Bohr Proton Research Articles

Solution NMR study of the structural basis of the Bohr effect in the monomeric hemoglobins from Chironomus thummi thummi.

The larva of the midge Chironomus thummi thummi possesses two monomeric hemoglobins. HbIII and HbIV, with extensive sequence homology, which exhibit marked but differential Bohr effects (pH influence on ligand affinity). These Hbs serve as ideal models for allosteric control of ligand affinity via tertiary-only structural changes. The cyanomet derivatives of these two Hbs have been shown to possess essentially indistinguishable heme cavity structures in solution at low pH (Zhang et al., 1996) that are also very similar to that of the low pH form of HbIII in the crystal (Steigemann & Weber, 1979). 2D 1H NMR has been utilized to elucidate the solution heme cavity structure of the alkaline form of the cyanomet derivatives of HbIII and HbIV to identify the Bohr proton binding site and characterize the nature of the structural changes that accompany the allosteric transition. Significant structural changes with pH have been identified in two regions of the heme cavity, near the axial His and at the junction of pyrroles B and C. The Bohr proton site is identified as His94, which at low pH makes a salt bridge to the terminal Met136. The rupture of this salt bridge at high pH leads to the expulsion of the Met136 side chain next to the His F8 ring where it serves as a spacer between the heme and F-helix, and leads to a cascade of side chain reorientations in the densely packed hydrophobic interior involving five Phe (65, 66, 128, 129, 133), Val132, and Ile69, all on the E- and H-helices. The terminal member of the cascade, Phe65, which acts as a spacer between the E- and F-helices at low pH, is rotated toward the heme plane. The conversion of the low pH, low-affinity "tense" to the high pH, high-affinity "relaxed" state is primarily due to the removal of the Met136 and Phe65 spacers. A central residue in transmitting the Bohr effect from His94 to Phe65 is residue 132. In HbIV, Val132 provides a cavity in the hydrophobic core to readily accommodate the initial step in rotating the Phe129 side chain. In HbIII, the Ile132 provides tight packing to all neighboring side chains and hence would inhibit the rotation of the Phe129 side chain. It is proposed that the lone internal residue difference between HbIII (Ile132) and HbIV (Val132) is the primary basis for the different amplitudes of their Bohr effect.

Effect of haemoglobin oxygenation on Bohr proton release and CO 2 excretion in the rainbow trout

CO 2 excretion in trout is dependent upon the Haldane effect and the release of protons (Bohr protons) from haemoglobin during oxygenation of the blood. Oxygenation of whole blood from trout in vitro, resulted in a non-linear release of Bohr protons over the haemoglobin-oxygen (HbO 2) equilibrium curve, where the majority of Bohr protons were released between 60 and 100% of Hb oxygen saturation (S O 2 ). Oxygenation of the blood over this region of the HbO 2 equilibrium curve elevated the HCO − 3 flux rate across the HCO 3/Cl exchanger on the red cell membrane by about 30% during CO 2 excretion in vitro. Oxygenation of the Hb between 0 and 60% S O 2 did not elevate CO 2 excretion rate in vitro. These data indicate that utilization of different regions of the HbO 2 dissociation curve in trout in vivo will influence CO 2 excretion by altering the rate of HCO − 3 entry into the red cell during gill blood transit.

The interaction between oxygen and carbon dioxide movements in fishes

Abstract Many teleost fishes have haemoglobins which possess a Root effect, a large Haldane effect and a low buffer capacity. This combination of characteristics influences the interaction between movements of oxygen and carbon dioxide in the red cell, in the respiratory epithelium, and in the tissues. The presence of the Root effect may limit oxygen uptake at the gills due to an accumulation of Bohr protons released upon oxygenation. However, the Root effect is probably important in maintaining or elevating blood PO 2 during muscle capillary transit, enhancing oxygen delivery to the tissues. Bohr protons are reversibly bound to haemoglobin. The release of Bohr protons during oxygenation facilitates bicarbonate dehydration at the gills, while Bohr proton binding facilitates CO 2 hydration at the tissues. In some teleost fishes, most of the Bohr protons are released and bound to haemoglobin, between 50 and 100% of haemoglobin-oxygen saturation (27). This trait is probably significant in maximizing oxygen uptake at the gills and in conserving body CO 2 stores during exposure to hypoxia and exercise, when the lower reaches of the haemoglobin-oxygen equilibrium curve are used.

