Structural transitions upon ligand binding in a cooperative dimeric hemoglobin.

Comparison of the 2.4 angstrom resolution crystal structures of dimeric clam hemoglobin in the deoxygenated and carbon-monoxide liganded states shows how radically different the structural basis for cooperative oxygen binding is from that operative in mammalian hemoglobins. Heme groups are in direct communication across a novel subunit interface formed by the E and F helices. The conformational changes at this interface that accompany ligand binding are more dramatic at a tertiary level but more subtle at a quaternary level than those in mammalian hemoglobins. These findings suggest a cooperative mechanism that links ligation at one subunit with potentiation of affinity at the second subunit.

The dimeric assembly of Photobacterium leiognathi and Salmonella typhimurium SodC1 Cu,Zn superoxide dismutases is affected differently by active site demetallation and pH: an analytical ultracentrifuge study.

To establish whether the species-specific variations at the subunit interface of bacterial Cu,Zn superoxide dismutases affect dimer assembly, the association state of the Photobacterium leiognathi (PlSOD) and Salmonella typhimurium (StSOD) enzymes, which differ in 11 out of 19 interface residues, was investigated by analytical ultracentrifugation. The same linkage pattern correlates quaternary assembly, active site metallation, and pH in the two enzymes albeit with quantitative differences. Both holo-enzymes are stable dimers at pH 6.8 and 8.0, although their shape is altered at alkaline pH. In contrast, dimer stability is affected differently by metal removal. Thus, apo-StSOD is a stable dimer at pH 6.8 whereas apo-PlSOD is in reversible monomer-dimer equilibrium. In both apoproteins a pH increase to 8.0 favors monomerization. These effects prove the existence of long-range communication between the active site and the subunit interface and provide a structural explanation for the known functional differences between the two enzymes.

Kinetics of iron reduction of ferritin cores and of a low molecular weight hydroxy-iron polymer.

The kinetics of reduction by dithionite of iron cores in ferritin and of iron cores after removal of the protein shell (isolated cores) has been analysed along with that of a low molecular weight hydroxy-iron polymer. Spectrophotometric measurements have shown that the time course of the reaction approximates a pseudo first-order behaviour. In the case of ferritin cores the pseudo first-order rate constant reaches an asymptotic value which markedly increases when the cores are isolated. In contrast, in the low molecular weight hydroxy-iron polymer, no clear asymptotic value is reached. Thus, the rate of reduction appears to be determined both by the presence of the protein shell and by the crystallite surface area. Kinetic light scattering experiments show that in ferritin and in the isolated cores a rapid drop in molecular weight occurs during the first stages of reduction, suggesting a fragmentation of the iron crystallite.

The distal heme pocket of Escherichia coli flavohemoglobin probed by infrared spectroscopy.

The infrared absorption spectra of ferric cyanide and ferrous carbonmonoxy Escherichia coli flavohemoglobin have been measured in order to probe the fine structural properties of the distal heme pocket, characterized by the presence of a tyrosine in position B10 and a glutamine in position E7. The stretching frequency of iron bound cyanide occurs at 2136 cm(-1), an unusually high value if compared to other heme proteins. The infrared spectrum of the CO bound derivative displays two peaks centered at 1960 cm(-1) and at 1909 cm(-1) respectively. H(2)O effects have been studied in both the ferric cyanide and ferrous CO derivatives in order to establish the presence of a distal hydrogen bonding to the iron bound ligand. The observed isotope shifts indicate that in the ferric cyanide derivative a hydrogen bond is donated from a residue in the distal pocket to the biatomic ligand whereas in the ferrous carbon monoxy derivative only the 1909 cm(-1) component is most likely hydrogen bonded to the phenolic group of TyrB10.

Dimeric and tetrameric hemoglobins from the mollusc Scapharca inaequivalvis. Reaction of the oxidized derivatives with azide and fluoride.

