) derivatives so far reported. This result suggests that the anomalous photodissociated form may be attributable to some structural distortion of porphyrin or a new electronic state of the haem with different spin state from that of deoxyhaemoglobin.     

Integrin cytoplasmic domains mediate inside-out signal transduction

We analyzed the binding of fibronectin to integrin alpha 5 beta 1 in various cells; in some cells fibronectin bound with low affinity (e.g., K562 cells) whereas in others (e.g., CHO), it bound with high affinity (Kd approximately 100 nM) in an energy-dependent manner. We constructed chimeras of the extracellular and transmembrane domains of alpha IIb beta 3 joined to the cytoplasmic domains of alpha 5 beta 1. The affinity state of these chimeras was assessed by binding of fibrinogen or the monoclonal antibody, PAC1. The cytoplasmic domains of alpha 5 beta 1 conferred an energy-dependent high affinity state on alpha IIb beta 3 in CHO but not K562 cells. Three additional alpha cytoplasmic domains (alpha 2, alpha 6A, alpha 6B) conferred PAC1 binding in CHO cells, while three others (alpha M, alpha L, alpha v) did not. In the high affinity alpha chimeras, cotransfection with a truncated (beta 3 delta 724) or mutated (beta 3(S752-->P)) beta 3 subunit abolished high affinity binding. Thus, both cytoplasmic domains are required for energy-dependent, cell type-specific affinity modulation. In addition, mutations that disrupted a highly conserved alpha subunit GFFKR motif, resulted in high affinity binding of ligands to alpha IIb beta 3. In contrast to the chimeras, the high affinity state of these mutants was independent of cellular metabolism, cell type, and the bulk of the beta subunit cytoplasmic domain. Thus, integrin cytoplasmic domains mediate inside-out signaling. Furthermore, the highly conserved GFFKR motif of the alpha subunit cytoplasmic domain maintains the default low affinity state.     

Use of nitric oxide as a probe for assessing the formation of asymmetrical hemoglobin hybrids. An attempted comparison between alphaNObetaNOalphadeoxybetadeoxy, alpha2NObeta2deoxy, and alpha2deoxybeta2NO hybrids

It has been recently demonstrated that some nitrosyl hemoglobin derivatives have different optical spectrum according to the nature of their quaternary structure (Cassoly, R. (1974) C. R. Seances Acad. Sci., Paris 278, 1417-1420; Salhany, J. M., Ogawa, S., and Shulman, R. G. (1974) Proc. Natl, Acad. Sci. U.S.A. 71, 3359-3362; Cassoly, R. (1975) J. Mol. Biol. 98, 581-595). This property has been used in order to detect the presence of asymmetrical hybrids alphaNObetaNOalpha'O2beta'O2 in a mixture of the two hemoglobins alpha2NObeta2NO and alpha2'O2beta2'O2. When one changes, by deoxygenation, the conformation of the hybrid, there is a characteristic modification in the optical spectrum of the nitrosyl subunits. Quantitative analysis of this phenomenon shows that asymmetrical alphaNObetaNOalphadeoxybetadeoxy and symmetrical alpha2NObeta2deoxy hybrids have distinct properties. The structure-linked optical transition is different in rate and amplitude; it is faster and larger for the asymmetrical molecule. Carbon monoxide binding kinetics performed in absence of phosphate have also indicated that the allosteric equilibrium is more displaced in favor of the T state for alphaNObetaNOalphadeoxybetadeoxy by comparison with the symmetrical deoxygenated intermediates.     

Allosteric interpretation of the oxygen-binding reaction of human hemoglobin tetramers

An allosteric model is presented that provides a simple explanation for the low population of triply ligated species, relative to the other species, in the oxygenation of human hemoglobin tetramers as found in high-concentration studies [Gill, S. J., Di Cera, E., Doyle, M. L., Bishop, G. A., & Robert, C. H. (1987) Biochemistry (preceding paper in this issue)]. The model is a quantitative interpretation of the Perutz mechanism [Perutz, M. F. (1970) Nature (London) 228, 726-739] and is based on a number of structural and thermodynamic findings so far reported in the analysis of hemoglobin properties. Human hemoglobin is assumed to exist in two quaternary states: the T or low-affinity state and the R or high-affinity state. An extreme chain heterogeneity in the T state is postulated so that oxygen binds only to the alpha chains. Nearest-neighbor interactions between the alpha chains may lead to cooperativity within the T state. The R state is noncooperative, and both the alpha and beta chains have equal oxygen affinity.     