Bohr Effects of the Partially-Ligated (CN-met) Intermediates of Hemoglobin as Probed by Quaternary Assembly

Free energies of quaternary assembly (dimers to tetramers) were determined for the 10 ligation species of CN-methemoglobin in the region of the alkaline Bohr effect (pH 7.0-9.5). Analysis of this database yielded the following principal findings: (1) At each pH, the nine CN-met species exhibit two distinct values of Bohr proton release and Bohr free energy. The two Bohr effects are found to distribute in a fashion that coincides with predictions of a symmetry rule (Ackers et al., 1992), i.e., the first value reflects a "tertiary Bohr effect" arising from ligation within the quaternary T tetramer and a second Bohr effect arises from the quaternary transition (T-->R) which occurs when both dimeric half-molecules acquire at least one ligated subunit. (2) The Bohr effects for CN-met ligation are in good agreement with previously-established Bohr effects for stepwise O2 binding under identical conditions (Chu et al., 1984). (3) In combination with recent studies which show that CN-met species [21] has a quaternary T structure (Daugherty et al., 1991; Doyle & Ackers, 1992; LiCata et al., 1993), the present results show that the "tertiary Bohr effect" within quaternary T exceeds the Bohr effect of dissociated dimers, as suggested by Lee and Karplus (1983). (4) The tertiary Bohr effect is found to account for the pH dependence of tertiary constraint energy, delta Gtc, which "pays" for ligand-binding cooperativity prior to the quaternary (T-->R) switchover. Possible origins of the tertiary Bohr effect and its relationship to the quaternary Bohr effect are considered.

Electron paramagnetic resonance study on crosslinked asymmetric Fe(II)-Co(II) hybrids of hemoglobin

Asymmetric Fe-Co hybrid hemoglobins (Hbs) containing one, two, and three Co(II) protoporphyrins IX per tetramer have been synthesized. These asymmetric Fe-Co hybrids have been prepared by appropriate combinations of subunits of HbA, HbS, Co(II)HbA, Co(II)HbS, and Fe-Co hybrid Hbs and crosslinking with bis(3,5-dibromosalicyl) fumarate, followed by purification with molecular sieve and ion-exchange chromatography. Electron paramagnetic resonance (EPR) spectra have been measured for the crosslinked asymmetric Fe-Co hybrids to investigate the intermediate ligation states of Hb. The electronic state of the Co(II) ion provides a useful probe to estimate how ligation to iron-containing subunits changes the structure of the cobalt-substituted heme vicinity as described previously ( T. Inubushi and T. Yonetani (1983) Biochemistry 22, 1894–1900). The crosslink does not affect the EPR spectra of Fe-Co hybrids. EPR spectra of the α(Co) subunits in crosslinked hybrids in the fully deoxy state exhibit a mixture of a broad EPR component and a narrow EPR component which correspond to distorted and undistorted Co(II) coordinate states, respectively, and the ratio of the components varies with pH. EPR spectra of the di-liganded asymmetric hybrid, [α(Co)β(Co)] A-[α(Fe-CO)β(Fe-CO)] SXL, and tri-liganded hybrid, [α(Co)β(Fe-CO)] A-[α(Fe-CO) β(Fe-CO)] SXL, show that the coordination state of the α(Co) ion in these hybrids is changed to the completely undistorted state in the pH range between 6.5 and 8.8 upon ligation of carbon monoxide (CO). Mono-liganded hybrids, [α(Co)β(Fe-CO)] A-[α(Co) β(Co)] SXL and [α(Fe-CO)β(Co)] A-[α(Co)β(Co)] SXL show intermediate EPR spectra at pH 6.5–7.6, which represent a mixture of the broad and narrow EPR components. The broad component is converted into the narrow one more extensively upon the ligation to the β(Fe) subunit than to the α(Fe) subunit. However, the coordination state of an α(Co) subunit in mono-liganded hybrids corresponds to a completely undistorted coordination structure at pH 8.2–8.8. The EPR signal of the α(Co) ion is sensitive to the release of alkaline Bohr proton(s) and consequently to the allosteric quaternary structural change of Hb.

Proton-magnetic-resonance investigation of the dynamics of the conformational transition in allosteric monomeric insect hemoglobins.