The reaction of the oxidized derivatives of the dimeric (HbI) and tetrameric (HbII) Scapharca inaequivalvis hemoglobins with azide and fluoride has been studied. The two oxidized hemoglobins have specific characteristics. Oxidized HbI consists of a dimeric high-spin aquomet form which is in a pH-dependent association-dissociation equilibrium with a monomeric low-spin hemichrome. In contrast, in HbII the high-spin aquomet derivative is only a transient species that converts itself into a tetrameric hemichrome which in turn dissociates into lower molecular weight forms. The reaction of oxidized HbI with azide and fluoride can be described in terms of a simple reaction scheme which assumes that external ligands bind only to the aquomet derivative. In the case of HbII, the reaction route is the same; however, the situation is complicated by the fact that in the dissociated hemichromes the internal protein ligand can no longer be displaced. Therefore, irreversible processes take place whose relevance depends primarily on the affinity of the external ligand for the ferric heme iron.

On the identity of the dissociation-linked chloride binding sites in human hemoglobin. Studies with hemoglobin modified with 4-isothiocyanatobenzenesulfonic acid and 4-isothiocyanatobenzenesulfonamide.

The binding of chloride ions to specific sites on the human hemoglobin molecule has well-known effects on the oxygen equilibrium and on the stability of the tetrameric structure. Several lines of evidence suggest that the oxygen-linked and the dissociation-linked chloride binding sites differ. Direct evidence for this difference has been obtained from the chloride dependence of the dimer-tetramer equilibrium of oxyhemoglobin modified with 4-isothiocyanatobenzenesulfonic acid, in which all the oxygen-linked chloride binding sites are blocked, or with 4-isothiocyanatobenzenesulfonamide, in which the linkage between chloride and oxygen is unperturbed. Thus, the chloride dependence of the dimer-tetramer assembly is unaffected by the chemical modification in both proteins and resembles that of unreacted hemoglobin. It is suggested that histidines alpha-103, alpha-122 and beta-97 may constitute, at least in part, the dissociation-linked chloride binding sites.

Aromatic amino-acids and subunit assembly in the hemoglobins from Scapharca inaequivalvis: a fluorescence and CD study of the apoproteins.

The dimeric and tetrameric hemoglobins from the mollusc Scapharca inaequivalvis have a unique assembly that places the heme-carrying E and F helices in the inside of the molecule. These helices form the intersubunit contact in the dimer, which represents the structural unit since the tetramer is a dimer of dimers. The E and F helices are highly conserved and contain about 70% of the phenylalanine and tyrosine residues, while the tryptophan residues are near the tetramer contact. The spectroscopic properties (circular dichroism and intrinsic fluorescence) of the aromatic amino-acid residues in the two globins indicate that heme removal brings about a larger conformational change in the tetrameric than in the dimeric protein and that the tryptophan residues acquire a more rigid conformation in the tetramer.

Subunit interactions in the dimeric and tetrameric hemoglobins from the mollusc Scapharca inaequivalvis.

The dissociation behaviour of oxygenated Scapharea inaequivalvis HbII, the tetrameric hemoglobin component contained in the erythrocytes of this bivalve mollusc, has been compared with that of oxygenated human hemoglobin, HbA. At neutral pH the molluscan protein dissociates reversibly into dimers as does HbA, although dissociation is less marked; moreover the dimer-tetramer association constant is not sensitive to the presence of inorganic phosphates and high salt concentrations. HbII dimers hybridize with HbA dimers in solution, pointing to an overall similarity of the dimer interfaces in these hemoglobins from distantly related species. The gel filtration behaviour of the dimeric hemoglobin component of the erythrocytes. HbI, indicates that at neutral pH the protein has very little tendency to dissociate into monomers even at micromolar concentrations. Hb I was found to contain small amounts (2-4%) of bound carbohydrates.

Amphitrite ornata erythrocruorin. II. Molecular controls of function.

