Buffer-anion-dependent Ca2+ leaching from horseradish peroxidase at low pH

Abstract

Despite highly conserved active-site structures, members of the plant peroxidase superfamily exhibit a wide range of pH optima. Horseradish peroxidase isozyme C (HRPC) is an ideal peroxidase to investigate the structural determinants of pH stability and activity in superfamily members. Conflicting reports exist on the low-pH stability of HRPC and consequently the pKa of the catalytic distal histidine, which is neutral in active peroxidases. Towards resolving such discrepancies, acid-induced changes in HRPC from two popular commercial suppliers were systematically analyzed. Specifically, FTIR v(CO) and Soret-CD spectra of HRPC-CO and Soret absorption of ferric HRPC were recorded to probe time-dependent heme-pocket changes at pH 3.0 in phosphate, citrate and formate buffers, while the FTIR amide I' and far-UV CD spectra were examined to probe changes in secondary structure. Both HRPC-CO samples exhibited identical pH 7.0 v(CO) bands at 1934 and 1905 cm-1. In the pH 3.0 spectrum of sample A, the 1934 cm-1 band was dominant while a broad 1969 cm-1 band appeared in sample B. The intensity of this band, which is assigned to solvent-exposed heme, was greater in citrate than phosphate buffer, but in formate the 1934 cm-1 band remained dominant. Other spectral changes mirrored the v(CO) trends. No time- or buffer-anion-dependent conformation changes were detected in 1 mM CaCl2, revealing that buffer-anion-dependent leaching of stabilizing Ca2+ from HRPC occurs at pH 3.0. Since the N-glycans present in HRPC are of the flexible protein-surface-shielding type, the variation in low-pH conformational stability of the HRPC samples could be attributed to heterogeneous glycosylation, which was detected by SDS-PAGE. It is further proposed that glycosylation patterns may affect the low-pH stability of class II and III plant peroxidases.

Ferric species equilibrium of the giant extracellular hemoglobin of Glossoscolex paulistus in alkaline medium: HALS hemichrome as a precursor of pentacoordinate species

“…The intense shoulder approximately at 363 nm, which is the spectral profile of the Soret band in the more drastic alkaline conditions evaluated in the present work, is very suggestive of a pentacoordinate species presenting a water molecule as fifth ligand [24,25,[34][35][36]. This species is in equilibrium with the typical pentacoordinate species, which shows the Soret band at 396 nm, indicating that the fifth ligand, in this case, is a histidine residue most likely from proximal site.…”

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.…”