Replacement of the proximal heme thiolate ligand in chloroperoxidase with a histidine residue

Chloroperoxidase is a versatile heme enzyme which can cross over the cataly tic boundaries of other oxidative hemoproteins and perform multiple functio ns. Chloroperoxidase, in addition to catalyzing classical peroxidative reac tions, also acts as a P450 cytochrome and a potent catalase. The multiple f unctions of chloroperoxidase must be derived from its unique active site st ructure. Chloroperoxidase possesses a proximal cysteine thiolate heme iron ligand analogous to the P450 cytochromes; however, unlike the P450 enzymes, chloroperoxidase possesses a very polar environment distal to its heme pro sthetic group and contains a glutamic acid residue in close proximity to th e heme iron. The presence of a thiolate ligand in chloroperoxidase has long been thought to play an essential role in its chlorination and epoxidation activities; however, the research reported in this paper proves that hypot hesis to be invalid. To explore the role of Cys-29, the amino acid residue supplying the thiolate ligand in chloroperoxidase, Cys-29 has been replaced with a histidine residue. Mutant clones of the chloroperoxidase genome hav e been expressed in a Caldariomyces fumago expression system by using gene replacement rather than gene insertion technology. C. fumago produces wild- type chloroperoxidase, thus requiring gene replacement of the wild type by the mutant gene. To the best of our knowledge, this is the first time that gene replacement has been reported for this type of fungus. The recombinant histidine mutants retain most of their chlorination, peroxidation, epoxida tion, and catalase activities. These results downplay the importance of a t hiolate ligand in chloroperoxidase and suggest that the distal environment of the heme active site plays the major role in maintaining the diverse act ivities of this enzyme.