Studies on cobalt myoglobins and hemoglobins. Preparation of isolated chains containing cobaltous protoporphyrin IX and characterization of their equilibrium and kinetic properties of oxygenation and EPR spectra.

Human hemoglobin containing cobalt protoporphyrin IX or cobalt hemoglobin has been separated into two functionally active alpha and beta subunits using a new method of subunit separation, in which the -SH groups of the isolated subunits were successfully regenerated by treatment with dithiothreitol in the presence of catalase. Oxygen equilibria of the isolated subunit chains were examined over a wide range of temperature using Imai's polarographic method (Imai, K., Morimoto, H., Kotani, M., Watari, H., and Kuroda, M. (1970) Biochim. Biophys. Acta 200, 189-196). Kinetic properties of their reversible oxygenation were investigated by the temperature jump relaxation method at 16 degrees. Electron paramagnetic resonance characteristics of the molecules in both deoxy and oxy states were studies at 77K. The oxygen affinity of the individual regenerated chains was higher than that of the tetrameric cobalt hemoglobin and was independent of pH. The enthalpy changes of the oxygenation have been determined as -13.8 kcal/mol and -16.8 kcal/mol for the alpha and beta chains, respectively. The rates of oxygenation were similar to those reported for iron hemoglobin chains, whereas those of deoxygenation were about 10(2) times larger. The effects of metal substitution on oxygenation properties of the isolated chains were correlated with the results obtained previously on cobalt hemoglobin and cobalt myoglobin. The EPR spectrum of the oxy alpha chain showed a distinctly narrowed hyperfine structure in comparison with that of the oxy beta chain, indicating that the environment around the paramagnetic center (the bound oxygen) is different between these chains. In the deoxy form, EPR spectra of alpha and beta chains were indistinguishable. These observations suggest that one of the inequivalences between alpha and beta chains might exist near the distal histidine group.     

Binding of CO to mutant alpha chains of hemoglobin M Iwate; evidence for distal imidazole ligation.

The optical contribution of the beta chains to the spectrum of hemoglobin M Iwate (alpha87his leads to tyr)2beta2a was subtracted with the aid of a computer so that the spectrum of ferric alpha chains was obtained. Tyrosinate binding to the heme is suggested from spectral resemblance to ferric heme phenolate in dimethyl sulfoxide. The slow reduction of the abnormal ferric alpha chains in hemoglobin M Iwate by dithionite was studied spectrophotometrically both in the presence and absence of CO. The rate of reduction was found to be dependent on the state of ligation of the normal beta chains. The CO-ligated form of the reduced alpha chains bears strong spectral resemblance to the CO-ligated form of the reduced beta chains suggesting similar structures for the heme-ligand complex. A model compound with similar optical properties to the CO-ligated protein can be prepared in dimethyl sulfoxide from hemin chloride, imidazole, and CO using chromous acetate as the heme reductant. Substitution of phenolate for imidazole produces a spectral entity so different from that observed in the protein as to rule out tyrosinate ligation to ferrous heme of the alpha chains when CO is bound.     

Ethylisocyanide equilibria of hemoglobins M Iwate, M Boston, M Hyde Park, M Saskatoon, and M Milwaukee-I in half-ferric and fully reduced states.

