Direct electrochemistry and biolelectrocatalysis of H2O2 reduction of recombinant tobacco peroxidase on graphite. Effect of peroxidase single-point mutation on Ca2+-modulated catalytic activity

Castillo, J; Ferapontova, E; Hushpulian, D; Tasca, F; Tishkov, V; Chubar, T; Gazaryan, I; Gorton, L


Direct electron transfer (DET) reactions and bio(electro)catalytic reduction of H2O2 catalysed by native and recombinant forms of tobacco peroxidase (nTOP and rTOP) were studied in homogeneous-phase catalysis and when TOPs were adsorbed on graphite electrodes. Non-glycosylated wild type and Glu141 -> Phe mutant forms of rTOP were produced using an Escherichia coli expression system. Mutation was introduced to explore the mechanisms for modulation of the catalytic activity of TOP by Ca2+ ions. At the pH optimum of 5.0, direct electrochemical Fe3+/2+ transformation of the peroxidase heme was characterised by potentials of -208 mV (nTOP) and -239 mV vs. Ag vertical bar AgCl (rTOP), and 0.9 +/- 0.1 and 1.1 +/- 0.4 pmoles of adsorbed nTOP and rTOP, correspondingly, were in DET contact with graphite. Kinetic analysis of amperometric (at +50 mV) data on H2O2 reduction at TOP-modified electrodes, placed in a wall-jet flow-through electrochemical cell, yielded 82% (nTOP) and 88% (rTOP) of adsorbed TOP molecules active in the DET reaction. The efficiency of DET (and bioelectrocatalysis) increased 3.5-fold when changing from glycosylated nTOP to rTOP. The Glu141 Phe mutation in the heme-binding pocket of rTOP enabled to achieve a Ca2+-tolerance of TOP in the reaction with H2O2, which is characteristic of other plant peroxidases, and to a large extent in heterogeneous DET and reaction with a second substrate catechol. The results promote further applications of TOP for biosensor- and solid-phase biocatalysts development. (c) 2005 Elsevier B.V. All rights reserved.

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Título según WOS: ID WOS:000235264000013 Not found in local WOS DB
Volumen: 588
Número: 1
Editorial: Elsevier
Fecha de publicación: 2006
Página de inicio: 112
Página final: 121


Notas: ISI