A Calorimetric Study of the CO Bohr Effect of Monomeric Haemoglobins

Abstract

A calorimetric study has been made of the heats of CO reaction with the monomeric haemoglobins of Chironornus thummi thummi I11 and IV as a function of pH. The number of Bohr protons released at pH 7.1 was determined from heats of reaction in different buffers as 0.19 and 0.31 mol H+/mol CO for haemoglobin I11 and IV respectively. The heat of the Bohr ionization process was found to be 6 and 8 kcal/mol H' (25 and 34 kJ/mol) for the haemoglobins 111 and IV. These values are consistent with values found for histidine groups. A pH-independent part of the reaction enthalpy was determined as -19.7 kcal/mol CO (-82.4 kJ/mol). The same reaction with myoglobin is less exothermic. From the combination of AGO and A H o values TASo values have been calculated. It was found for both haemoglobins that the entropy of reaction is greater by 2 cal K-' mol-' (8.4 J K-' mol-') at pH 9.5 as compared to pH 6.0.The Bohr effect discovered in two monomeric haemoglobins of Chironomus thummi thummi [l, 21 shows that linked functions [3] or allosteric effects can exist in monomeric proteins. The pH dependence of the measured equilibrium half-saturation pressure was also found to depend upon the particular ligand (0, or CO) involved in the reaction to the haem [4]. The largest effect was noted for oxygen. In this protein the Bohr proton ionization site has been identified by nuclear magnetic resonance as a single histidine [4,5]. The X-ray structural studies by Huber et al. [6,7] and the results of chemical shifts observed by nuclear magnetic resonance [5] indicate that this histidine forms a salt bridge with the C-terminal carboxyl group of methionine-H22. The distance between the Bohr proton site and the iron of the haem group is about 1.5 nm. At low pH values the salt bridge is stabilized by the presence of the proton. Under these conditions the ligand affinity is found to be smaller than that at high pH values. The observed shift in pK value for the Bohr proton binding group, identified as histidine-G2, has been fully correlated to the pH dependence of the half-saturation pressure [4,5]. A suggested molecular mechanism for the long-range interaction between the ligand reaction and Bohr proton site depends upon the movement of the proximal histidine in Van der Waals contact with the C-terminal methionine residue [5].The structural and spectroscopic information obtained with these monomeric haemoglobins offer a unique opportunity to study the thermodynamic aspects of intra-chain allosterism. We have therefore carried out a series of direct calorimetric measurements on the CO binding reaction at different values of pH. The results add further quantitative confirmation to the previously noted Bohr effect. They also show that the heat of the Bohr proton release indicates the involvement of a histidine group. MATERIALS AND METHODS Preparation of SamplesThe monomeric haemoglobins 111 and IV from Chironomus thummi thummi were prepared as described previously [5]. The lyophilized salt-frce material was dissolved in 0.2 M maleate, bis-Tris, o...

Solution 1H‐NMR Structure of the Heme Cavity in the Low‐Affinity State for the Allosteric Monomeric Cyano‐Met Hemoglobins from Chironomus thummi thummi

“…Solution 'H-NMR studies of the cyanomet form of C. thummi thummi Hbs have shown that the allosteric transition can be detected both in the titration of a His side chain, which is the site of the Bohr proton [5], and in the hyperfine shifts for the heme in this low-spin ferric form [12, 131. One property that makes the C. thummi thummi Hbs poor candidates for structural studies is that they exhibit heterogeneities that plague both solution and crystal structures [S, 14,151.…”

Resonance Raman study of deoxy and ligated (O2 and CO) mesoheme IX-reconstituted myoglobin, hemoglobin and its α and β subunits

“…405 (12) 405 (9) 405 (9) 405 ( EPR (electron paramagnetic resonance spectroscopy) data [31,43]. The m(Fe-His) mode of the natural abundance and d 12 -labeled deoxy meso-a and -b subunits is observed at 222 and 225 cm À1 , respectively, i.e.…”

“…by 15 cm À1 , upon the d 12 substitution (Fig. 2) is assigned to a C b -propionate bending mode [20,14,25,43], one of the most characteristic heme modes. Almost the same frequency of this mode is observed in the deoxy mesoHb of CTT [5].…”

Dynamic properties of monomeric insect erythrocruorin III from Chironomus thummi-thummi: relationships between structural flexibility and functional complexity

“…We have chosen this protein because it is unique in many respects. Its ligand affinity increases with pH (Gersonde et al, 1986), and a pronounced conformational change accompanies the transition from the low-affinity to the high-affinity state (Gersonde et al, 1976;La Mar et al, 1978;Peyton et al, 1991), such that soaking crystals of CTT-Hb-III obtained at acidic pH in alkaline buffer causes them to crack (Steigemann and Weber, 1979). Thus, despite its relatively simple monomeric structure, CTT-Hb-III undergoes pH-induced conformational transitions and is therefore particularly well suited to pinpointing possible relationships between protein dynamics and functional complexity.…”