The ethylisocyanide equilibria of all the five known hemoglobins M, namely Hb M Iwate (alpha287 Tyrbeta2), Hb M Boston (alpha258 Tyrbeta2), Hb M Hyde Park (alpha2beta292 Tyr), Hb M Saskatoon (alpha2beta263 tyr), and Hb M Milwaukee-I (alpha2beta267 Glu), were studied both in the half-ferric and fully reduced heme states. In the half-ferric state, no heme-heme interaction was observed for Hb M Iwate, Hb M Boston, and Hb M Hyde Park, but Hb M Saskatoon and Hb M Milwaukee-I show small but definite heme-heme interaction with Hill's n of 1.3. The beta chain mutants, Hb M Hyde Park and Hb M Saskatoon, have almost normal affinity for ethylisocyanide and a normal Bohr effect, whereas the alpha chain mutants, Hb M Iwate and Hb M Boston, have abnormally low affinity and almost no Bohr effect. Hb M Milwaukee-I showed a large Bohr effect and low affinity. These results are consistent qualitatively with those on oxygen equilibria reported previously. In the fully reduced state, in which all four hemes were in the ferrous state and capable of binding ethylisocyanide distinct differences were found in the extent of heme-heme interaction. Namely, the n values for proximal histidine mutants, Hb M Iwate and Hb M Hyde Park, were 1.1 and 1.0, respectively, whereas the distal histidine mutants, Hb M Boston and Hb M Saskatoon, showed high n values of 2.4 and 1.6, respectively. Hb M Milwaukee-I also exhibited a high n value of 2.0 The ethylisocyanide affinity of the four histidine mutants was high compared with that of Hb A, while that for Hb M Milwaukee-I was almost normal. All five Hbs M had approximately normal magnitudes of Bohr effect. In the half-ferric state, the proximal and distal histidine mutants of the same chain showed similar affinity for ethylisocyanide and Bohr effect, rather different from those of the mutants of the opposite chain. These differences seem to be derived from the difference of abnormal bonding of ferric iron to tyrosine or glutamic acid. On the other hand, the reduction of iron, which abolished the abnormal bonding and made all of the chains capable of binding ligand, extinguished the differences of alpha and beta chains, and the effect of amino acid side chains close to iron on ligand binding properties became clear. Proximal histidine, which is considered to trigger the transition between the T and R states, seems to be essential to the heme-heme interaction.     

Structure of deoxy-quaternary haemoglobin with liganded beta subunits

We have determined the structure of a T-state haemoglobin in which the haem groups of the beta subunits have carbon monoxide bound, and the alpha subunits have nickel replacing the haem iron and are ligand-free. The structural adjustments on binding ligand in the T state are in the same direction as those associated with the quaternary transition, and a translational shift of the haem is severely restricted. We explain how these observations may account for the low ligand affinity of the beta haem of T-state haemoglobin.     

The Root effect

Considering the presently available data it is clear that the Root effect represents an exaggerated alkaline Bohr effect which occurs in the absence of a normal acid Bohr effect and is associated with a loss of oxygen binding capacity at low pH. Undoubtedly at the molecular level the presence of a Ser residue at position F9(94) beta in these haemoglobin is of primary importance. No Root effect haemoglobin has yet been identified which lacks this substitution. On the other hand however many haemoglobins are known which possess this Ser residue and at the same time lack a Root effect. Other factors arising from interactions at other sites in the haemoglobin molecule are obviously sufficient to negate the otherwise stabilizing effect of this critical Ser residue. The loss of cooperativity of Root effect systems as the pH is lowered is readily explained as due to stabilization of the low affinity T state to such a degree that the switch to the high affinity R state is suppressed even in the fully liganded molecule. The observation of Hill coefficients of less than unity requires that within the T state chain heterogeneity exists such that the alpha and beta chain haems demonstrate significantly different affinities for ligand. The physiological role of Root effect haemoglobins is demonstrably not inevitably linked to the swim bladder but more probably arose from the need to oxygenate the poorly vascularized retina of many fishes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hb Santa Clara (beta 97His-->Asn), a human haemoglobin variant: functional characterization and structure modelling

This study examines the functional and structural effects of amino acid substitution at alpha(1)beta(2) interface of Hb Santa Clara (beta 97His-->Asn). We have characterized the variation by a combination of electrospray ionisation mass spectrometry and DNA sequence analysis followed by oxygen-binding experiments. Functional studies outlined an increased oxygen affinity, reduced effect of organic phosphates and a reduced Bohr effect with respect to HbA. In view of the primary role of this interface in the cooperative quaternary transition from the T to R conformational state, a theoretical three-dimensional model of Hb Santa Clara was generated. Structural investigations suggest that replacement of Asn for His beta 97 results in a significant stabilization of the high affinity R-state of the haemoglobin molecule with respect to the low affinity T-state. The role of beta FG4 position has been further examined by computational models of known beta FG4 variants, namely Hb Malm? (beta 97His-->Gln), Hb Wood (beta 97His-->Leu), Hb Nagoya (beta 97His-->Pro) and Hb Moriguchi (beta 97His-->Tyr). These findings demonstrate that, among the various residues at the alpha(1)beta(2) (and alpha(2)beta(1)) intersubunit interface, His beta FG4 contributes significantly to the quaternary constraints that are responsible for the low oxygen affinity of human deoxyhaemoglobin.     

Different functional modulation by heterotropic ligands (2,3-diphosphoglycerate and chlorides) of the two haemoglobins from fallow-deer (Dama dama).

