Spectroscopic evidence for a conformational transition in horseradish peroxidase at very low pH.

Resonance Raman (RR), electronic absorption, and circular dichroism (CD) spectroscopies of the ferric, ferrous, and ferrous-CO forms of horseradish peroxidase (HRP-C) at pH 3.1 are reported. The CD spectra in the UV region show only a small decrease in the alpha-helical content upon pH lowering, whereas dramatic changes are observed in the Soret region. The final form of ferric HRP-C is 5-coordinate high-spin heme whose histidine ligand is replaced by a water ligand with a polar character. The electronic and CD spectra show the presence of an intermediate form with a 6-coordinate heme. Therefore, the cleavage of the proximal Fe-imidazole bond is preceded by the binding of a distal water molecule. For the ferrous form of HRP-C, the pH-dependence of the absorption spectra revealed only the native form in the range pH 5-7 and an unfolded form with a Soret maximum at 383 nm at pH 3.1. An intermediate state, characterized by a Soret maximum at 424 nm, was observed only in a transient way, within a few milliseconds. A metastable and a final species are observed also for the ferrous-CO complex at pH 3.1, as proved by isosbestic points in the electronic absorption spectra. The two forms show different RR nu(Fe-C) and IR nu(CO) modes. The metastable form corresponds to a heme where histidine is replaced by water. The final form is due to the displacement of the water ligand by the proximal histidine. We propose a kinetic model to account for our results at pH 3.1 for the ferric, ferrous, and ferrous-CO forms.