Spectroscopic Evidence for a Conformational Transition in Horseradish Peroxidase at Very Low pH

Abstract

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.

Novel Conformational Transitions of Human Cytochrome P450 2C8 during Thermal and Acid‐induced Unfolding

“…415 nm, suggesting a remaining attached heme. 56 The results supported the suggestion that the heme in the Fe 3＋ -P420 2C8 acidic form was released from the heme pocket due to disruption of the heme iron-Cys435 coordination bond and the disintegration of the tertiary structure around heme. Since the heme was driven away from the cavity, such acidic form could not return to its native state but only precipitated.…”

“…in myoglobin, peroxidase, cytochrome b 5 etc.). 52,56,57 Although the conversion of P450 cam to its P420 form was extensively studied, little was known about the purified human P450 proteins and their P420 forms.…”

“…Although the absorption spectrum of the Fe 2＋ -P420 2C8-CO acidic form at pH 3.0 (λ max , 422, 540, 568 nm) closely resembled that of the Fe 2＋ -HRP-CO at pH 3.1 (λ max , 422, 542, 570 nm), the CD spectra were different from each other. 56 Fe 2＋ -HRP-CO had the prominent negative and positive ellipiticity peaks at ca. 433 and ca.…”

Estudo espectroscópico do equilíbrio entre espécies hexacoordenada e pentacoordenada dos monômero d nativo e reconstituído da hemoglobina extracelular gigante de Glossoscolex paulistus em meio alcalino

“…Visca e colaboradores 49 empregam, inclusive, a determinação da razão entre as intensidades das bandas de Soret do composto estudado e de uma espécie atribuída como hexacoordenada para determinar a presença da espécie pentacoordenada, uma vez que este estado de coordenação tem uma absortividade molar muito menos intensa. De qualquer maneira, os dois fenômenos espectrais acima discutidos, ou seja, o deslocamento hipsocrômico e a diminuição de absortividade molar são fortes indicativos da formação de uma espécie pentacoordenada, conforme uma série de trabalhos envolvendo hemoproteínas, por meio de uma variada série de técnicas espectroscópicas 27,28,[50][51][52][53][54][55][56][57][58][59][60][61] . Ademais, surge uma banda de transferên-cia de carga do ligante para o metal (TCLM) em 620 nm, que também corresponde a uma espécie de spin alto, como é o caso do pentacoordenado.…”

Hemes férricos pentacoordenados e hexacoordenados dos monômeros d nativo e reconstituído da hemoglobina extracelular de Glossoscolex paulistus: estudos espectroscópicos em meio ácido

“…A formação de uma banda relativamente larga em 371 nm foi atribuída a uma espécie pentacoordenada 19,[37][38][39] . De fato, vários autores sugerem que, partindo-se de um heme hexacoordenado, a formação de um heme pentacoordenado implica em uma diminuição significativa da absortividade molar e um deslocamento hipsocrômico da banda de Soret, sendo que a grandeza deste deslocamento é peculiar para cada hemoglobina 37,[40][41][42][43][44][45][46] .…”