Two haemoglobin components have been identified and purified from fallow-deer (Dama dama) erythrocytes. They are present in similar amounts and the two tetrameric molecules share the same alpha chain, while two different beta chains are detected in the two components. The beta chains differ by 14 residues, even though they both have 145 amino-acid residues, which account for a molecular mass of 16,023 and 16,064 Da, respectively, while alpha chain has 141 residues, yielding a molecular mass of 15,142 Da. Compared with human Hb, the N-terminal region of both beta chains shows deletion of Val beta 1 and the replacement of His beta 2 by a methionyl residue, a modification which is common to most ruminant haemoglobins. Although both isolated components show a low intrinsic affinity for oxygen, meaningful differences between the two haemoglobins have been found with respect to the effect of heterotropic effectors, such as 2,3-diphosphoglycerate and chloride ions. In view of the high sequence homology between the two components, the different effect of heterotropic ligands has been tentatively correlated to possible localized structural variations between beta chains of the two haemoglobin components.     

A comparative approach to protein-and ligand-dependence of the Root effect for fish haemoglobins

Ligand-binding equilibria, kinetics and (13)C n.m.r. spectra of bound (13)CO, of the haemoglobins from two fishes that are very distant on the evolutionary scale, i.e. the fourth haemoglobin component from Salmo irideus and the single component from Osteoglossum bicirrhosum, were studied. The C-terminal sequence was also determined for the haemoglobin from Osteoglossum. The results show that (i) the C-terminal residues of both chains are not directly responsible for the characteristic heterotropic effect known as Root effect, since for both fish haemoglobins these residues are identical with those of human haemoglobins. (ii) In all haemoglobins characterized by the Root effect a dependence of the (13)CO n.m.r. resonances on pH is observed. However, the extent of the shift(s) depends on the particular protein, and is probably the result of a combination of both tertiary and quaternary conformational changes. (iii) Both haemoglobins from trout and Osteoglossum manifest a functional heterogeneity between the two types of chains in the tetramer, which increases with proton activity. For CO, the effect is very small for trout haemoglobin IV, and very marked for Osteoglossum haemoglobin; for O(2) strongly heterogeneous binding curves were obtained at approx. pH6.2 with both haemoglobins. (iv) Estimations of the relative values of the affinity constants for the alpha and beta chains in the tetramer were obtained for both haemoglobins from (13)CO n.m.r. spectra at low fractional saturation. On the basis of these findings the molecular mechanism underlying the Root effect is discussed.     

Structure of haemoglobin in the deoxy quaternary state with ligand bound at the alpha haems

We report the X-ray crystal structure of two analogues of human haemoglobin in the deoxy quaternary (T) state with ligand bound exclusively at the alpha haems. These models were prepared from symmetric, mixed-metal hybrid haemoglobin molecules. The structures of alpha Fe(II) beta Co(II), its carbonmonoxy derivative alpha Fe(II)CO beta Co(II), and alpha Fe(II)O2 beta Ni(II) are compared with native deoxy haemoglobin by difference Fourier syntheses at 2.8, 2.9 and 3.5 A resolution, respectively, and the refined alpha Fe(II)CO beta Co(II) structure is analysed. In both the native deoxy and liganded T molecules, the mean plane of the alpha-subunit haem is parallel with the axis of the F helix, but this plane is tilted with respect to the helix axis in the oxy-quaternary R state. The side-chains of LeuFG3 and ValFG5 sterically restrict haem tilting in the T state. We propose that strain energy develops at the contact between the haem and these residues in the liganded T-state haemoglobin, and that the strain is, in part, responsible for the low affinity of the T-state alpha haem.     

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.     

