Panulirus Interruptus Hemocyanin Research Articles

Structure of arthropod hemocyanin

Hemocyanins are large multi-subunit copper proteins that transport oxygen in many arthropods and molluscs. The amino acid sequence of subunit a of Panulirus interruptus hemocyanin (657 residues) has been completed and fitted to the electron-density map (3.2 Å resolution). Comparison of amino acid sequence data for several different hemocyanin subunits of arthropod species indicated that the general features of the polypeptide architecture as found in spiny lobster hemocyanin occur in all arthropods. This structure must therefore be at least as old as the estimated time of divergence of crustaceans and chelicerates, 540–600 million years ago.

Structure determination of Panulirus interruptus haemocyanin at 3.2 Å resolution : Successful phase extension by sixfold density averaging

The three-dimensional structure of Panulirus interruptus haemocyanin has been determined at 3.2 Å resolution by X-ray diffraction techniques. Starting from a double isomorphous replacement map at 4 Å resolution, the phases of the 32,709 reflections were first improved by six cycles of sixfold molecular averaging and solvent flattening. This also generated phase for 3078 reflections with no isomorphous replacement data. Next, phases for the reflections between 4.0 Å and 3.2 Å were obtained by a stepwise expansion procedure. In each expansion step 2000 to 3000 new reflections were added to the set of already phased reflections, followed by a few cycles of density averaging and solvent flattening at constant resolution. The eventual map at 3.2 Å was calculated with 63,843 reflections with an average Sim weight of 0.75 and an overall R-factor of 23.5%. The polypeptide chain could be traced without any problems, while the dinuclear copper site, disulphide bridges and the first three moieties of the carbohydrate chain were clearly visible. Each subunit consists of three distinct domains. The first domain is mainly helical, containing one disulphide bridge and the carbohydrate chain. The second domain is also predominantly helical and contains the dinuclear copper site at its centre. The core of the third domain is an anti-parallel β-barrel with the same topology as in the immunoglobulins and Cu,Zn-superoxide dismutase. This domain contains two disulphide bridges. Two long loops extend from the β-barrel and have numerous interactions with the other two domains. The two copper ions are at ~ 3.7 ± 0.25 Å from each other and co-ordinated by six histidines, which are strictly conserved in all seven arthropodan haemocyanins with known amino acid sequences. No bridging protein ligand is present in the electron density distribution. Hydroxyl groups from tyrosines, which are invariant among arthropodan haemocyanins, are 17.4 Å or more removed from the centre of the two copper ions. The closest Panulirus tyrosine hydroxyl is 10.6 Å from the copper ions. So, it appears unlikely that tyrosine is involved in the co-ordination of the coppers either in the deoxy or in the oxy form of arthropodan haemocyanins.

Panulirus Interruptus Hemocyanin : The amino-acid sequence of the region containing one copper-binding site and the sites susceptible to limited proteolysis

The amino-acid sequence of a part of Panulirus interruptus hemocyanin subunit a comprising the three ligands of the first copper ion has been determined. By fitting this sequence to the electron density map (Gaykema W.P.J.; Hol, W.G.J.; Vereijken J.M; Soeter N.M.; Bak H.J.; Beintema J.J. (1984) Nature (Lond.) 309, 23–29), all three ligands were found to be histidines. Comparison of the sequence with other sequenced subunits of arthropodan hemocyanins showed that a part of the residues, including the liganding histidines, has been conserved during evolution. The sequenced part of the molecule also contained the cleavage sites for proteolytic enzymes, located at the surface of the native molecule.

3.2 Å structure of the copper-containing, oxygen-carrying protein Panulirus interruptus haemocyanin

For the first time, the three-dimensional structure of a member of the copper-containing class of oxygen-carrying proteins—the haemocyanins—has been determined. The structure of Panulirus interruptus haemocyanin at 3.2 A resolution shows that each subunit of the 450,000-molecular weight hexamer is folded into three domains, and that the binuclear copper site is situated in the centre of the second domain. Amino acid sequence comparisons suggest that the polypeptide fold of P. interruptus haemocyanin is common to all arthropod haemocyanins.

Raman spectroscopic analysis of the secondary structure in Panulirus interruptus hemocyanin

The secondary structure content of Panulirus interruptus hemocyanin crystals is estimated to be 29±5% helix and 42±4% ß-strand. These estimates are obtained from an analysis of the Raman amide I spectrum. This report constitutes a test of this method of analyzing Raman spectra since the X-ray structure of P. interruptus, which is unknown to us, is under intensive study. Also, the amide I spectrum of P. interruptus in solution, at a somewhat higher pH, is significantly different from the spectrum of crystals which indicates that the secondary structure content may be different in the solution sample.

