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

Abstract

Chloroperoxidase is a versatile heme enzyme which can cross over the catalytic boundaries of other oxidative hemoproteins and perform multiple functions. Chloroperoxidase, in addition to catalyzing classical peroxidative reactions, also acts as a P450 cytochrome and a potent catalase. The multiple functions of chloroperoxidase must be derived from its unique active site structure. 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 prosthetic group and contains a glutamic acid residue in close proximity to the 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 hypothesis 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 have 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, epoxidation, and catalase activities. These results downplay the importance of a thiolate ligand in chloroperoxidase and suggest that the distal environment of the heme active site plays the major role in maintaining the diverse activities of this enzyme.

Monooxygenation of an Aromatic Ring by F43W/H64D/V68I Myoglobin Mutant and Hydrogen Peroxide

“…Recently it has been found that these mechanisms may not be entirely valid, and the actual mechanism may involve two electrophilic oxidants and/or two spin states of the iron oxo species (6,8,14). Although P450cam has been shown to lose its hydroxylation activity when an imidizole is substituted for the axial thiolate ligand (15,16), in contrast, chloroperoxidase retains its chlorination, peroxidation, epoxidation, and catalase activities when its thiolate ligand is mutated to histidine (17). Also, studies with iron porphyrin compounds have demonstrated that imidazole ligation can replace the thiolate in P450 type reactions under certain conditions (18).…”

A Lifetime of Playing with Enzymes

“…Our similar studies with CPO mutants indicated that the thiolate ligand (Cys 29 ) could be replaced with a histidine residue and retain most of the chlorination, peroxidation, epoxidation, and catalase activities (68). C. fumago, the fungus that produces wild-type CPO, was used for the expression of the Cys-to-His mutant.…”

Tailoring Heme-Thiolate Proteins into Efficient Biocatalysts with High Specificity and Selectivity

“…However, in contrast to the results of P450cam C357H, the thiolate coordination in chloroperoxidase is suggested to be not as important as in P450. After substituting the proximal thiolate ligand (Cys 29) with a histidine, the CPO C29H was found to retain most (around 80%) of its chlorination, peroxidation, epoxidation and catalase activities compared to those of the wild type protein (114). These are surprising observations in consideration of many suggestions for the significance of the proximal thiolate ligand in P450.…”

“…Glu183 then protonates the distal oxygen of Cpd 0 and the peroxide O-O is cleaved heterolytically to generate Cpd I and release a water molecule, similar to other peroxidase mechanisms (107,108). CPO is adept in catalyzing a number of chiral oxidations with high yields and high enantioselectivity, in addition to alkyne hydroxylation and heteroatom dealkylation (109)(110)(111)(112)(113)(114). The stereoselective ability of CPO in catalysis is suggested to be based on its unique active site structure and unique catalytic mechanism, which make it the promising candidate for industrial catalysts.…”