Carbon dioxide binding to human hemoglobin cross-linked between the alpha chains

The binding of carbon dioxide to human hemoglobin cross-linked between Lys alpha 99 residues with bis(3,5-di-bromosalicyl) fumarate was measured using manometric techniques. The binding of CO2 to unmodified hemoglobin can be described by two classes of sites with high and low affinities corresponding to the amino-terminal valines of the beta and alpha chains, respectively (Perrella, M., Kilmartin, J. V., Fogg, J., and Rossi-Bernardi, L. (1975b) Nature 256, 759-761. The cross-linked hemoglobin bound less CO2 than native hemoglobin at all CO2 concentrations in deoxygenated and liganded conformations, and the ligand-linked effect was reduced. Fitting the data to models of CO2 binding suggests that only half of the expected saturation with CO2 is possible. The remaining binding is described by a single affinity constant that for cross-linked deoxyhemoglobin is about two-thirds of the high affinity constant for deoxyhemoglobin A and that for cross-linked cyanomethemoglobin is equal to the high affinity constant for unmodified cyanomethemoglobin A or carbonmonoxyhemoglobin A. The low affinity binding constant for cross-linked hemoglobin in both the deoxygenated and liganded conformations is close to zero, which is significantly less than the affinity constants for either subunit binding site in unmodified hemoglobin. Comparing the low affinity sites in this modified hemoglobin to native hemoglobin suggests that cross-linking hemoglobin between Lys alpha 99 residues prevents CO2 binding at the alpha-subunit NH2 termini.     

High-altitude respiration of birds. Structural adaptations in the major and minor hemoglobin components of adult Rüppell's Griffon (Gyps rueppellii, Aegypiinae): a new molecular pattern for hypoxic tolerance

The primary structures of the hemoglobins Hb A, Hb A', Hb D and Hb D' of Rüppell's Griffon (Gyps rueppellii), which can fly as high as 11,300 m, are presented. The globin chains were separated on CM-Cellulose in 8M urea buffers, the four hemoglobin components by FPLC in phosphate buffers. The amino-acid sequences of five globin chains were established by automatic Edman degradation of the globin chains and of the tryptic peptides in liquid-phase and gas-phase sequenators. The sequences are compared with those of other Falconiformes. A new molecular pattern for survival at extreme altitudes is presented. For the first time four hemoglobins are found in blood of a bird; they show identical beta-chains and differ in the alpha A- and alpha D-chains by only one replacement. These four hemoglobins cause a gradient in oxygen affinities. The two main components Hb A and Hb A' differ at position alpha 34 Thr/Ile. In case of Ile as found in Hb A' an alpha 1 beta 1-interface is interrupted raising oxygen affinity compared to Hb A. In addition the hemoglobins of the A- and D-groups differ at position alpha 38 Pro or Gln/Thr (alpha 1 beta 2-interface). Expression of Gln in Hb D/D' raises the oxygen affinity of these components compared to Hb A/A' by destabilization of the deoxy-structure. The physiological advantage lies in the functional interplay of four hemoglobin components. Three levels of affinity are predicted: low affinity Hb A, Hb A' of intermediate affinity, and high affinity Hb D/D'. This cascade tallies exactly with oxygen affinities measured in the isolated components and predicts oxygen transport by the composite hemoglobins over an extended range of oxygen affinities. It is contended that the mechanisms of duplication of the alpha-genome (creating four hemoglobins) and of nucleotide replacements (creating different functional properties) are responsible for this remarkable hypoxic tolerance to 11,300 m. Based on this pattern the hypoxic tolerances of other vultures are predicted.     

Structure-related changes of the electron spin resonance spectra of the monomeric nitrosyl haemoglobin IV from Chironomus thummi thummi

The monomeric haemoglobin IV from Chironomus thummi thummi (CTT IV) is an allosteric protein characterized by pH-dependent ligand affinities (Bohr-effect). The ligand-linked proton dissociation gives rise to a t r conformational transition. Furthermore, the Bohr-effect is ligand-dependent and decreases in magnitude following the order of ligands, O₂ > CO > NO. Although the Bohr-effect for NO is smallest, the electron spin resonance (ESR) spectra of frozen solutions of ¹⁵NO-ligated CTT IV measured as higher derivatives at 77 K reflect this pH-dependent conformation change. g Tensor and hyperfine constants coinciding with the principal directions of the g tensor have been evaluated for ⁵⁷Fe, ¹⁵NO, ¹⁴N_E-imidazole, and ¹⁴N-pyrroles.Hyperfine parameters and g values of both conformation states of this haemoglobin, i.e., of the t state at low pH with low ligand affinity and of the r state at high pH with high ligand affinity, are characteristic for a hexacoordinated nitrosyl haem complex. The change in pH leads to a variation of the Fe-N-O bond angle which is larger at high pH (r conformation) than at low pH (t conformation). Furthermore, the spin transfer from NO into iron orbitals is larger at high pH than at low pH. These results are consistent with the assumption that the interaction of proximal imidazole and iron is smaller in the r conformation than in the t conformation.Binding of anionic detergents to nitrosyl CTT IV causes a conversion of the native (t, r) into a denatured (super-r) structure. The latter, on the basis of hyperfine and g values, apparently contains a pentacoordinated nitrosyl haem complex. Because of the extreme displacement of the proximal imidazole in the super-r structure, the Fe-N-O gouping is nearly linear and a large spin transfer from NO into iron orbitals occurs. Removal of anionic detergents from the protein leads to a full reconversion of the super-r into the native conformations.These structure-related changes of hyperfine constants and g tensor further support the assumption that the trans-effect of the proximal imidazole is an important link of allosteric interactions in haemoglobins.     

