The present study investigates whether compound I and compound II of manganese peroxidase from the white-rot fungus Phanerochaete chrysosporium utilize the same Mn-binding site for catalysis. Manganese peroxidase was expressed from its cDNA in Escherichia coli and refolded from inclusion bodies to yield fully active enzyme. Three mutants of the enzyme were generated by site-directed mutagenesis. Each of the three amino acid residues proposed to be involved in Mn2+ binding, E35, D179, and E39, was mutated. The acidic side chains of E35 and E39 were shortened by one carbon to the acidic group D, and the acidic side chain of D179 was shortened by one carbon to the alkyl group A. These mutants, E35D, D179A, and E39D, were used to determine whether Mn2+ reacts at the same site with both compound I and compound II of manganese peroxidase and to determine whether phenolic substrates for the enzyme react at this site. Our results conclusively demonstrate that E35 and D179 residues are involved not only in Mn2+ binding but also in electron transfer from Mn2+ to the enzyme for both compound I and compound II. In contrast, E39 is not critically important to either process. None of the three residues is involved in reactions with phenolic substrates or with H2O2.