Kinetic and thermodynamic parameters for oxygen binding to the allosteric states of panulirus interruptus hemocyanin

The temperature dependence of the oxygen binding equilibria and kinetics of Panulirus interruptus hemocyanin has been analyzed within the context of the two-state allosteric model. Oxygenation of the T-state is characterized by a more negative value of DeltaH than that of the R-state; therefore, cooperative effects in oxygen binding to P. interruptus hemocyanin are thermodynamically governed by favorable entropy changes. The allosteric transition in the unliganded derivative shows an enthalpy-entropy compensation effect. The activation enthalpies for oxygenation and deoxygenation of the T-state are larger than those for the R-state, while the activation entropies are favorable for the T-state and unfavorable for the R-state. Thus, the activation free energies for oxygen binding to the T- and R-states are similar, while for the deoxygenation reaction DeltaG++ is smaller for the T-state. The analysis reported confirms the applicability of the Monod-Wyman-Changeux two-state allosteric model to P. interruptus hemocyanin and yields a complete thermodynamic characterization of oxygen binding under both equilibrium and dynamic regimes.

Characterization of cation-binding sites on Panulirus interruptus hemocyanin by 43Ca and 23Na NMR.

The binding of Na+ and Ca2+ ions to Panulirus interruptus hemocyanin has been studied by nuclear magnetic resonance. The combined use of 43Ca and 23Na NMR has allowed two different classes of sites to be distinguished. The sites are characterized by a relatively fast chemical exchange, local mobility and binding constants of the order of 10(3)-10(4) M-1 for Ca2+ and Mg2+ and approximately equal to 10(2) M-1 for Na+. These features and the observed pH dependence of the Ca2+ binding are consistent with the protein ligands being carboxylate groups, some of which may occur in clusters.

Limited proteolysis of the 94 000-dalton subunit of Panulirus interruptus hemocyanin; the carbohydrate attachment site.

Limited proteolysis of the native monomeric 94-kDa subunit of Panulirus interruptus hemocyanin by trypsin, plasmin and subtilisin produces an 18-kDa fragment, a 71-kDa fragment and a small glycopeptide. In the plasmin digest a 23-kDa precursor of the 18-kDa fragment has been observed. Automatic Edman degradations demonstrated that the 18-kDa fragments have their origin at the N terminus of the 94-kDa subunit and incubations with carboxypeptidase A showed that the 71-kDa fragments originate from the C terminus. The glycopeptide is situated in between. The amino acid sequence of the glycopeptide has been determined. Its carbohydrate content accounts for the total carbohydrate of the 94-kDa subunit. All three proteases cleave the subunit at two positions within a very restricted area of the polypeptide chain, which indicates the presence of an exposed loop, carrying the carbohydrate chain, in the corresponding part the native molecule.

Reaction of carbon monoxide with hemocyanin: Stereochemical effects of a non-bridging ligand

The carbon monoxide binding equilibria and kinetics of a number of molluscan and arthropodal hemocyanins have been investigated employing the visible luminescence of the carbon monoxide-copper complex. Proteins from both phyla, in oligomeric and monomeric form, bind carbon monoxide non-co-operatively; the reaction is largely enthalpy driven is associated with a small unfavourable entropy change. Molluscan hemocyanins display a carbon monoxide affinity ( p 50 = 1 to 10mm Hg) higher than that of arthropodal hemocyanins ( p 50 = 100 to 700mm Hg), and only Panulirus interruptus hemocyanin, among those studied here, exhibits a small Bohr effect. The observed differences in equilibrium constant are kinetically reflected in differences in the carbon monoxide dissociation rate constant, which ranges from 20 to 70 s −1 for molluscan hemocyanins and from 200 to 9000 s −1 for arthropodal hemocyanins; on the other hand the differences in the combination rate constants between the two phyla are considerably smaller. A comparison of the equilibrium and kinetic results shows some discrepancies between the two sets of data, suggesting that carbon monoxide binding may be governed by a complex mechanism. The correlation between the ligand binding properties and the stereochemistry of the active site is discussed in the light of the knowledge that, while oxygen is bound to both copper atoms in a site, carbon monoxide is a “non-bridging” ligand, being bound to only one of the metals.

Interaction of lanthanide ions with Panulirus interruptus hemocyanin: Evidence for vicinity of some of the cation binding sites

The interaction of a number of lanthanide ions (namely terbium, praseodymium, erbium, lanthanum, gadolinium and europium) with Panulirus interruptus hemocyanin has been studied. Results from O 2-binding experiments indicate that all these ions may substitute for calcium as allosteric effectors of hemocyanin. Addition of the lanthanides to deoxygenated Panulirus hemocyanin saturated with Tb 3+ results in a quenching of the terbium luminescence. The highly efficient quenching observed in the case of Eu 3+ may indicate energy-transfer between Tb 3+ and Eu 3+. Since energy-transfer between lanthanides is only effective over very short distances, the data suggest that some of the cation binding sites of Panulirus hemocyanin are clustered.