In the marine terebellid worm Amphitrite ornata the vascular fluid contains a high molecular weight erythrocruorin, while cells of the coelom contain a monomeric hemoglobin. The structural integrity of the erythrocruorin molecule is known to be dependent on the presence of a minimal concentration of divalent cations (1-3 mM) in the medium. The functional properties of Amphitrite erythrocruorin are also affected by cations. The oxygen affinity tends to increase with increasing cation concentration and the degree of cooperative interactions, expressed in the kinetics and equilibria of ligand binding, goes through a maximum. Maximal Hill coefficients of 3-4 are observed with 50 mM CaCl2, 50 mM MgCl2 or 1 M NaCl in measurements at the physiological pH of 7.75. Only 2 mM CaCl2 is required for maximal cooperativity at pH 8.5. This suggests partial deprotonation of the cation binding site at high pH. It is somewhat unusual that pH effects on cooperativity are reversible, since this is not a common feature of the giant erythrocruorin molecules. The oxygen binding experiments revealed a marked effect of divalent cations of Amphitrite erythrocruorin at high pH and cation concentration. Above pH 8.5, at 50 mM CaCl2 and 12 degrees C, the erythrocruorin will form a polymer upon deoxygenation. This polymerization is readily reversible by bringing the temperature for 12 to 20 degrees C or by oxygenation. Under physiological conditions of pH and cation concentration and at 12 degrees C, the erythrocruorin and the monomeric coelomic hemoglobin require a similar oxygen pressure for half saturation. However, the allosteric regulation of function is absent for the coelomic protein.

Oxidation reaction of human oxyhemoglobin with nitrite: a reexamination.

The oxidation reaction of human oxyhemoglobin with nitrite is complex and is characterized by a lag period followed by an autocatalytic phase. On the basis of contradictory experimental results, in order to describe the time-course of the reaction, two different mechanisms have been proposed, involving either hydrogen peroxide or the superoxide anion as reaction intermediates. This paper reports a careful reinvestigation of this reaction carried out as a function of reagent concentration, buffer composition, presence of enzymatic scavengers of oxygen radicals or of other compounds which may affect the intermediate steps of the reaction. The results obtained show that: hydrogen peroxide can be definitely identified as the reaction intermediate, in agreement with the mechanism proposed by Kosaka et al. (Biochim. Biophys. Acta 702 (1982) 237-241); the reaction time-course depends in a different way on the concentrations of hemoglobin and nitrite, a finding that cannot be explained on the basis of this mechanism. A more complex reaction scheme is proposed, that provides a satisfactory description in quantitative terms for all the available experimental data.

Oxidation reaction of Scapharca inaequivalvis hemoglobins with nitrite.

The oxidation reaction with nitrite of the dimeric and tetrameric hemoglobins from the mollusc Scapharca inaequivalvis has been studied kinetically and at equilibrium. In line with previous findings obtained with ferricyanide as oxidant, in both proteins the stable oxidation product is a hemichrome, although the nitrite-methemoglobin complex is formed in significant amount when excess nitrite is employed. The reaction kinetics are characterized by a lag period followed by an autocatalytic phase, as in the case of human hemoglobin. However, with respect to human hemoglobin, in the two molluscan proteins the lag phase is prolonged significantly due to the instability of their met-form, an obligatory intermediate for the onset of autocatalysis. All the data obtained in spectrophotometric, EPR and sedimentation velocity experiments under a variety of experimental conditions conform to the reaction mechanism proposed for human hemoglobin (Spagnuolo et al., Biochim. Biophys. Acta 911 (1987) 59-63) provided hemichrome formation and nitrite binding are taken into account.

Nucleation of the iron core occurs at the three-fold channels of horse spleen apoferritin: an EXAFS study on the native and chemically-modified protein.

