Harry A. Kuiper

Thermodynamics of ligand binding and allosteric transition in hemoglobins. Reaction of Hb trout IV with CO

The reaction of hemoglobin trout IV with carbon monoxide has been studied in three parallel sets of experiments comprising: (1) microcalorimetric measurements in different buffers at two pH values (7.1 and 8.5) and two temperatures (20 °C and 5 °C) to determine the overall value of the enthalpy change of ligand binding as well as its point values as a function of fractional saturation with CO; (2) a study of the CO binding curves at three pH values (from 5.7 to 7.4) and at 23 °C and 5 °C in order to explore the pH and temperature dependence of the model-independent binding parameters which describe the system; (3) differential titrations in 0.2 m-sodium chloride at 23 °C and 5 °C of the deoxy and CO saturated forms of the molecule in order to determine the overall CO Bohr effect as a function of temperature. Analysis of the results of these three different sets of experiments at a purely phenomenological level shows that they are in satisfactory agreement. Further analysis in terms of a modified two-state allosteric model shows how the three model parameters kT, kR, and L vary with pH and temperature. From this it appears that the essential feature of the functional behavior of trout IV hemoglobin, which is characteristic of hemoglobins of teleost fish in general, lies in a proton-induced shift in the allosteric equilibrium constant. The data indicate, semiquantitatively, that the apparent enthalpy change corresponding to the allosteric equilibrium constant is pH-dependent and positive.

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

Abstract 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 × 107 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 (kon = 4.6 × 107 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.

Luminescence of the copper—carbon monoxide complex of Neurospora tyrosinase

Spectroscopic studies of tyrosinase and hemocyanin have indicated that the copper-sites of both proteins are very similar. Thus the stoichiometry of oxygen binding [ I2], EPR meas~lre~~~ents on various derivatives [3,4], magnetic susceptibility measurements [5.6] and resonance Raman spectroscopy [7] suggest the presence of a pair of antiferromagnetically coupled copper-ions separated by 36 8. Moreover hernocyanin and tyrosinase both bind carbon monoxide [8,9]. We have reported that the carbon monoxide complex of hemocyanins is luminescent [ 10.1 11. Upon excitation in the ultraviolet region, a strong emission band is observed above 500 nm. Excitation and difference absorption spectra, as well as CO titrations, clearly indicate that the emission originates from the copper-CO chromophore. In view of the above similarities between hemocyanin and tyrosinase, we have studied the binding of CO to ~e~~r~sp~~~ tyrosinase by fluorescence spectroscopy, and observed that the luminescence characteristics of CO-tyrosinase are similar to those reported for CO-hemocyanins [IO,1 I]. This result emphasizes the close structural relationships existing between the active site of these two classes of proteins, notwithstandmg the overall functional differences; moreover it confirms the potentialities of fluorescence spectroscopy of the CO complexes of these proteins, to investigate the structure of their binuclear copper sites. 2. Materials and methods

The replacement of calcium by terbium as an allosteric effector of hemocyanins

The role of ligands, such as H*, Ca2* and Na+ on the association-dissociation and oxygen binding properties of hemocyanins is well established [ 1,2]. , h f3] we presented evidence that in the case of Funfairs ~~te~~~~ hemocyanin these ions react with the macromolec~e in a linked fashion exerting .a control on its biolo~c~ function. Since Ca2’ are silent to a great number of spectroscopic tec~iques~ we have investigated the pos~b~ity to employ lantha~ides as substitutes for Ca”‘. In this study we report: (i) Fluorimetric measurements with Panulinrs hemocyanin indicating that terbium (Tb3’) competes with Ca2’ for the same sites; (ii) Oxygenbinding experiments showing that Tb3’ has an effect on the oxygen-bind~g properties of this protein similar to that demonstrated for Ca’” [?I. We conclude that Tb3+ may be used as a replacement species for Ca2* in h~~y~n, thus providing direct i~f~~atlon on the effects exerted by various ions on the control of the biological function of the macromolecule,

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

Abstract 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 (p50 = 1 to 10mm Hg) higher than that of arthropodal hemocyanins (p50 = 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.

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.

Kinetics of the co-operative reaction of Helix pomatia hemocyanin with oxygen. Oxygen binding at low and intermediate oxygen saturations.

The kinetics of oxygen binding of Helix pomatia α-hemocyanin has been studied at low and intermediate levels of ligand saturation, under conditions in which oxygen binding is highly co-operative. Temperature-jump relaxation spectra are heterogeneous and can be resolved into a slow and a fast phase. The latter is related to a bimolecular reaction, i.e. the binding of oxygen. At very low degrees of fractional saturation (<0.15) the reactant concentration-dependence of the faster relaxation rate allows the combination and dissociation rate constants of the low affinity or T-state to be estimated as 1.3 × 106m−1 s−1 and 300 s−1, respectively. A possible interpretation of the slow component in the relaxation spectrum is discussed. In stopped-flow experiments, after mixing deoxyhemocyanin with oxygen-containing buffer, most of the binding process to the T-state is lost in the dead time. The observed initial rates of oxygen binding are between 15 and 120 s−1. depending on the oxygen concentration, and may reflect the rate of the allosteric change from a low to a high affinity state (T→R transition), which is slower than oxygen binding. Similarities and differences in the overall kinetic properties of small and giant respiratory proteins, i.e. hemoglobin and hemocyanin, are discussed.

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 O2-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 Tb3+ results in a quenching of the terbium luminescence. The highly efficient quenching observed in the case of Eu3+ may indicate energy-transfer between Tb3+ and Eu3+. 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.

Kinetics of the Bohr effect in the reaction of Helix pomatia β-hemocyanin with oxygen

Abstract The timecourse of the uptake of Bohr protons during oxygenation of β-hemocyanin from the vineyard snail Helix pomatia has been studied. It may be concluded that all the Bohr protons are taken up during the conformational change of the protein from the low- to the high oxygen affinity state, a process which appears to be rate limiting at high ligand concentration.

Displacement of O2 and CO by cyanide from the active site of helix pomatia β-hemocyanin.: Formation of a mixed-liganded species

Abstract Addition of KCN to Helix pomatia β-hemocyanin fully saturated with either O 2 or CO results in a decrease of the spectroscopic properties of the protein (absorbance at 340 nm and luminescence at 550 nm) due to the displacement of the gaseous ligands (O 2 or CO) from the active site. The anionic form of cyanide (CN − ) is supposed to bind to the active site; its intrinsic affinity for the protein, as calculated from independent O 2 and CO displacement experiments, is between 2 and 6 × 10 6 M −1 . The replacement of O 2 or CO shows some differences which may be correlated with the different modes of binding at the active site. Thus, while displacement of oxygen by cyanide is hyperbolic, addition of cyanide to carbonylated hemocyanin shows a lag phase. This finding suggests the formation of a mixed liganded complex at the active site. The simultaneous presence of CO and CN − at the active site of hemocyanin is also supported by the experiment in which addition of small amounts of KCN to hemocyanin partially saturated with O 2 and CO gives rise to an increase of emission intensity and a concomitant decrease of the O 2 absorption band. The mixed-liganded species displays luminescence properties similar to those of CO-saturated hemocyanin, and the formation of the complex is reversible on dialysis or oxygenation.