Oxygen binding by hemocyanin from Levantina hierosolima. I. Exclusion of subunit interactions as a basis for cooperativity.

The effect of oxygen on the distribution of hemocyanin from Levantina hierosolima among the three sedimenting species 20, 60, and 100 S was determined under two sets of experimental conditions: (a) at pH 7.63 in the absence of Ca2+, where oxygen binding in noncooperative; (b) at pH 8.20 in the presence of 2 x 10-3 M Ca2+, where oxygen binding is cooperative. A comparison of the results in the two cases eliminates the possibility that equilibrium between species with different oxygen affinities is responsible for the cooperative behavior. Cooperative oxygen binding was demonstrated for the 20S subunits at pH 8.80 and 1 x 10-3 M Ca2+. Under these conditions, the concentration of calcium is sufficient to affect the oxygen affinity, but the concentration of calcium plus proton is not sufficient to bring about association. The findings exclude interactions among 20S subunits as a basis for cooperativity in hemocyanin.     

The oxygen-binding modulation of hemocyanin from the Southern spiny lobster <Emphasis Type="Italic">Palinurus gilchristi</Emphasis>

Arthropod hemocyanins transport and store oxygen and are composed of six subunits, or multiples thereof depending on the species. Palinurus gilchristi hemocyanin is found only as 1 × 6-mers, as normally occurs in spiny lobsters. An alkaline pH and removal of calcium ions induce a wholly reversible dissociation into monomers. The oxygen-binding properties of 1 × 6-meric hemocyanin from P. gilchristi were investigated with respect to pH and modulating effect exerted by calcium, lactate and urate. The oxygen affinity was highly affected by pH in the presence of calcium ions, while in its absence the Bohr coefficient became 60% lower. The protein is insensitive to lactate, but affected by urate which markedly increased hemocyanin–oxygen affinity, acting as the physiological major positive effector. Calcium ions decrease oxygen affinity at low concentration range (0–1 mM), while as concentration becomes higher than 100 mM, the oxygen affinity increases, indicating the presence of two independent types of calcium-binding sites with high and low affinity, respectively. The previous hypothesis, that the presence of high-affinity binding sites in addition to low affinity ones could be a characteristic feature of Palinuran hemocyanins, has been tested by analyzing, with respect to calcium–hemocyanin interaction, three other species belonging to Palinura.     

Binding of urate and caffeine to hemocyanin of the lobster Homarus vulgaris (E.) as studied by isothermal titration calorimetry

Hemocyanin serves as an oxygen carrier in the hemolymph of the European lobster Homarus vulgaris. The oxygen binding behavior of the pigment is modulated by metabolic effectors such as lactate and urate. Urate and caffeine binding to 12-meric hemocyanin (H. vulgaris) was studied using isothermal titration calorimetry (ITC). Binding isotherms were determined for fully oxygenated hemocyanin between pH 7.55 and 8.15. No pH dependence of the binding parameters could be found for either effector. Since the magnitude of the Bohr effect depends on the urate concentration, the absence of any pH dependence of urate and caffeine binding to oxygenated hemocyanin suggests two conformations of the pigment under deoxygenated conditions. Urate binds to two identical binding sites (n = 2) each with a microscopic binding constant K of 8500 M(-1) and an enthalpy change DeltaH degrees of -32.3 kcal mol(-1). Caffeine binds cooperatively to hemocyanin with two microscopic binding constants: K(1) = 14 100 M(-1) and K(2) = 40 400 M(-1). The corresponding enthalpy changes in binding are as follows: DeltaH degrees (1) = -23.3 kcal mol(-1) and DeltaH degrees (2) = -27.1 kcal mol(-1). The comparison of urate and caffeine binding to the oxygenated pigment indicates the existence of two protein conformations for oxygen-saturated hemocyanin. Since effector binding is not influenced by protons, four different conformations are required to create a convincing explanation for caffeine and urate binding curves. This was predicted earlier on the basis of the analysis of oxygen binding to lobster hemocyanin, employing the nesting model.     