Effects of mercuric chloride on the structural and functional properties of Panulirus interruptus hemocyanin

1. 1. The effects of mercuric chloride on Panulirus interruptus hemocyanin has been studied. 2. 2. Binding experiments indicate that a fairly large number of mercuric ions may be bound to Panulirus hemocyanin. 3. 3. At low concentrations of mercury (when no loss of copper occurs) cooperative interactions in oxygen binding are almost completely abolished. 4. 4. At higher concentrations, mercury may labilize copper from the active site, with a concomitant decrease in the oxygen binding capacity of the protein. 5. 5. EPR spectroscopy indicates that mercury induces a distortion of the geometry of the active site. 6. 6. Since hemocyanin is a widely distributed oxygen carrier, a study of its interactions with mercury may be of general interest to elucidate the molecular mechanism of toxicity by heavy metals in a large number of marine organisms.

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.

Luminescence of carbon monoxide hemocyanins.

The effect of carbon monoxide on the luminescence properties of Helix pomatia alpha-hemocyanin and Panulirus interruptus hemocyanin has been studied. These proteins, when saturated with carbon monoxide, show, besides the intrinsic fluorescence arising from the aromatic amino acid residues, emission in the visible region with a maximum between 540 and 560 nm. Results of carbon monoxide titration experiments and data from absorption and excitation-emission spectra provide convincing evidence that the observed emission originates from a fluorescent copper(I)-carbon monoxide complex, and this emission is interpreted as charge-transfer luminescence.

Sodium and calcium binding to Panulirus interruptus hemocyanin as studied by 23Na nuclear magnetic resonance.

Addition of Panulirus hemocyanin to NaCl solutions produces marked changes in the 23Na relaxation parameters; they show that sodium ions interact with binding sites on the protein and exchange rapidly with the bulk. The observed non-lorentzian lineshapes and the non-exponential decay of the transverse magnetization indicate that non-extreme narrowing conditions apply and give information on the dynamics of the interaction. Panulirus hemocyanin has at least two classes of Na+ binding sites; the binding constant of the more strongly bound sodium ions is in the order of 1 X 10(2) M-1. Competition between Na+ and Ca2+ for protein binding sites is demonstrated by the effect of Ca2+ on the 23Na relaxation parameters. However, only the more strongly bound Na+ are displaced by Ca2+. The number of Ca2+ needed to displace these sodium ions is 3--5 per oxygen binding site. The 23Na relaxation parameters are influenced also by the state of oxygenation of the protein, indicating a linkage between Na+ and oxygen binding. The simplest interpretation of the data is that sodium ions bind more strongly to oxyhemocyanin in agreement with oxygen equilibrium experiments.

Kinetic control of co-operativity in the oxygen binding of Panulirus interruptus hemocyanin

The kinetics of the oxygen reaction of Panulirus interruptus hemocyanin have been studied at pH 9.6 under conditions where the protein exists in the undissociated, co-operative state and in the dissociated, non-co-operative state. Temperature-jump relaxation measurements of the undissociated protein at high oxygen saturation levels show one relaxation process which has been assigned to the high oxygen affinity (R) state, the on and off kinetic constants being 3.1 × 10 7 m −1 s −1 and 60 s −1, respectively. Stopped-flow measurements of the oxygen dissociation reaction show (1) an autocatalytic time-course of the reaction at pH 9.6 and (2) an increase in the overall oxygen dissociation rate constant, as the pH is decreased from 9.6 to 7.0. Temperature-jump relaxation measurements of the dissociated protein show one relaxation process characterized by a very high oxygen dissociation rate constant (1500 s −1) and a combination constant which is of the same order of magnitude as reported for undissociated protein ( k on = 4.6 × 10 7 m −1 s −1 ). The behaviour of dissociated protein can be considered as characteristic of the low oxygen affinity (T) state. The results presented in this paper, together with data available for other hemocyanins as well as hemoglobins, lead to the conclusion that respiratory proteins show a common feature in the kinetic control of co-operative oxygen binding: the stability of the oxygen-protein complex is largely determined by the value of the dissociation rate constant, the oxygen combination process very often appearing to be diffusion controlled.

Subunit composition, X-ray diffraction, amino acid analysis and oxygen binding behaviour of Panulirus interruptus hemocyanin

The subunit composition of Panulirus interruptus hemocyanin has been studied. Equilibrium sedimentation, gel chromatography and sodium dodecyl sulphate-polyacrylamide electrophoresis reveal a hexameric structure for this hemocyanin, with a molecular weight of 450,000 for the undissociated protein and 75,000 for the subunits. Amino acid analysis data suggest homogeneity of the polypeptide chain. X-ray diffraction gives the cell parameters a = 119·8, b = 193·1 and c = 122·2 , β = 118·1o. The asymmetric unit contains one molecule. Oxygen binding data show the undissociated protein to be co-operative while the dissociated protein binds oxygen non-co-operatively with a low oxygen affinity compared to that of whole molecules.