Acid-induced transformations of myoglobin. Characterization of a new equilibrium heme-pocket intermediate.

The pH dependence of the absorption and resonance Raman (RR) spectra of the deoxy and met forms of myoglobin (Mb) has been examined in detail. The spectral data were acquired at a number of different pHs (12) in the 2.6-7.6 range. RR spectra were obtained for both the low- and high-frequency regions by using a variety of excitation wavelengths ranging from the UV to the green. The data obtained for deoxyMb indicate that a spectroscopically distinct intermediate (I') exists at equilibrium in the pH 3.5-4.5 range. The I'-form of metMb could not be identified. The Soret absorption maximum of the I'-form of deoxyMb is at approximately 426 nm compared with the value of 435 observed for the native (N) form and 383 nm observed for the so-called unfolded (U') form which occurs in the pH 2.6-3.5 range. The absorption and vibrational spectra of the I'-form of deoxyMb observed at equilibrium are very similar to those of the intermediate that appears within a few milliseconds in pH-jump experiments. The RR data indicate that the structure of the heme group in the I'-form is distinctly different from that of either N- or U'-forms. The iron-histidine bond, characteristic of the N-form, is ruptured in both the I'- and U'-forms as is evidenced by the absence of the RR band due to the stretching vibration of this unit. In the I'-form, the histidine ligand is replaced by a relatively strongly bound, exchangeable water molecule. This ligand is absent in the U'-form. The aquo ligand of the five-coordinate heme in the I'-form is identified by a RR band at 411 cm-1 which undergoes a 15-17 cm-1 downshift in deuteriated buffer solutions. In contrast, none of the RR bands of the N- and U'-forms exhibit any significant isotope sensitivity. The properties of the I'-form and the conditions under which it is generated strongly suggest that this form corresponds to the molten globule intermediate of apoMb.