Locking the beta3 integrin I-like domain into high and low affinity conformations with disulfides

Although integrin alpha subunit I domains exist in multiple conformations, it is controversial whether integrin beta subunit I-like domains undergo structurally analogous movements of the alpha7-helix that are linked to affinity for ligand. Disulfide bonds were introduced into the beta(3) integrin I-like domain to lock its beta6-alpha7 loop and alpha7-helix in two distinct conformations. Soluble ligand binding, ligand mimetic mAb binding and cell adhesion studies showed that disulfide-bonded receptor alpha(IIb)beta(3)(T329C/A347C) was locked in a low affinity state, and dithiothreitol treatment restored the capability of being activated to high affinity binding; by contrast, disulfide-bonded alpha(IIb)beta(3)(V332C/M335C) was locked in a high affinity state. The results suggest that activation of the beta subunit I-like domain is analogous to that of the alpha subunit I domain, i.e. that axial movement in the C-terminal direction of the alpha7-helix is linked to rearrangement of the I-like domain metal ion-dependent adhesion site into a high affinity conformation.     

Spectral transitions of nitrosyl hemes during ligand binding to hemoglobin.

Human deoxyhemoglobin has been titrated with nitric oxide at several pH values ranging from 6.0 to 9.0, in the presence and absence of the allosteric effector inositol hexaphosphate at 25 degrees C. Samples were frozen for EPR measurements or analyzed optically within 30 s after mixing to ensure a kinetic population of intermediates. Fractions of pentacoordinate alpha-NO heme groups were determined by fitting EPR and absorbance difference spectra in terms of linear combinations of standard signals. Equivalent results were obtained by these techniques. The fraction of alpha-NO heme exhibiting pentacoordinate character in Hb4NO increases from 0.07 to 0.73 in going from pH 9 to 6. The fraction of alpha hemes which are pentacoordinate in fully saturated nitrosyl hemoglobin, Hb4(NO), increases from 0.0 to 0.41 over the same pH range. Only in the presence of bound inositol-P6 are all 4 the alpha-NO hemes pentacoordinate. Thus, the expression of modified NO heme character is not simply a reflection of the formation of low affinity quaternary conformations. Rather, within this conformation the alpha chain iron atoms exhibit an equilibrium between hexa- and pentacoordinate structures which is perturbed markedly by both proton and phosphate binding. No intermediate coordination structure of the type suggested by Chevion et al. (Chevion, M., Stern, A., Peisach, J., Blumberg, W.E., and Simon, S. (1978) Biochemistry 17, 1745-1750) appears to occur since the observed alpha-NO heme spectra can always by represented quantitatively as a linear combination of the normal hexacoordinate and pentacoordinate signals. The formation of pentacoordinate alpha-NO causes this subunit to exhibit a higher affinity for nitric oxide. Thus on standing at low levels of saturation, there is a slow (t1/2 approximately equal to 8 min at pH 7, 25 degrees C) re-equilibration of ligand from beta to alpha subunits. The final ratio of alpha-NO to beta-NO is 2 to 1 in the absence of phosphates and greater than 10 to 1 in the presence of inositol hexaphosphate.     

Xenopus laevis hemoglobin and its hybrids with hemoglobin A+

Isolated alpha and beta chains from Xenopus laevis hemoglobin have been purified. The isolation procedure yields native alpha chains whose functional behavior has been characterized and compared with that of human alpha chains. Isolated beta chains in the presence of oxygen are characterized by low stability, and hence their functional characterization was limited to the CO binding kinetics. When stoichiometric amounts of the isolated alpha and beta chains are mixed, a tetramer characterized by heme-heme interactions and oxygen affinity comparable to that of the native molecule is readily reconstituted. Moreover, both chains, under appropriate conditions, form stable hybrid tetramers with the partner subunits from human hemoglobin; results on the functional properties of these hybrid hemoglobins are presented and discussed in relation to the stereochemical model of the Root effect.