Oxygen-binding modulation of hemocyanin from the slipper lobster Scyllarides latus

The oxygen-binding properties of hexameric hemocyanin (Hc) from Scyllarides latus were investigated with respect to pH, temperature, and modulating effect exerted by calcium, lactate, and urate. The oxygen affinity decreased at higher temperature, was slightly affected by pH, and was insensitive to lactate. Nevertheless, urate markedly increased Hc-oxygen affinity and its temperature sensitivity, acting as the physiological major positive effector: four urate sites per hexamer with an overall affinity constant of 1 x 10(4) M(-1) were found and the exothermic contribution of their binding was found to be about 30 kJ mol(-1). Calcium ions largely influenced oxygen affinity: their effect, which has an opposite sign at low (0-1 mM) and high (0.1-1 M) concentration ranges, indicates the presence of two independent types of binding sites with high and low affinity, respectively; however, only the former ones seem to be operative in vivo because, at physiological calcium concentrations, they are already saturated and the oxygen affinity is reduced.     

Cooperative transition in the conformation of 24-mer tarantula hemocyanin upon oxygen binding

Hemocyanins are large respiratory proteins of arthropods and mollusks, which bind oxygen with very high cooperativity. Here, we investigated the relationship between oxygen binding and structural changes of the 24-mer tarantula hemocyanin. Oxygen binding of the hemocyanin was detected following the fluorescence intensity of the intrinsic tryptophans. Under the same conditions, structural changes were monitored by the non-covalently bound fluorescence probe Prodan (6-propionyl-2-(dimethylamino)-naphthalene), which is very sensitive to its surroundings. Upon oxygen binding of the hemocyanin a red shift of 5 nm in the emission maximum of the label was observed. A comparison of oxygen binding curves recorded with tryptophan and Prodan emission revealed that structural changes in tarantula hemocyanin lag behind oxygen binding at the beginning of oxygenation. Analyses based on the nested two-state model, which describes cooperative oxygen binding of hemocyanins, indicated that the transition monitored by Prodan emission is closely related to one of the four conformations (rR) predicted for the allosteric unit. Earlier, the allosteric unit of tarantula hemocyanin was found to be the 12-mer half-molecule. Here, fluorescence titration revealed that the number of Prodan binding sites/24-mer tarantula hemocyanin is approximately 2, matching the number of allosteric units/hemocyanin. Based on the agreement between oxygen binding curves and fluorescence titration we concluded that Prodan monitors a conformational transition of the allosteric unit.     

Oxygen binding by the hemocyanin of Busycon canaliculatum

• 1.1. This study examined the effect of the monoamines dopamine and octopamine, as well as tyrosine on the oxygen affinity and cooperativity of oxygen binding by the hemocyanin of the marine gastropod Busycon canaliculatum. The effect of temperature on hemocyanin oxygen affinity was also examined.
• 2.2. Freezing Busycon hemocyanin did not affect the binding of oxygen.
• 3.3. Dopamine, octopamine and tyrosine had no significant effect on the oxygen affinity or cooperativity of oxygen binding by the hemocyanin of B. canaliculatum.
• 4.4. It was concluded that Busycon hemocyanin either has no binding sites for the two monoamines or for tyrosine, or that binding of the molecules has no functional significance.
• 5.5. Both temperature sensitivity and affinity of hemocyanin-oxygen binding were similar to values previously reported for hemocyanin of Busycon from other localities.

     

Unusual oxygen binding behavior of a 24-meric crustacean hemocyanin

Hemocyanins from Crustacea usually are found as 1 × 6 or 2 × 6-meric assemblies. An exception is the hemocyanin isolated from thalassinidean shrimps where the main component is a 24-meric structure. Our analysis of oxygen binding data of the thalassinidean shrimp Upogebia pusilla based on a three-state MWC-model revealed that despite the 24-meric structure the functional properties can be described very well based on the hexamer as allosteric unit. In contrast to the hemocyanins from other thalassinidean shrimps the oxygen affinity of hemocyanin from U. pusilla is increased upon addition of l-lactate. A particular feature of this hemocyanin seems to be that l-lactate already enhances oxygen affinity under resting conditions which possibly compensates the rather low intrinsic affinity observed in absence of l-lactate. The fast rate of oxygen dissociation might indicate that in this hemocyanin a higher cooperativity is less important than a fast response of saturation level to changes in oxygen concentration.     

