ACID-INDUCED TRANSFORMATIONS OF MYOGLOBIN .2. EFFECT OF IONIC-STRENGTH ON THE FREE-ENERGY AND FORMATION RATE OF THE 426-NM ABSORBING DEOXYHEME INTERMEDIATE

The acid unfolding of deoxymyoglobin (deoxyMb) from the native (N) for m to the unfolded (U) form proceeds through at least two spectroscopic ally distinct heme intermediates. The 426-nm absorbing heme intermedia te (I'-form) occurs in the pH similar to 3.5-4.5 range. In the I'-form , the iron-proximal histidine bond is broken; however, the heme is fiv e-coordinate due to binding of a water molecule. The I'-form was first observed in pH-jump (neutral to acid conditions) experiments, where i t was characterized as a transient species which rapidly forms (10 ms) and dissipates. Recently, however, it was shown that the I'-intermedi ate also forms under equilibrium conditions. To elucidate the factors which control the formation of the I'-intermediate, a detailed series of equilibrium and slow kinetic ( >2-s) experiments were performed. Eq uilibrium pH titrations reveal that the I'-intermediate forms at succe ssively higher pH as the ionic strength increases. pH-jump experiments (pH 6.9 to 3.2 and pH 4.4 to 3.2) indicate that the rate of formation of the intermediate is dramatically affected by the ionic strength co nditions. If the ionic strength is held constant during the pH-jump, t he I'-intermediate forms slowly (similar to 35 s) and the formation ra te is independent of ionic strength. If the ionic strength is jumped f rom low to high values during the pH-jump, the formation rate of the I '-intermediate monotonically increases. Conversely, if the ionic stren gth is jumped from high to low values during the pH-jump, the rate mon otonically decreases. The former result explains the finding of early pH-jump experiments wherein the I'-intermediate was found to form very rapidly. In these experiments, the ionic strength was also jumped fro m low to very high values during the pH-jump. In both types experiment s where the pH and ionic strength are simultaneously jumped, the rate of formation of the I'-intermediate is independent of the initial and final ionic strength and depends only on the difference. The kinetic a nd equilibrium data are well accounted for with a simple three-state m odel in which the N-form is transformed into the I'-form via a single transition (T) state, and the free energy of the various forms depends linearly on the ionic strength. The model predicts that both the N-fo rm and the T-state are stabilized with increasing ionic strength and t hat the extent of stabilization is approximately the same for both (-4 .84 cal/mol per mM). The I'-form is also stabilized with increasing io nic strength; however, the extent of stabilization is greater than for the N-form. This picture is qualitatively consistent with a simple Bo rn model which predicts that a medium with higher dielectric constant should impart greater stabilization to a species with higher overall c harge. The I'-form is stabilized relative to the N-form at higher ioni c strength (higher dielectric constant) because it is formed in a pH r egion where several of the histidine residues in the protein titrate, thus increasing the net positive charge on the protein relative to the N-form at neutral pH. Collectively, the studies provide a self-consis tent picture of the factors which control the acid-induced transformat ion of deoxyMb from the N- to I'-forms. (C) 1997 John Wiley & Sons, In c.