Extended X-ray absorbance fine structure measurements have been carried out on the initial Fe(III)-apoferritin complex at a Fe/subunit ratio of 2 in native and modified horse spleen apoferritin. Analysis of the data indicates that in the native protein the iron forms a protein-bound polynuclear cluster (Fe-Fe distance 3.4 A) with a first coordination sphere constituted by 5-6 low-Z atoms, e.g., nitrogen atoms, carboxylate-like ligands or oxo bridges between the iron atoms. Modification of Cys-126, a residue localized on the outer surface of the hydrophilic three-fold channels, with p-chloromercuribenzoate (PMB) or phenylmercuric acetate (PMA) brings about distinctive differences. In particular, in the PMB-reacted protein the feature assigned to the iron-iron interaction disappears from the spectrum, whilst in the PMA-reacted protein the main differences with respect to the native protein are observed at the level of the first coordination sphere. These results confirm the formation of protein-Fe(III)-clusters and localize these sites at the hydrophilic three-fold channels of horse spleen apoferritin.

Root effect of Panulirus interruptus hemocyanin.

Panulirus interruptus hemocyanin exhibits a progressive decrease in oxygen affinity and a parallel loss of cooperativity with decrease in pH, resulting in an apparent loss of the oxygen-binding capacity of the protein. For a characterization of this system, oxygen-binding curves have been determined over the complete range of oxygen saturation, applying a special technique which involves high-pressure spectrophotometry. Although the oxygen-binding behavior as a function of pH is complex and cannot be described within the frame of a simple two-state Monod-Wyman-Changeux model, the observed Root effect is clearly related to a progressive stabilization of a low oxygen affinity state of the protein and functional heterogeneity is not apparent.

Studies on erythrocruorin. VII. Reconstitution of earthworm erythrocruorin from the apoprotein.

Apoerythrocruorin prepared from the giant respiratory hemoprotein of the earthworm (60 S, Mr = 3 X 10(-6)) is an electrophoretically homogeneous molecule which sediments as a single peak of low molecular weight (3.5 S) and has a lower alpha-helical content (approx. 30%) than the native protein. Titration of globin with ferric heme indicates the presence of different binding sites; however, after purification by ion exchange chromatography, the reconstitution product contains 1 haem/23 000 g of protein as the native molecule. Reconstituted ferric erythrocruorin is a low molecular weight hemichrome with the same optical and physicochemical properties of the hemichrome formed by natural ferric erythrocruorin. Reconstituted ferrous erythrocruorin reacquires the alpha-helical content and the quaternary structure of the native molecule. Reassociation into 10-S speices (1/12 of the whole molecules) is fast and easy, while that into whole molecules is slow and somewhat erratic. The functional properties of reconstituted ferrous erythrocruorin (oxygen affinity, cooperativity in oxygen binding, magnitude of Bohr effect) are very similar to those of the "stable" low cooperativity form of the undissociated protein.

Amphitrite ornata erythrocruorin. I. Structural properties and characterization of subunit interactions.

A high molecular weight erythrocruorin (Mr approx. 3 . 10(6)) is found in the vascular system of the marine terebellid worm Amphitrite ornata, while a low molecular weight hemoglobin is contained in the coelomic cells. Polyacrylamide gel electrophoresis indicates that Amphitrite erythrocruorin contains three different types of polypeptide chain, of molecular weight approx. 15,000, whereas the molecular weight per heme group is approx. 20,000. These data suggest that only two of three polypeptide chains may be associated with a heme group. The coelomic hemoglobin, which occurs as a monomer, has an apparent molecular weight of approx. 14,000. The circular dichroism spectra of Amphitrite erythrocruorin and of the coelomic protein reveal marked differences in the heme environment, while the alpha-helical contents are not very different (60% and 70%, respectively). Amphitrite erythrocruorin is unusual in its dissociation behavior. Divalent cations are required for maintaining the quaternary structure. In the pH range 7.75--8.5, when the Ca2+ concentration is reduced below 1 mM, the whole molecule (57 S) dissociates into a number of lower molecular weight species (25, 15, 10 and 3 S) which have been correlated with specific subunit structures by electron microscopy. Whole molecules and 25 S subunits are not in equilibrium with the lower molecular weight species and can be isolated from partially dissociated mixtures. In contrast, the lower molecular weight subunits are themselves in a state of rapid equilibrium which is sensitive to cations, protons and oxygen. Of special interest is the dimerization reaction of the 10 S subunits, which appears to be mediated by Ca2+ and conforms to the predictions of the Cann and Goad theory on ligand mediated equilibria. The dissociation of Amphitrite erythrocruorin is readily reversible when the Ca2+ concentration is increased. The subunits obtained at physiological (7.8) or slightly acid (6.5) pH completely reassemble into whole molecules. Reassembly, however, is only partial when dissociation occurs at high pH. The presence of stable intermediates, such as the 15 S species, may facilitate the reassociation process.