Metal ion interactions with Limulus polyphemus and Callinectes sapidus hemocyanins: stoichiometry and structural and functional consequences of calcium(II), cadmium(II), zinc(II), and mercury(II) binding

Hemocyanins are oligomeric metalloproteins containing binuclear copper centers that reversibly combine with oxygen molecules. The structural stability and functional properties of these proteins are modified by divalent cations. Equilibrium dialysis was used to study the reversible interaction of Callinectes sapidus and Limulus polyphemus hemocyanins with the divalent cations calcium, cadmium, zinc, copper, and mercury. The number of binding sites and association constants for each cation were obtained from an analysis of the binding data by a nonlinear least-squares minimization procedure. Spectral analysis showed Limulus hemocyanin to possess two mercury-reactive sulfhydryl groups per subunit (Kassoc = 2.02 X 10(45) M-1). Callinectes hemocyanin contains only one such group (Kassoc = 2.29 X 10(34) M-1). Cadmium and zinc are shown to substitute for calcium ions. Oxygen binding studies with Limulus hemocyanin showed that all five divalent metal ions increase its oxygen affinity. Calcium ions increase cooperativity of oxygen binding, while heavy-metal ions have an opposite effect. Binding of two mercuric ions per Limulus hemocyanin subunit irreversibly fixes the 48 subunit aggregate in a high-affinity noncooperative conformational state. These results offer a striking contrast to the functional consequences of heavy-metal ion interactions with Callinectes hemocyanin [Brouwer, M., Bonaventura, C., & Bonaventura, J. (1982) Biochemistry 21, 2529-2538]. The functional alterations associated with metal ion interactions are discussed within the context of an extension of the two-state model for allosteric transitions of Monod et al. [Monod, J., Wyman, J., & Changeux, J.P. (1965) J. Mol. Biol. 12, 88-118]. Incubation of Limulus oxy- or deoxyhemocyanin with mercuric chloride results in the conversion of 60% of the binuclear copper sites to stable half-apo sites. The remaining active sites are stable with respect to mercury-induced copper displacement when oxygen is bridging both coppers. In the absence of oxygen these sites will eventually lose both copper atoms. Under the same conditions 50% of the binuclear copper sites of Callinectes deoxyhemocyanin are converted to half-apo sites. In this case oxygen completely protects against copper displacement [Brouwer, M., Bonaventura, C., & Bonaventura, J. (1982) Biochemistry 21, 2529-2538]. The binuclear copper center of Busycon carica is not affected at all, demonstrating profound differences between the active sites of hemocyanins of a chelicerate arthropod (Limulus), a crustacean arthropod (Callinectes), and a gastropod mollusc (Busycon).     

Quaternary structure and functional properties of Penaeus monodon hemocyanin

The hemocyanin of the tiger shrimp, Penaeus monodon, was investigated with respect to stability and oxygen binding. While hexamers occur as a major component, dodecamers and traces of higher aggregates are also found. Both the hexamers and dodecamers were found to be extremely stable against dissociation at high pH, independently of the presence of calcium ions, in contrast to the known crustacean hemocyanins. This could be caused by only a few additional noncovalent interactions between amino acids located at the subunit-subunit interfaces. Based on X-ray structures and sequence alignments of related hemocyanins, the particular amino acids are identified. At all pH values, the p50 and Bohr coefficients of the hexamers are twice as high as those of dodecamers. While the oxygen binding of hexamers from crustaceans can normally be described by a simple two-state model, an additional conformational state is needed to describe the oxygen-binding behaviour of Penaeus monodon hemocyanin within the pH range of 7.0 to 8.5. The dodecamers bind oxygen according to the nested Monod-Whyman-Changeaux (MWC) model, as observed for the same aggregation states of other hemocyanins. The oxygen-binding properties of both the hexameric and dodecameric hemocyanins guarantee an efficient supply of the animal with oxygen, with respect to the ratio between their concentrations. It seems that under normoxic conditions, hexamers play the major role. Under hypoxic conditions, the hexamers are expected not to be completely loaded with oxygen. Here, the dodecamers are supposed to be responsible for the oxygen supply.     