1H-NMR spectra of the monomeric insect (Chironomus thummi thummi) hemoglobins CTT III and CTT IV were investigated in the pH range 5-10 to gain insight into the dynamics of the tense to relaxed (t in equilibrium r) conformational transition in the deoxy (at 200 MHz) and cyano-met (at 360 MHz) form. These hemoglobins exhibit a pH-sensitive O2 affinity (Bohr effect) which is linked to the conformational transition. Both hemoglobins are comprised of two components which show heme rotational disorder due to a 180 degrees rotation of the heme group about the alpha,gamma-meso axis. The heme rotational components differ remarkably in their Bohr effects [Gersonde, K. et al. (1986) Eur. J. Biochem. 157, 393-404]. Several of the hyperfine-shifted heme proton resonances in these hemoglobin derivatives show pH-induced line-broadening which is largest at pK = 7.46 (for the heme rotational component with large Bohr effect) or pK = 7.06 (for the heme rotational component with small Bohr effect) determined from the plots of chemical shift versus pH. The line broadening at pK approximately equal to 7.5, shown for the heme rotational component of cyano-met CTT IV with the largest Bohr effect, decreases in the following order in parallel with the pH-induced shift change: 4-H beta-c (1.20 ppm) greater than 3-CH3 (0.80 ppm) approximately greater than 4H alpha (0.76 ppm) approximately greater than 4H beta-t (0.73 ppm) greater than 8-CH3 (0.35 ppm). Decrease in temperature at the pK value also leads to line-broadening. At 4 degrees C the hyperfine-shifted resonance attributed to 3-CH3 is split into two resonances assigned to the t (low-pH form) and r (high-pH form) conformation, respectively. This temperature dependence confirms the t in equilibrium r exchange process as the origin of the pH-induced line-broadening. The Bohr proton exchange rate at the allosteric site is orders of magnitude larger than the t in equilibrium r exchange rate. Therefore, the proton-linked t in equilibrium r transition appears as a first-order reaction. The rate constant ktr for the t in equilibrium r transition at the pK in different hemoglobin derivatives ranges over 0.2-7 ms-1. Surprisingly, ktr is identical for deoxy and cyano-met CTT IV.(ABSTRACT TRUNCATED AT 400 WORDS)

A quantitative model for the cooperative mechanism of human hemoglobin.

A quantitative model has been developed for the cooperative oxygenation of human hemoglobin. The model correlates the structural and energetic features of ligand-linked subunit interactions within the tetrameric molecule and the coupling of these interactions to the binding of oxygen and Bohr protons. Recent findings are incorporated regarding (i) the sites of regulatory energy change within the tetrameric molecule, (ii) the nature of the Bohr effect for tetramers and dimers, (iii) the fractional Bohr proton release at each stage of oxygenation, (iv) relative probabilities of binding to the alpha and beta chains within the tetramer, and (v) an extensive data base recently obtained on the linked processes of oxygenation, proton binding, and subunit interactions [Chu, A. H., Turner, B. W. & Ackers, G. K. (1984) Biochemistry 23, 604-617]. Least squares minimization was used to evaluate from these data the free energies for the various processes. A special feature of the model lies in the synchronization of Bohr proton release with changes in quaternary structure. This leads to the striking prediction that a major fraction (as much as 30%) of tetramers are in the oxy quaternary structure after the first oxygen is bound. The model provides a rationale for the essential features of regulatory energy control, and it defines several kinds of additional information that are needed for a more complete understanding of the hemoglobin mechanism.

Model for cooperativity of biological membranes.

We present a mathematical model for the complex cooperativity observed in biological membranes. In our model, it is assumed that the proteins bound on the membrane are noncooperative and possess a Bohr proton. It is further assumed that the net charge of the unliganded state of the protein is different from that of the liganded state owing to the structural change upon binding the ligand. With this model, we show how an all-or-none response, a graded response, and a noncooperative response arise in the binding curve of such biological membranes. In addition, we show how an effector, which can alter the pKa involved in the binding site, induces a complex cooperativity.

Proton nuclear magnetic resonance investigation of the nature of solution conformational equilibria of monomeric insect deoxyhemoglobins.

