The crystal structure of the major peroxidase of barley grain (BP 1) has been solved by molecular replacement and phase combination and refined to an R-factor of 19.2% for all data between 38 and 1.9 Å. The refined model includes amino acid residues 1-309, one calcium ion, one sodium ion, iron-protoporphyrin IX, and 146 solvent molecules. BP 1 has the apparently unique property of being unable to catalyze the reaction with the primary substrate hydrogen peroxide to form compound I at pH values > 5, a feature investigated by obtaining crystal structure data at pH 5.5, 7.5, and 8.5. Structural comparison shows that the overall fold of inactive barley grain peroxidase at these pH values resembles that of both horseradish peroxidase C and peanut peroxidase. The key differences between the structures of active horseradish peroxidase C and inactive BP 1 include the orientation of the catalytic distal histidine, disruption of a hydrogen bond between this histidine and a conserved asparagine, and apparent substitution of calcium at the distal cation binding site with sodium at pH 7.5. These profound changes are a result of a dramatic structural rearrangement to the loop region between helices B and C. This is the first time that structural rearrangements linked to active site chemistry have been observed by crystallography in the peroxidase domain distal to heme.All living organisms utilize peroxidases in a variety of biosynthetic or degradable processes and in defense against pathogens or oxidative pressure. The majority of known peroxidases belong to the plant peroxidase superfamily, which is characterized by a central heme group sandwiched between a distal and a proximal protein domain. The plant peroxidase superfamily is subdivided into three classes based on structural divergence (1). Class I constitutes intracellular peroxidases of prokaryotic origin, and class I peroxidases are found in yeast (cytochrome c peroxidase; CCP), 1 plants (pea cytosolic ascorbate peroxidase), and bacteria. Class II and III peroxidases are found in fungi and plants, respectively, and are largely extracellular. Both class II and class III peroxidases contain two conserved calcium ions, one in each of the distal and proximal domains. Class II includes the lignin-degrading, manganese-dependent (MnP), and Coprinus peroxidases (synonymous with Arthromyces ramosus peroxidase). Class III includes the classic horseradish peroxidase (HRP C); peanut peroxidase (PNP), for which the first crystal structure of a class III peroxidase was solved (9); and the major peroxidase from barley grain (BP 1).In common with other heme-containing peroxidases, BP 1 catalyzes the following multistep reaction.Still, with its unique pH and calcium dependent activity profile (2, 3), BP 1 is the most interesting and atypical of the class III peroxidases. In the presence of calcium and following a pHinduced conformational change with a pK a Ͻ 3.7, BP 1 has a rate constant for compound I formation comparable with the rate constant for HRP C. The similarities between BP ...