Direct and mediated electron transfer catalyzed by anionic tobacco peroxidase - Effect of calcium ions

The properties of anionic tobacco peroxidase (TOP) adsorbed on graphite ele ctrode have been studied in direct and mediated electron transfer in a wall -jet flow injection system. The percentage of tobacco peroxidase molecules active in direct electron transfer is about 83%, which is higher than that for horseradish peroxidase (40-50%). This observation is explained in terms of the lower degree of glycosylation of TOP compared with horseradish pero xidase and, therefore, a reduced interference from the oligosaccharide chai ns with direct electron transfer. Calcium ions cause an 11% drop in the rea ction rate constant toward hydrogen peroxide. The detection limit of calciu m chloride has been estimated as 5 mM. The results obtained by means of bio electrochemistry, stopped-flow kinetics, and structural modeling provide ev idence for the interaction between calcium cations and negatively charged r esidues at the distal domain (Glu-141, heme propionates, Asp-79, Asp-80) bl ocking the active site. The observation that both soluble and immobilized e nzyme undergo conformational changes resulting in the blockade of the activ e site indicates that the immobilized enzyme preserves conformational flexi bility. An even stronger suppressing effect of calcium ions on the rate con stant for mediated electron transfer was observed. Ln the case of direct el ectron transfer, this could mean that there is no direct contact between th e electrode and the active site of TOP. The electrons are shuttled from the active site to the surface of the electrode through electron transfer path ways in the protein globule that are sensitive to protein conformational ch anges.