CONSTRUCTION OF A BISAQUO HEME ENZYME AND BINDING BY EXOGENOUS LIGANDS

The crystal structure of the His-175 --> Gly (H175G) mutant of cytochr ome-c peroxidase (EC 1.11.1.5), missing its only heme ligand, reveals that the histidine is replaced by solvent to give a bisaquo heme prote in. This protein retains some residual activity, which can be stimulat ed or inhibited by addition of exogenous ligands. Structural analysis confirms the binding of imidazole to the heme at the position of the w ild-type histidine ligand. This imidazole complex reacts readily with hydrogen peroxide to produce a radical species with novel properties. However, reactivation in this complex is incomplete (approximate to 5 %), which, in view of the very similar structures of the wild-type and the H175G/imidazole forms, implies a critical role for tethering of t he axial ligand in catalysis. This study demonstrates the feasibility of constructing heme enzymes with no covalent link to the protein and with unnatural Ligand replacements. Such enzymes may prove useful in s tudies of electron transfer mechanisms and in the engineering of novel heme-based catalysts.