Allosteric properties and the association equilibria of hemocyanin from Callianassa californiensis.

The influence of magnesium ions, hydrogen ions, and oxygen on the monomer (17 S)-tetramer (39 S) equilibrium of the hemocyanin from Callianassa californiensis has been investigated. Data have been interpreted in terms of a theory integrating the allosteric equilibria and association equilibrium. Binding of oxygen and divalent cations by the hemocyanin has also been studied in terms of the theory, and the parameters in the model have been determined. The 17 S monomer, which contains six polypeptide chains, is found to be the allosteric unit which behaves as a self-contained co-operative system with allosteric properties. Both magnesium and hydrogen ions are shown to affect the association directly, whereas the effect of oxygen binding can be explained in terms of a difference in the allosteric equilibrium constant L′ in the different associated states of the protein. It is shown that whereas the effect of oxygen on the monomer-tetramer equilibrium is easily observable, the converse effect of the association equilibrium on the oxygen binding curve is at the limit of detectability.     

Hemocyanin from E. californicum encapsulated in silica gels: oxygen binding and conformational states

Cooperativity depends on the existence of equilibria among functionally distinct conformational states that are affected by homo and heterotropic effectors. In order to isolate the quaternary conformations of hemocyanin from E. californicum, the 24-meric giant protein was encapsulated in wet, nanoporous silica gels, either in the absence or presence of oxygen. The deoxy- and oxy-hemocyanin gels exhibit a p50 for oxygen of 11 and 2.5 torr, respectively, values in close agreement with those for hemocyanin in solution. The observed Hill coefficients are lower than unity, indicating a conformational heterogeneity within each locked conformational state, a finding in agreement with the assumption that at least four conformational states are required to explain the oxygen binding properties of E. californicum hemocyanin in solution.     

Lipid binding capacity of spider hemocyanin.

The spider hemocyanin capacity to bind different lipid classes was evaluated by measuring some binding kinetic parameters. A very high lipoprotein (VHDL) which contains hemocyanin, was isolated from Polybetes pythagoricus hemolymph plasma and delipidated. Hemocyanin was bound separately to labelled palmitic acid, phosphatidylcholine, cholesterol, and triolein resulting in several artificial lipoprotein structures. It was possible to corroborate in vitro the lipid-hemocyanin interactions which had been previously observed and, consequently, the apolipoprotein role played by the respiratory pigment of spiders. Lipoproteins were analysed by gel filtration chromatography, and three subfractions with different hemocyanin structures were obtained. The four lipid classes were only bound to the hexameric structure (420 Kda), possibly to low polarity sites. Upon radioactivity measurements of the protein-associated lipids, maximal binding ratios (Mr), dissociation constants (Kd), and the maximal binding effectiveness at low lipid concentrations (Eo) were calculated. Lipid/protein ratios were increased proportionally to each available lipid concentration, following a hyperbolic binding model. Values of saturation, affinity, and maximal binding efficiency to hemocyanin were found to be different for each lipid class assayed. The highest lipid/protein ratio (41.5) was obtained with the free fatty acid and the lowest (7.2) with triolein. Phosphatidylcholine and cholesterol showed the highest relative affinities for hemocyanin (Kd = 63 x 10(-5) M and 74 x 10(-5) M, respectively). Phosphatidylcholine at low concentrations, similar to the physiological ones, presented the highest Eo value. Maximal lipid/protein ratios reached in vitro, were greater than those in P. pythagoricus VHDL, pointing out that hemocyanin could play the apolipoprotein role even under physiological conditions with a very high plasma lipid concentration. J. Exp. Zool. 284:368-373, 1999.     

Tubular polymers derived from Helix pomatia beta-hemocyanin.