NMR and stopped-flow studies of metal ion binding to alpha-lactalbumins.

1H-NMR spectroscopy and stopped-flow techniques have been used to investigate the binding of a host of metal ions to alpha-lactalbumins from bovine, goat, and human sources. We have identified two 1H-NMR markers diagnostic of metal ion binding to the high-affinity Ca2+-binding site of bovine alpha-lactalbumin, namely the signals corresponding to the delta-CH3 groups of Met-90, and a leucine, tentatively assigned to Leu-96. A number of metal ions other than Ca2+ bind to this site in either slow (La3+, Lu3+, Y3+, Sr2+, Sc3+) or fast (Cd2+, Ba2+, Pb2+) exchange. From competition experiments using this approach, we have determined an affinity series for metal ion binding at this site, in which lanthanides and Y3+ bind the strongest (Y3+>La3+, Lu3+>Ca2+>Sr2+>Cd2+, Pb2+, Ba2+>Sc3+). Several metal ions do not alter the 1H spectrum of bovine alpha-lactalbumin, retaining the protein in an 'apo-like' state. Evidence is given to support the notion that the paramagnetic divalent metal ions Co2+ and Cu2+ bind to a second distinct site, termed the 'zinc site', and that His-68 is involved in metal ion coordination. Finally, stopped-flow techniques using the indicator Xylenol orange were employed to obtain lanthanide off-rates for bovine, human, and goat alpha-lactalbumins (bovine and goat alpha-LA: k(off)(s-1) approximately 0.2 to 0.01 from La3+ to Lu3+; human alpha-LA: k(off)(s-1) approximately 0.02 to 0.001 from La3+ to Lu3+). In each case, we found that k(off) values decreased by an order of magnitude across the series, meaning that the dissociation constants for these metal ions are relatively constant. Data for the bovine and goat proteins are virtually identical, while the off-rates for human alpha-lactalbumin are appreciably slower. In addition, these rates are much slower than the Ca2+ off-rate for the bovine protein (k(off)(s-1) approximately 2 to 5), determined using the fluorescent indicator, BAPTA.

Proton-NMR investigation of the heme cavity in the cyanomet derivative of the cooperative homodimeric hemoglobin from Scapharca inaequivalvis.

The active-site structure of the paramagnetic cyanomet complex of the cooperative homodimeric hemoglobin from Scapharca inaequivalvis has been investigated by solution homonuclear NMR. In spite of the large size (32 kDa), the residues on the key proximal F- and distal E-helices could be sequence-specifically assigned and placed in the heme pocket in a manner common to diamagnetic systems. These backbone assignments were greatly facilitated by the significant dispersion of backbone chemical shifts by the highly anisotropic paramagnetic susceptibility tensor of the low-spin ferric state. The remainder of the residues in contact with the heme are assigned based on unique contacts to the heme predicted by the crystal structure and the observations of scalar connectivities diagnostic for the residues. The magnitude of the dipolar shifts for non-ligated residues was used to determine the anisotropy and orientation of the paramagnetic susceptibility tensor, and the major axis found tilted from the normal in a manner similar to that found for the Fe-CO unit in the crystal structure. The combination of NOESY inter-residue and heme-residue contacts, paramagnetic-induced relaxation and correlation between observed and dipolar shifts provide a description of the heme cavity in cyanomet Hb that is essentially the same as found in the carbonmonoxy Hb crystal structure. The pattern of both the heme methyl dominant contact shifts and the heme meso-proton dominant dipolar shifts are shown to be consistent with the orientation of the axial His. It is concluded that the present homonuclear NMR methods allow effective solution structure determination in the cyanomet form for dimeric Hb and suggest profitable extension to the tetrameric vertebrate hemoglobins.

