Catalase-peroxidases have a two-domain structure. The N-terminal domain contains the bifunctional active site, but the function of the C-terminal domain is unknown. We produced catalase-peroxidase containing only its N-terminal domain (KatG(Nterm)). Removal of the C-terminal domain did not result in unexpected changes in secondary structure as evaluated by CD, but KatG(Nterm) had neither catalase nor peroxidase activity. Partial recovery of both activities was achieved by incubating KatG(Nterm) with the separately expressed and isolated KatG C-terminal domain. Spectroscopic measurements revealed a shift in heme environment from a mixture of high-spin species (wtKatG) to exclusively hexacoordinate, low-spin (KatG(Nterm)). Moreover, a > 1000-fold lower k(on) for CN- binding was observed for KatG(Nterm). EPR spectra for KatG(Nterm) and the results of site-specific substitution of active site histidines suggested that the distal histidine was the sixth ligand. Thus, one important role for the C-terminal domain may be to support the architecture of the active site, preventing heme ligation by this catalytically essential residue. (C) 2004 Elsevier Inc. All rights reserved.