SPECTROSCOPIC EVIDENCE FOR A CONFORMATIONAL TRANSITION IN HORSERADISH-PEROXIDASE AT VERY-LOW PH

Resonance Raman (RR), electronic absorption, and circular dichroism (C D) spectroscopies of the ferric, ferrous, and ferrous-CO forms of hors eradish peroxidase (HRP-C) at pH 3.1 are reported. The CD spectra in t he UV region show only a small decrease in the ct-helical content upon pH lowering, whereas dramatic changes are observed in the Soret regio n. 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 f orm with a 6-coordinate heme. Therefore, the cleavage of the proximal Fe-imidazole bond is preceded by the binding of a distal water molecul e. 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 unfol ded form with a Soret maximum at 383 nm at pH 3.1. An intermediate sta te, characterized by a Sorer maximum at 424 nm, was observed only in a transient way, within a few milliseconds. A metastable and a final sp ecies are observed also for the ferrous-CO complex at pH 3.1, as prove d by isosbestic points in the electronic absorption spectra. The two f orms show different RR nu(Fe-C) and IR v(CO) modes. The metastable for m corresponds to a heme where histidine is replaced by water. The fina l 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.