Upon trypsinolysis Helix pomatia beta-hemocyanin forms long tubular structures, which appear to be linear polymers of hemocyanin molecules from which the collar structure has been removed. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate shows that only few peptide bonds are hydrolyzed by trypsin. The structure of the polymers has been investigated by electron microscopy, combined with optical diffraction. Preliminary X-ray diffraction data are presented. Functional properties of the polymers are similar to those of the native protein. Both show a calciumion-dependent co-operativity of oxygen binding and a Bohr effect. The results suggest that the collar of a hemocyanin molecule has no special function in the process of (co-operative) oxygen binding, different from that of the wall of the molecule.     

Terbium ion binding to Limuluspolyphemus hemocyanin

Terbium ion binds to calcium-free $\text{Limulus}$ hemocyanin at pH 7.0 and 8.9, and promotes the aggregation of hemocyanin subunits, a phenomenon associated with calcium binding. An excitation maximum for the bound terbium at 293 nm and the results of treating the hemocyanin with N-bromosuccinimide indicate that energy transfer from tryptophan to the bound terbium is responsible for the enhancement of terbium fluorescence. At pH 8.9, addition of calcium to hemocyanin containing bound terbium results in only a partial loss of terbium fluorescence, suggesting heterogeneity in the terbium binding sites. Titration of hemocyanin with terbium also indicates multiple binding sites.     

The oxygenation-linked binding of neutral red to spiny lobster hemocyanin. A structural study of the partially oxygenated protein

The structural change of lobster hemocyanin in cooperative O2 binding was studied by the dye-binding method. It was found that neutral red shows an O2-linked binding to hemocyanin with a higher affinity for the oxy form. The number of the dye-binding sites was estimated to be three in the hexameric molecule of oxyhemocyanin. The course of the structural change in the partially oxygenated hemocyanin was examined using the absorbance change of the bound dye as a measure. It was found that the fractional change in the dye binding was considerably greater than the degree of O2 saturation of hemocyanin. The three-state allosteric model, which was proposed for explanation of the O2 binding properties of lobster hemocyanin [N. Makino (1986) Eur. J. Biochem. 154, 49--55], was also consistent with the effects of the dye on the O2 binding to the native hemocyanin. On the basis of this model, the dye binding to partially oxygenated hemocyanin could be connected with the populations of the affinity states. It was inferred that the binding of neutral red reflects the quaternary structure of the protein. In contrast, O2 binding to the stripped (EDTA-treated) hemocyanin showed a considerable decrease in the cooperativity in the presence of the dye. The O2-binding isotherms could not be explained by the three-state model. It is suggested that the subunit interaction is partially blocked by the dye in the absence of divalent cations.     

Binding of oxygen and carbon monoxide to arthropod hemocyanin: an allosteric analysis

The binding of oxygen and carbon monoxide to hemocyanin from the mangrove crab Scylla serrata and the lobster Homarus americanus has been studied by thin-layer optical absorption and front face fluorescence techniques. Three types of experiments were performed on subunit and oligomeric preparations of each hemocyanin: oxygen binding, carbon monoxide binding, and oxygen-carbon monoxide competition studies. The results obtained from the subunit preparations of dissociated oligomers from both hemocyanins show that the binding site can be ligated by either one oxygen or one carbon monoxide. The binding results obtained with the oligomeric samples of hemocyanin from both species cannot be described by the two-state MWC model [Monod, J., Wyman, J., & Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118] since the data from the three types of binding experiments cannot be fit with a single set of binding constants. The MWC model has been extended by including a third allosteric form, and an analysis based on the three-state model is able to fit the data from the three types of experiments with the same set of binding constants. The comparison of the oxygen to carbon monoxide affinity ratios (kO2/kCO) indicates that the structure around the binding site of subunits in the T form oligomer is similar to that of the free subunits. The oligomeric forms of both these hemocyanins bind carbon monoxide with a weak but definite positive cooperativity. An analysis of the affinity ratios for the T, S, and R forms suggests that the high affinity of the R form results from a specific interaction between oxygen and binding site.     