The proton nuclear magnetic resonance spectra of the three monomeric deoxyhemoglobins of the insect larva Chironomus thummi thummi have been recorded, assigned, and analyzed. In the two allosteric hemoglobins, the heme methyls and vinyl protons were assigned by specific deuterium labeling. The hyperfine-shifted proximal histidyl imidazole exchangeable protons for the three native and two deuteroheme-reconstituted hemoglobins were assigned by comparison of spectra in H2O and 2H2O. Both native and reconstituted allosteric hemoglobins exhibit two sets of interconvertible resonances indicative of two heme orientations differing by a 180 degrees rotation about the alpha-gamma-meso axis, as previously found for the met-cyano analogues [La Mar, G. N., Smith, K. M., Gersonde, K., Sick, H., & Overkamp, M. (1980) J. Biol. Chem. 255, 66]. The relative pH sensitivities of the heme resonance hyperfine shifts for the two allosteric hemoglobins and the apparent pK approximately 8 indicate that the t in equilibrium r allosteric transition, as modulated by the Bohr proton, is being observed. For the native hemoglobins, the t in equilibrium r conformational transition was found to be centered at the heme periphery, with the proximal histidyl imidazole environment insensitive to both pH and the rotational position of the heme, consistent with the absence of a pH influence on the ligation on-rate. For the deuteroheme-reconstituted allosteric hemoglobins, both the heme and axial imidazole environments sense the t in equilibrium r transition, and the histidine environments for the two components for each hemoglobin can be clearly distinguished, suggesting that the ligation on-rates may depend on both pH and heme orientation.

Energetics of subunit assembly and ligand binding in human hemoglobin

An extensive and self-consistent set of thermodynamic properties has recently been established for the coupled processes of subunit assembly and ligand binding (oxygen and protons) in human hemoglobin. The resulting thermodynamic values permit a consideration of the possible sources of energetic terms accounting for stability of the tetrameric quaternary structures at different stages of ligation, and of the possible sources of cooperative energy. The analysis indicates that: (a) The change in buried surface ara upon oxygenation (i.e., hydrophobic stabilization) does not play a dominant role in stabilizing the unliganded tetramer relative to the liganded tetramer. (b) The pattern of enthalpic and entropic contributions to the free energies of dimer-tetramer. (c) The thermodynamic results are consistent with a dominant role of increased hydrogen bond formation in the deoxy quaternary structure. (d) Within tetramers the variation in free energy for successive oxygenation steps arises from both enthalpic and entropic contributions and the enthalpic contributions are almost entirely attributable to the heats of Bohr proton release. At pH 7.4 the pattern of thermodynamic values suggests that a large contribution to the free energy of cooperativity may arise from the energetics of Bohr proton release. It is suggested that a combination of proton ionization and hydrogen bonding may account for the main energetic features of cooperativity. Possible contributions from fluctuation behavior cannot presently be evaluated.

Ligand-dependent allosteric transformation of hemoglobins from the blood clam, Anadara Broughtonii

1. 1. Oligomeric Hb I and II of Anadara broughtonii, which are unusual with respect to having no Bohr effect, were shown to have a R-T transformation on ligand-binding on the basis of the following experimental results. (a) Iodoacetamide reacted preferentially with the oxyforms of the hemoglobins. (b) CD spectra at the far-ultraviolet regions significantly changed on ligand-binding. (c) 1-Anilinonaphthalene-8-sulfonate bound to the hemoglobins with a preference for deoxyforms. From these results and previous findings [1], it is concluded that the absence of the Bohr effect in these hemoglobins is due to the lack of the Bohr proton ionizing groups in the molecules. 2. 2. Hb I and II treated with p-chloromercuribenzoate, designated as PMB-I and PMB-II, showed greatly increased oxygen affinity and decreased cooperativity. CD spectra at the far-ultraviolet of the PMB-Hb in the oxygen liganded state gave similar patterns to those of native oxygenated Hb. However no changes in the spectra were observed on deoxygenation. These findings suggest that the PMB-I and PMB-II retain their native oxy conformation even in the deoxy states. The PMB-modification might prevent the initial ligand-induced conformational change within the protomers.

Isomeric incorporation of the Haem into monomeric haemoglobins of Chironomus thummi thummi. 2. The Bohr effect of the component III explained on a molecular basis and functional differences between the two isomeric structures.

