Thermodynamic analysis of carbon monoxide binding by hemoglobin trout I.

Calorimetric measurements at 25 degrees of the differential heat of CO binding by hemoglobin trout I have been examined together with the CO binding isotherms for the protein at 4 degrees and 20 degrees. Simultaneous treatment of these data sets by a statistically rigorous technique permits evaluation of all the thermodynamic parameters for both the Adair and the Monod, Wyman, Changeux (MWC) models. The results show the details of the unusual temperature dependent cooperativity which this hemoglobin exhibits. In the Adair formalism the increasingly favorable free energy change for successive steps of ligand binding are nearly linearly paralleled by increasingly negative enthalpy changes for these steps. This causes the enhanced cooperativity observed as the temperature is decreased. For the MWC case, lowering the temperature increases the stability of the unligated T state relative to the unligated R state since the enthalpy of the T leads to R transition is 29.4 kcal mol-1. Simultaneously, the favorability of ligating R forms relative to T is enhanced since R form ligation is 14.1 kcal (mol CO)-1 more exothermic than that of T. The balance between these opposing effects is to increase ligand binding cooperativity at low temperatures. The predicted temperature dependence of the Hill coefficient for the MWC and Adair models is identical at low and intermediate temperatures, but, interestingly, would show a strong divergence at high temperatures where negative cooperativity is suggested for the Adair case and positive cooperativity for the MWC case.     

Stereochemistry of carbon monoxide binding to myoglobin and hemoglobin

The binding of carbon monoxide to myoglobin and hemoglobin is examined to determine the origin of the deviation of the FeCO geometry from that found in model systems. Possible distortions due to protein-ligand interactions are analyzed with special attention to protein relaxation. It is estimated that the protein can support a strain of less than 10 kcal per mole; this may be sufficient to produce a displacement of a linear FeCO unit from the heme normal.     

Oxidation-reduction reactions of hemoglobin A, hemoglobin M Iwate, and hemoglobin M Hyde Park

The kinetics and equilibrium of the redox reactions of hemoglobin A, hemoglobin M Iwate, and hemoglobin M Hyde Park using the iron (II) and iron (III) complexes of trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetate (CDTA4-) as the reducing and oxidizing agents have been studied. With respect to the equilibrium it was found that hemoglobin M Iwate (where the beta chains were reduced) was more readily reduced than hemoglobin M Hyde Park (where the alpha chains are reduced). This difference was shown to be a result of a difference in the rate constant for reduction but not oxidation. The observed rate contants for the reduction of all three hemoglobins were shown to decrease with increasing pH. This was attributed to a decrease in the [T]/[R] ratio. The observed rate contants for the oxidation reaction were shown to increase with increasing pH. Accompanying this increase was a change in the kinetic profile for hemoglobin A from pseudo first order to one in which the rate increased as the extent of reaction increased. Inositol hexaphosphate had no effect on the rate of oxidation of deoxyhemoglobin A. This was a result of binding of FeCDTA2- or HCDTA3- to the protein. However, in the presence of inositol hexaphosphate, the reduction of methemoglobin A exhibited biphasic kinetics. This result was interpreted in terms of the production of a small amount of a conformation which was more readily reduced.     

The relation between carbon monoxide binding and the conformational change of hemoglobin.

The spectral difference between normal and rapidly reacting deoxyhemoglobin (Sawicki and Gibson (1976), J. Biol Chem. 251:1533-1542) is used to study the relationship between CO binding to hemoglobin and the conformational changes to the rapidly reacting form in a combined flow-laser flash experiment. In both pH 7 phosphate buffer and pH 7 bis(2-hydroxy-ethyl)imino-tris (hydroxymethyl)methane buffer (bis-Tris) with 500 muM 2,3-diphosphoglycerate (DPG), the conformational change lags far behind CO binding; rapidly reacting hemoglobin is not observed until more than 10% of the hemoglobin is liganded. In pH 9 borate buffer the formation of rapidly reacting hemoglobin leads CO binding by a significant amount. A simple two-state allosteric model (Monod et. al. (1965), J. Mol. Biol. 12:88-118) which assumed equivalence of the hemoglobin subunits in their reaction with CO was used to simulate the experimental results. In terms of the model, the conformational change lead observed at pH 9 suggests that significant conformational change has occurred after binding of only one CO molecule per tetramer. In the presence of phosphates good agreement between experimental results and simulations is obtained using parameter values suggested by previous experimental studies. The simulations suggest that the conformational change occurs after binding of three CO molecules.     

Quaternary structure dynamics and carbon monoxide binding kinetics of hemoglobin valency hybrids.

