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

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• 作者：John Castillo^a ; Elena Ferapontova^a ; ^{elena.ferapontova@ed.ac.uk} ; Dmitri Hushpulian^b ; Federico Tasca^a ; Vladimir Tishkov^b ; Tatiana Chubar^b ; Irina Gazaryan^b ; Lo Gorton^a
• 关键词：Enzymatic catalysis ; Bioelectrocatalysis ; Recombinant tobacco peroxidase ; Single-point mutation ; Refolding ; Graphite electrodes ; Heterogeneous direct electron transfer
• 刊名：Journal of Electroanalytical Chemistry
• 出版年：2006
• 出版时间：1 March 2006
• 年：2006
• 卷：588
• 期：1
• 页码：112-121
• 全文大小：419 K

文摘

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 ?#xA0;Phe mutant forms of rTOP were produced using an m>Escherichia colim> 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 Fe^3+/2+ transformation of the peroxidase heme was characterised by potentials of ?08 mV (nTOP) and ?39 mV vs. Ag|AgCl (rTOP), and 0.9 ¡À 0.1 and 1.1 ¡À 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 ?#xA0;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.