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Issue 22, 2010
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Cytochrome c covalently immobilized on mesoporous silicas as a peroxidase: Orientation effect

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Abstract

Cytochrome c (cyt c), a heme protein with positive electric charge and global dimensions of 2.5 × 2.5 × 3.7 nm, is immobilized by covalent bonding in the nanochannels and on the surface of IBN4 (pore size = 5.3–7.1 nm) mesoporous silicas. The composite material behaves as a peroxidase in the oxidation of organic molecules in the presence of hydrogen peroxide. The surface of IBN4 was first modified with three different linkers, glutaric anhydride (GAC), glutaraldehyde (GAH) and succinimido-3-maleimidopropanoate (SMP), to facilitate the binding (bioconjugation) with cyt c. Different linkers expose the catalytic active site (Fe-heme) to different environments, which allows us to examine the orientation effect imposed by the binding linker. Molecular modeling further allows us to assess the orientation effect on the catalytic activity arising from the distribution of electric charges of cyt c immobilized in different surface-modified nanochannels. The accessibility of the Fe active center in immobilized cyt c is found to be in the following order: IBN4-N-SMP-cyt c > IBN4-N-GAH-cyt c > IBN4-N-GAC-cyt c, which is correlated to the measured trend of the initial specific peroxidase-like activities of immobilized cyt c in three different modified surfaces towards the oxidation of 4-aminoantipyrine. The surface-modified nanochannels of mesoporous silica provide the confining spaces that could prevent cyt c from protein unfolding and orient the active site in a favorable location in the pores to facilitate its activity. However, there is much more structural decay after hydrothermal treatment and the activities diminish accordingly: the IBN4-N-GAH-cyt c sample lost most of its activity, the IBN4-N-SMP-cyt c lost its activity due to less protection of the active center of Fe-heme, and the IBN4-N-GAC-cyt c retained good activity. These temperature effects are further confirmed in the UV-Vis spectra and EPR studies. Cyt c immobilized on functionalized IBN4 surfaces exists in high spin state, as inferred from EPR and UV-Vis studies, which differs from the primarily low spin state of native cyt c. The high spin state arises from the replacement of Met-80 ligands of heme Fe(III) by water or silanol groups on the silica surface, which could open up the heme groove for easy access of oxidants to the iron center and facilitate the catalytic activity. Finally, we apply the covalently immobilized cyt c in the oxidation of a representative polycyclic aromatic hydrocarbon (PAH)—pyrene. The trend in activity can be understood from the design principle we learned in this work.

Graphical abstract: Cytochrome c covalently immobilized on mesoporous silicas as a peroxidase: Orientation effect

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Supplementary files

Article information


Submitted
02 Dec 2009
Accepted
01 Mar 2010
First published
12 Apr 2010

J. Mater. Chem., 2010,20, 4653-4662
Article type
Paper

Cytochrome c covalently immobilized on mesoporous silicas as a peroxidase: Orientation effect

K. Kao, C. Lee, T. Lin and C. Mou, J. Mater. Chem., 2010, 20, 4653
DOI: 10.1039/B925331A

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