The Bohr effect in the monomeric haemoglobin component III of Chironomus thummi thummi is described on a molecular basis using various pH-dependent proton resonances. One of the three titratable histidines changes its pK from 7.9 in the unligated state to 7.7 upon ligation with CO and up to 7.0 upon ligation with O2. It can be concluded from an analysis of the C-2 proton resonance intensities that these shifts in pK occur in only one of the two conformational isomers of the component III, whereas in the second isomer these pK shifts are not observed. The differences between these functional properties are also derived from pH-dependent changes of the mesoproton resonances as well as of the methyl resonances of Ile-E11. The conformational change of the component III, which is connected with the Bohr effect, influences a variety of other resonances which could not be assigned. The pK values derived from the pH dependence of these changes agree exactly with the pK value of the Bohr proton donating group, which is supposed to be His-G2. Also the corresponding signal intensities indicate the presence of two conformational isomers in the haemoglobin component III. Although the change in chemical shift of the His-G2 C-2 proton resonance is only small upon deprotonation of the imidazole ring, it has been shown with potentiometric and calorimetric methods that this histidine is deprotonated in this pH range. Considering the tertiary structure obtained from an X-ray study of Steigemann and Weber we can readily explain the conformational changes in the Bohr active isomer of the component III. With binding of ligands dislocations of side chains at the proximal site produce a change in the interactions of His-G2 with the carboxyl group of the C-terminal methionine-H22.

Beta-chain allostery in the frozen quaternary T-structure of haemoglobin M Iwate.

Two hybrid species of haemoglobin M Iwate exist: α2Mmetβmetβdeoxy and α2Mmetβ2deoxy These species differ in their ligand and effector binding properties.The α2Mmetβmetβdeoxy hybrid is characterized by a Bohr effect, while the Hill coefficient is n= 1.00. The energy of the interaction between the Bohr protons and the ligand‐binding site corresponds to the interaction energy found in monomeric haemoglobins and amounts to 0.55 kcal/mol. One molecule of 2, 3‐bisphosphoglycerate per mole is bound at neutral pH, while at alkaline pH two co‐operative molecules are bound. Binding of 2,3‐bisphosphogly‐cerate reduces the ligand affinity and shifts the pK value of the Bohr proton binding site to the alkaline region.The α2Mmetβ2deoxy hybrid shows a pH‐dependent co‐operativity of the ligand‐binding sites (n= 1.8 at pH 9). The co‐operativity is weakened by binding of protons or 2,3‐bisphospho‐glycerate. The latter lowers the ligand affinity, but in the alkaline region no influence is observed. Furthermore the Bohr effect curve is shifted to the alkaline region by this compound but the amplitude remains unchanged.The results of the present paper are interpreted in terms of intra‐chain and β‐β inter‐chain interactions, the latter being responsible for a consecutive change of the ligand affinity in a “frozen” T‐state molecule.

Reduced Bohr Effect in NO‐Ligated Chironomus Haemoglobin

The low‐temperature electron‐spin‐resonance spectra of the NO‐derivatives of the monomeric Chironomus haemoglobins I, III, and IV being nearly identical to each other, indicate rhombic symmetry of the innermolecular electric field. At gz= 2.003 nine hyperfine lines (ΔH= 7 G) of nearly identical intensity are observed in the presence of 14NO, while 15NO produces two hyperfine lines (ΔH= 30 G) both showing a further splitting into three superhyperfine lines (ΔH= 7 G). These hyperfine structures represent the interaction of the unpaired electron with the N‐nuclei of the 5th and 6th ligand.No changes of the electron‐spin‐resonance spectra were found in the range of pH 5 to 10, although haemoglobin III and IV are known to possess an alkaline Bohr effect. Thus in case of NO‐binding the Bohr effect is apparently reduced. With the aid of proton‐magnetic‐resonance studies of the Bohr proton binding site, His‐G2, in NO‐haemoglobin III it was demonstrated that the pK value of this group is shifted by only 0.15 units from that of deoxy‐haemoglobin. This shift is considerably lower as compared with that induced by CO‐ligation.

The effects of 2,3-diphosphoglycerate on the kinetics of deoxygenation of human hemoglobin

Using a stopped flow apparatus, the effects of 2,3-diphosphoglycerate (DPG), and pH on the hemoglobin-oxygen dissociation kinetic constants were determined. The order of kinetics was pseudo-first order between 85 and 40% saturation. Increasing the concentration of DPG increased the kinetic constants in a manner which is related to the extent of binding of DPG to hemoglobin. The dependence of the kinetic constants on pH was, however, not explained in terms of the total number of DPG bound to oxyhemoglobin. The DPG was affecting the kinetic constants in such a manner that the binding was functionally linked to the Bohr proton.