Studies on Scapharca hemoglobins. Properties of the dimeric protein reconstituted with Fe- or Co-porphyrin.

A native globin from the dimeric hemoglobin, hemoglobin I, of the mollusc Scapharca inaequivalvis has been obtained with the acid-acetone method. The globin has a lower sedimentation coefficient than the native protein at neutral pH; its reconstitution product with natural heme has the same physicochemical and functional properties as the native protein. proto- and meso-cobalt hemoglobin I have been prepared and characterized. proto-Cobalt hemoglobin I binds oxygen reversibly with a lower affinity and a lower cooperativity than native hemoglobin I; thus, the changes in the functional properties brought about by substitution of iron with cobalt are similar to those observed in human hemoglobin A. The EPR spectra of deoxy-proto-cobalt hemoglobin I and of the photolysis product of oxy-meso-cobalt hemoglobin I indicate that two histidine residues are the apical heme ligands. The broad signal at g = 2.38 in deoxy-proto-cobalt hemoglobin I points to a constrained structure of the heme site in this derivative which results from a distorted coordination of the hindered proximal histidine. A similar structure has been proposed previously for the alpha chains in deoxy-cobalt hemoglobin A.

A 35Cl(-)-NMR study of the singular anion-binding properties of dromedary hemoglobin.

35Cl(-)-NMR measurements of chloride binding to carbonmonoxy- and deoxy-dromedary hemoglobin reveal the existence of two classes of chloride-binding sites, one of high and the other of low affinity. Although this situation resembles that described for human hemoglobin, it was found that the number of binding sites as well as the association equilibrium constant for chloride binding are significantly higher in the dromedary protein. This difference may be due to the greater number of basic residues exposed to solvent and to the higher flexibility of dromedary hemoglobin. The two oxygen-linked polyanion-binding sites characteristic of this hemoglobin show competition for some of the high-affinity chloride-binding sites in keeping with their location in the cleft enclosed by the beta chains and between the alpha chains termini. It is suggested that the observed anion-binding properties of dromedary hemoglobin may contribute to the control of the physiological osmotic shock after rehydration.

Physicochemical and functional properties of Perinereis cultrifera (Grübe) erythrocruorin.

Perinereis erythrocruorin has the following physicochemical properties: So20,w = 55S, corresponding to a molecular weight around 2.7-10(6); minimum molecular weight (on the basis of the heme content) 23 700 +/- 500; isoelectric point 5.1; alpha-helix content approximately 40%. At alkaline pH values in the oxygenated form the 55-S molecules dissociate into subunits with a weight average sedimentation coefficient of 3S, corresponding to a molecular weight approximately 35 000. Deoxygenation of partially dissociated samples promotes association of the 3-S subunits into a 9S component. The functional properties of Perinereis erythrocruorin are characterized by a low cooperativity in oxygen binding (n 1/2 = 1.5) at neutral pH. Cooperativity increases reversibly towards both the acid and alkaline pH range, irrespective of changes in molecular weight. This finding, taken together with the ultracentrifuge results, suggests that a subunit may represent the functional unit of the protein. The pH dependence of the oxygen affinity can be accounted for in terms of a single oxygen linked group with a pK of 8.