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

Abstract

IH-NMR spectra of the monomeric insect (Chironomus thummi thummi) hemoglobins CTT 111 and CTT IV were investigated in the pH range 5-10 to gain insight into the dynamics of the tense to relaxed ( t e r ) conformational transition in the deoxy (at 200 MHz) and cyano-met (at 360 MHz) form. These hemoglobins exhibit a pH-sensitive Oz 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" rotation of the heme group about the a,y-meso axis. The heme rotational components differ remarkably in their Bohr effects [Gersonde, K. et al. (1986) Eur. J. Biochem. 157,393-4041.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 % 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-Hp-, (1.20 ppm) > 3-CH3 (0.80 ppm) 5 4H, (0.76 ppm) F4Hp-t (0.73 ppm) > 8-CH3 (0.35 ppm). Decrease in temperature at the pK value also leads to line-broadening. At 4°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+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+r exchange rate. Therefore, the proton-linked t e r transition appears as a first-order reaction. The rate constant k,, for the t+r transition at the pK in different hemoglobin derivatives ranges over 0.2-7 ms-l. Surprisingly, k,, is identical for deoxy and cyano-met CTT IV. The difference in k,, is very small between the cyano-met forms of CTT 111 and CTT IV. The heme rotational component with large Bohr effect exhibits a smaller k,, than that with small Bohr effect. Replacement of 'HZ0 by HzO in the solvent or photoheme-IX by deuteroheme-IX in CTTs results in an increase of kIr. The activation energy of the t e r exchange reaction is %70 kJ/mol. Allosteric control of ligand binding in hemoglobins (Hbs) is implemented by the interconversion of the protein between two (or more) distinct structural states. For tetrameric Hbs, the two alternative quaternary structures, T (low-affinity state) and R (high-affinity state), are considered as two functional structural units which differ primarily in the nature of the intersubunit contacts [l]. Since there are four ligand binding sites in tetrameric Hbs and each quaternary structure may exist with a variable number of ligands, the characterization of the dynamics of the structural interconversion is considerably complicated. Moreover, the degree...