The kinetics of CO binding and changes in quaternary structure for symmetric valency hybrids of human hemoglobin have been extensively studied by laser photolysis techniques. Both alpha+beta and alpha beta+ hybrids were studied with five different ferric ligands, over a broad range of CO concentrations and photolysis levels. After full CO photolysis, the hybrid tetramers switch extensively and rapidly (< 200 microseconds) to the T quaternary structure. Both R --> T and T --> R transition rates for valency hybrid tetramers with 0 and 1 bound CO have been obtained, as well as the CO association rates for alpha and beta subunits in the R and T states. The results reveal submillisecond R reversible T interconversion, and, for the first time, the changes in quaternary rates and equilibria due to binding a single CO per tetramer have been resolved. The data also show significant alpha-beta differences in quaternary dynamics and equilibria. The allosteric constants do not vary with the spin states of the ferric subunits as predicted by the Perutz stereochemical model. For the alpha beta+CN hybrid the kinetics are heterogeneous and imply partial conversion to a T-like state with very low (seconds) R reversible T interconversion.     

Thin-layer microcalorimetric studies of oxygen and carbon monoxide binding to hemoglobin and myoglobin.

A thin-layer gas-solution microcalorimeter has been developed to study the binding reactions of gaseous ligands with ligand binding macromolecules. We have measured the enthalpy of binding oxygen and carbon monoxide to horse myoglobin, human hemoglobin A0 and sperm whale myoglobin in phosphate buffer at pH 7.6, with the enzyme reducing system of Hayashi. Reactions of human hemoglobin were also done under various buffer conditions in order to elucidate the Bohr effect. These binding reactions were found not to exhibit a detectable enthalpy change over the temperature range of 10 degrees C to 25 degrees C. The enzyme reducing system was shown to react with oxygen in a manner that releases a substantial amount of heat. This problem was corrected by using a minimum amount and by placing the buffer and enzyme system in the reference cell effectively cancelling the oxygen enzyme reaction heat as well as the heat of gas dissolution. It was also demonstrated that glucose-6-phosphate, one of the reducing system components, in 50 mM concentrations can influence the heat of binding oxygen and carbon monoxide to hemoglobin. This effect was shown to be absent in the myoglobins and also with hemoglobin at glucose-6-phosphate concentrations less than 5 mM.     

Calorimetric studies of oxygen and carbon monoxide binding to human hemoglobin. Sequential binding heats for oxygen

Two high precision techniques, titration microcalorimetry and thin-layer optical binding measurements, have made possible the evaluation of enthalpy changes for the overall oxygenation reactions for human hemoglobin (HbAo). Although the heat of adding three oxygen molecules could not be evaluated due to the indeterminate contribution of this species to the oxygen binding curve of the protein (Gill, S. J., Di Cera, E., Doyle, M. L., Bishop, G. A., and Robert, C. H. (1987) Biochemistry, 26, 3995-4002), the heats for binding two and four oxygen molecules were found to be simple multiples of the first binding heat. A direct consequence of equal stepwise heats is invariance of the shape of the binding curve with temperature, as pointed out by Wyman (Wyman, J. (1939) J. Biol. Chem. 127, 581-599). Titration microcalorimetry was also performed for the binding of carbon monoxide to hemoglobin. While the tight binding of CO precludes high-precision binding measurements, it does allow one to accurately determine the heat of ligation as a function of the CO bound. In these titrations a uniform heat of reaction is not observed, but the heat of binding increases markedly near the end point. This implies that the stepwise binding enthalpy for adding the third CO molecule is anomalously endothermic and for adding the fourth strongly exothermic. A similar phenomenon cannot be ruled out in the case of oxygen because of imprecision intrinsic in the analysis of the weaker ligand binding.     

Allosteric kinetics and equilibria differ for carbon monoxide and oxygen binding to hemoglobin.

We have measured the forward and reverse rates of the allosteric transition between R (relaxed) and T (tense) quaternary structures for oxyhemoglobin A from which a single oxygen molecule was removed in pH 7, phosphate buffer, using the method of modulated excitation (Ferrone, F.A., and J.J. Hopfield. 1976. Proc. Natl. Acad. Sci. USA. 73:4497-4501 and Ferrone, F.A., A.J. Martino, and S. Basak. 1985. Biophys. J. 48:269-282). Despite the low quantum yield, which necessitated large light levels and an associated temperature rise, the data was of superior quality to the equivalent experiment with CO as a ligand, permitting comparison between the allosteric behavior of hemoglobin with different ligands. Qualitatively, the T structure is favored more strongly in triligated oxyhemoglobin than triligated carboxyhemoglobin. The rates for the allosteric transition with oxygen bound were essentially temperature independent, whereas for CO both the R----T and T----R rates increased with temperature, having an activation energy of 2.2 and 2.8 kcal, respectively. The R----T rate was higher for O2 than for CO being 3 x 10(3) s-1 vs. 1.6 x 10(3) s-1 for HbCO at 25 degrees C. The T----R rate for HbO2 was only 2 x 10(3) s-1, vs 4.2 x 10(3) s-1 for HbCO, giving an equilibrium constant between the structures greater than unity (L3 = 1.5). The data suggest that there may be some allosteric inequality between the subunits, but do not require (or rule out) ligand binding heterogeneity. The ligand-dependent differences are compatible with stereochemical studies of HbCO and HbO2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of carbon monoxide binding to fully and partially reduced human hemoglobin valency hybrids.

