pH-Dependent Peroxidase Activity of Yeast Cytochrome c and Its Triple Mutant Adsorbed on Kaolinite

Abstract

The peroxidase activity of wild-type yeast cytochrome c and its triple mutant K72AK73AK79A adsorbed onto kaolinite was investigated as a function of pH and temperature. Both adsorbed proteins displayed an appreciable catalytic activity, which remained constant from pH 7 to pH 10, decreased below pH 7, and showed a remarkable increase at pH values lower than 4. In the whole pH range investigated the catalytic activity of the adsorbed wild-type cytochrome c was higher than that of the mutant. Both diffuse-reflectance UV-vis and resonance Raman spectroscopies applied on solid samples were used to probe the structural features responsible for the catalytic activity of the immobilized proteins. At neutral and alkaline pH values a six-coordinate low-spin form of cytochrome c was observed, while at pH < 7 the formation of a high-spin species occurred whose population increased at decreasing pH. The orientation and exposure of the heme to the substrate-strictly dependent on adsorption-was found to affect the peroxidase activity.

Enhancing Biocatalysis: The Case of Unfolded Cytochrome c Immobilized on Kaolinite

“…These results suggest that the large differences in the PA observed between native and urea‐unfolded cytc (see below) are due to conformational changes in the catalytic pocket, which, however, do not alter the molar ratio of the low‐ and high‐spin forms on the Nak surface. Notably, at high pH values, native cytc is imparted with PA, although no high‐spin forms are present 6. Cytc does not desorb from Nak during catalysis, as indicated by the invariance of the spectra recorded before and after the oxidation of Gc (data not shown).…”

“…Cytc is able to strongly and quantitatively adsorb onto the surface of Nak through electrostatic interactions 13. This bioinorganic interface was found to exhibit appreciable catalytic activity in the oxidation of guaiacol (Gc) to tetraguaiacol (tetraGc) in the presence of H 2 O 2 6. The aim of this paper is to find conditions under which the catalytic properties are amplified and improved.…”

“…[b] Ref. 6. [c] Error in V max and K M is 5 and 8 %, respectively; error in V 0 and PA is 5 %; error in V max / K M is 12 %.…”

“…The diffuse‐reflectance UV‐Vis spectra (DR UV‐Vis) recorded at pH 7 (Supporting Information S5) in the presence and in the absence of 7 M urea [Figures 2 and S6 (Supporting Information), respectively] are very similar and consist of three bands at approximately 410, 530, and 628 nm, plus a shoulder at 558 nm. The band at 628 nm indicates the presence of a high‐spin ferric heme 6…”

Surface-enhanced infrared absorption spectroscopy and electrochemistry reveal the impact of nanoparticles on the function of protein immobilized on mimic biointerface

“…7b), which further induces the conformational changes of the β-sheet and the changes of its microenvironment indicated by the shift of band frequency. Such changes could also affect the microenvironment of heme since these residues are connected with heme propionate, which might facilitate the coordinate of H2O2, resulting in the enhanced electrocatalytic activity of adsorbed cyt c towards H2O2 reduction [42][43][44]. Moreover, such interaction could slightly change the adsorption orientation of adsorbed cyt c with heme more perpendicular to the surface as shown in Fig.…”