Hemocyanin from Tachypleus gigas. I. Oxygen-binding properties

Hemocyanin was prepared from an Asian horseshoe crab, Tachypleus gigas. The hemocyanin was found to be similar to Limulus hemocyanin in the size of native molecules (48-mer) and dissociation under nonphysiological conditions. It also showed the reverse Bohr effect. The O2 affinity of the dissociated monomer was higher than that of the native molecule. Equilibrium O2 binding to T. gigas hemocyanin was studied with special attention to the effect of inorganic ions. Neutral salts decreased the O2 affinity of the associated hemocyanin. In the presence of CaCl2 the strength of the effect was in the order of Na+ greater than Cs+ not equal to K+ for the series of chlorides, and Br- not equal to Cl- greater than SO4(2-) for the series of Na+ salts. A high concentration of CaCl2 (50-500 mM) considerably increased the Hill coefficient. The O2 binding data obtained under various ionic conditions were analyzed by model fitting. The two-state concerted model could be fitted to the data, if the ligand affinity of the states was allowed to vary. Statistical tests of the fitting showed that the hexameric structure can be regarded as the functional unit under physiological conditions.     

Quantum mechanical analysis of oxygenated and deoxygenated states of hemocyanin: theoretical clues for a plausible allosteric model of oxygen binding.

Abstract: In this work with ab initio computations, we describe relevant interactions between protein active sites and ligands, using as a test case arthropod hemocyanins. A computational analysis of models corresponding to the oxygenated and deoxygenated forms of the hemocyanin active site is performed using the Density Functional Theory approach. We characterize the electron density distribution of the binding site with and without bound oxygen in relation to the geometry, which stems out of the crystals of three hemocyanin proteins, namely the oxygenated form from the horseshoe crab Limulus polyphemus, and the deoxygenated forms, respectively, from the same source and from another arthropod, the spiny lobster Panulirus interruptus. Comparison of the three available crystals indicate structural differences at the oxygen binding site, which cannot be explained only by the presence and absence of the oxygen ligand, since the geometry of the ligand site of the deoxygenated Panulirus hemocyanin is rather similar to that of the oxygenated Limulus protein. This finding was interpreted in the frame of a mechanism of allosteric regulation for oxygen binding. However, the cooperative mechanism, which is experimentally well documented, is only partially supported by crystallographic data, since no oxygenated crystal of Panulirus hemocyanin is presently available. We address the following question: is the local ligand geometry responsible for the difference of the dicopper distance observed in the two deoxygenated forms of hemocyanin or is it necessary to advocate the allosteric regulation of the active site conformations in order to reconcile the different crystal forms? We find that the difference of the dicopper distance between the two deoxygenated hemocyanins is not due to the small differences of ligand geometry found in the crystals and conclude that it must be therefore stabilized by the whole protein tertiary structure.     

Allosteric models for multimeric proteins: oxygen-linked effector binding in hemocyanin

In many crustaceans, changing concentrations of several low molecular weight compounds modulates hemocyanin oxygen binding, resulting in lower or higher oxygen affinities of the pigment. The nonphysiological effector caffeine and the physiological modulator urate, the latter accumulating in the hemolymph of the lobster Homarus vulgaris during hypoxia, increase hemocyanin oxygen affinity and decrease cooperativity of oxygen binding. To derive a model that describes the mechanism of allosteric interaction between hemocyanin and oxygen in the presence of urate or caffeine, studies of oxygen, urate, and caffeine binding to hemocyanin were performed. Exposure of lobster hemocyanin to various pH values between 7.25 and 8.15 resulted in a decrease of p50. In this pH interval, p50 decreases from 95 to 11 Torr without effectors and from 49 to 6 Torr and from 34 to 5 Torr in the presence of 1 mM urate or caffeine, respectively. Thus, the allosteric effects induced by protons and urate or caffeine are coupled. In contrast, isothermal titration calorimetry did not reveal any differences in binding enthalpy (DeltaH degrees ) for urate or caffeine under either normoxic or hypoxic conditions at different pH values. Despite these apparently conflicting results, they can be explained by the nested MWC model if two different types of modulator binding sites are assumed, an allosteric and a nonallosteric type of site. Simulations of in vivo conditions with this model indicate that the naturally occurring modulator urate is physiologically relevant in H. vulgaris only during hypoxic conditions, i.e., either during environmental oxygen limitation or extensive exercise.