The kinetics of carbon monoxide binding following fast reduction of the valency hybrids alpha2+betaCO2 and alphaCO2beta+2 by hydrated electrons have been studied at different degrees of reduction. The results show that at pH 6.0 and 7.0 reduction of one heme group yields a species which reacts fast with carbon monoxide (rate constant of the order of 10(6) M-1S-1). At pH 6.0 the intermediates alphaCO2beta2 and alpha2betaCO2 bind carbon monoxide with a rate characteristic of the T state. At pH 7.0 alphaCO2beta2 is for the greater part in the T state, while in the case of alpha2betaCO2 the R and the T state are about equally populated.     

Studies on carbon monoxide binding by shark haemoglobin.

The kinetics of the reactions of Pacific-porbeagle haemoglobin with CO were studied by flash-photolysis and stopped-flow methods, and the equilibrium binding curves for CO were measured in spectrophotometric titrations. Measurements were made in the pH range 6-8 and in the temperature range 0-40 degrees C. The results are discussed in terms of the allosteric model proposed by Monod, Wyman & Changeux [(1965) J. Mol. Biol. 12, 88-118]. Within this framework the results indicate that in the R-state the haem groups fall into two classes of different reactivity with different spectral characteristics, but that in the T-state the groups may be essentially equivalent. The physiological importance of the temperature-insensitivity of the equilibrium ligand-binding curves for porbeagle haemoglobin is discussed.     

Stoichiometry of carbon monoxide binding by cytochrome c oxidase.

The stoichiometry of carbon monoxide binding to beef heart cytochrome c oxidase has been reinvestigated both by titration of the reduced oxidase with CO and by measuring the amount of carboxyhemoglobin that is formed after adding oxyhemoglobin to a solution of the CO-enzyme complex. In the titration experiments the ratio of CO bounds to total heme a present was always less than 0.50 while in the experiments where oxyhemoglobin was added the results were variable and of lower accuracy. These observations do not agree with the recent conclusion of Volpe, J.A., O'Toole, M.C., and Caughey, W.S. (1975) Biochem. Biophys. Res. Commun. 62, 48-53 that CO is bound in a 1:1 ratio with heme a. An explanation for their results is suggested.     

Enthalpy changes for inositol hexaphosphate binding to hemoglobins A and M Iwate.

Enthalpies of inositol hexaphosphate (IHP) binding to deoxy and carbonmonoxy (CO) HbA and HbM Iwate have been determined calorimetrically and compared as functions of pH. Values for deoxy HbA and for deoxy HbM Iwate are similar with CO HbM Iwate yielding slightly less heat of reaction. The results support the existence of both deoxy and CO HbM Iwate in T-like structures with only minor modifications occurring upon CO binding. For HbA observed heats of IHP binding have been corrected for heats of extraction of reacting protons from buffer. The resulting intrinsic IHP binding enthalpies show consistent values of ?7 to ?11 kcal/mol proton absorbed in binding. We suggest that a major driving force for organic phosphate binding is the exothermic protonation of histidine and/or a α-amino nitrogens induced by proximity of phosphate negative charges.     

Influence of carbon monoxide on hemoglobin-oxygen binding.

The oxygen dissociation curve and Bohr effect were measured in normal whole blood as a function of carboxyhemoglobin concentration [HbCO]. pH was changed by varying CO2 concentration (CO2 Bohr effect) or by addition of isotonic NaOH or HCl at constant PCO2 (fixed acid Bohr effect). As [HbCO] varied through the range of 2, 25, 50, and 75%, P50 was 26.3, 18.0, 11.6, and 6.5 mmHg, respectively. CO2 Bohr effect was highest at low oxygen saturations. This effect did not change as [HbCO] was increased. However, as [HbCO] was increased from 2 to 75%, the fixed acid Bohr factor increased in magnitude from -0.20 to -0.80 at very low oxygen saturations. The effect of molecular CO2 binding (carbamino) on oxygen affinity was eliminated at high [HbCO]. These results are consistent with the initial binding of O2 or CO to the alpha-chain of hemoglobin. The results also suggest that heme-heme interaction is different for oxygen than for carbon monoxide.