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Direct and mediated electron transfer catalyzed by anionic tobacco peroxidase
Authors:Florentina-Daniela Munteanu  Lo Gorton  Annika Lindgren  Tautgirdas Ruzgas  Jenny Emnéus  Elisabeth Csöregi  Irina G Gazaryan  Igor V Ouporov  Elena A Mareeva  L Mark Lagrimini
Institution:(1) Department of Analytical Chemistry Chemical Centre, Lund University, POB 124, S-22 100 Lund, Sweden;(2) Department of Biotechnology, Chemical Centre, Lund University, POB 124, S-22 100 Lund, Sweden;(3) Department of Chemical Enzymology, Chemical Faculty, Moscow State University, 119899 GSP Moscow, Russia;(4) Department of Agriculture and Crop Science, Ohio State University, 43210-1096 Columbus, OH
Abstract:The properties of anionic tobacco peroxidase (TOP) adsorbed on graphite electrode have been studied in direct and mediated electron transfer in a wall-jet flow injection system. The percentage of tobacco peroxidase molecules active in directelectron transfer is about 83%, which is higher than that for horeradish peroxidase (40–50%). This observation is explained in terms of the lower degree of glycosylation of TOP compared with horseradish peroxidase and, therefore, a reduced in terference from the oligosaccharide chains with direct electron transfer. Calcium ions cause an 11% drop in the reaction rate constant toward hydrogen peroxide. The detection limit of calcium chloride has been estimated as 5 m M. The results obtained by means of bioeletrochemistry, stopped-flow kinetics, and structural modeling provide evidence for the interaction between calcium cations and negatively charged residues at the distal domain (Glu-141, heme propionates, Asp-79, Asp-80) blocking the activesite. The observation that both soluble and immobilized enzyme under go conformational changes resulting in the blockade of the active site indicates that the immobilized enzyme preserves conformational flexibility. An even stronger suppressing effect of calcium ions on the rate constant for mediated electron transfer was observed. In the case of direct electron transfer, this couldmean that there is nodirect contact between the electrode and the active site of TOP. The electrons are shuttled from the active site to the surface of the electrode through electron transfer pathways in the protein globule that are sensitive to protein conformational changes.
Keywords:Direct electron transfer  mediated electron transfer  orientation effect  conformational changes  model structure  tobacco peroxidase  biosensor  calcium effect  rate constant
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