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 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 peroxidase and, therefore, a reduced interference 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 mM. The results obtained by means of bioelectrochemistry, 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 active site. The observation that both soluble and immobilized enzyme undergo 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. Ln the case of direct electron transfer, this could mean that there is no direct 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.