Chloroperoxidase (CPO) from Caldariomyces fumago is a versatile heme enzyme with great potential for environmental and pharmaceutical applications. It catalyzes a plethora of reactions including halogenation, dismutation, epoxidation, and oxidation. The diverse catalytic capabilities of CPO have long been attributed to the protein’s distinct active site that combines structural features of peroxidases and cytochromes P450. Particularly, the role of the axial thiolate ligand in CPO catalysis has been much debated. Furthermore, no data are available on the role of hydrogen bonding between Arg 26-Asn 37 and Ala 27-Asn 33 of the proximal helix in defining the structural and catalytic properties of CPO. In order to investigate the influence of the proximal thiolate and the proximal hydrogen bond network on the structural and biochemical properties of CPO, several mutant CPOs were constructed and characterized using various spectroscopic techniques and enzymatic assays. Cysteine 29, which coordinates to the heme, was replaced with a His (C29H) to mimic the proximal ligation of classical peroxidases. The UV-Vis spectrum of the carbon monoxide complex of ferrous C29H mutant remained essentially identical to that of wild type (WT) CPO and P450 although the ferric state of the variant enzyme showed a spectral pattern reminiscent of a classical histidine ligated heme peroxidase. Histidine ligation was further confirmed by paramagnetic NMR spectroscopy. Contrary to a previous report, the specific chlorination activity of C29H was essentially abolished (less than 1% of that of WT CPO) but the epoxidation and peroxidation activities were enhanced 10-fold and 55-fold, respectively. These findings demonstrate for the first time that the heme ligand, Cys 29 in CPO, is not a prerequisite for CPO’s unique P450-like spectroscopic signatures but is constitutive for the protein’s versatile catalytic activities. Arginine 26 and Asparagine 33 in the proximal heme pocket were replaced with Ala (R26A, N33A, and R26A/N33A) to disrupt hydrogen bonding. Tertiary structures and heme environments of R26A, N33A, and R26A/N33A differed from those of WT CPO as determined by CD spectroscopy. The specific chlorination and dismutation activities of all mutants were almost abolished but the peroxidation and epoxidation rates were increased. These results show that the proximal hydrogen bond network plays an important role in maintaining the structure and catalytic diversity of CPO.

Chloroperoxidase (CPO) is one of the most versatile heme enzyme isolated from the marine fungus, Caldariomyces fumago. Functionally, CPO can catalyze four types of reactions: peroxidation (peroxidase-like), dismutation (catalase-like), halogenation (halogenase-like), and peroxygenation (P450-like). Structurally, CPO has a distal and proximal pockets that can be best described as a hybrid of classical peroxidase and P450s. As a heme-thiolate protein, CPO contains the conserved proximal Pro28-Cys29-Pro30 stretch found in other members of the family. However, the structural and functional roles of these proline residues remain poorly understood. Site-directed mutagenesis was undertaken to generates three CPO mutants, P28A-, P30A-, P28A/P30A-CPO. The replacement of the rigid proline with a more flexible alanine residue, freed up the back bone amide for the formation of additional amide-sulfur hydrogen bond, allowing the investigation of the importance of these residues in CPO catalysis. The three CPO mutants displayed dramatic difference in ligand binding affinity and catalytic activities relative to WT-CPO. Any mutations on the proline resides within the proximal loop eliminated the halogenation and dismutation activities but enhanced the vii epoxidation and peroxidation activities by 4-14 fold. As the binding affinity for cyanide, the CPO mutants displayed significantly higher dissociation constant relative to WT-CPO. Our results revealed that Pro28 and Pro30 play important roles in maintaining the versatility of CPO. As a versatile enzyme, CPO has great application potential in pharmaceutical and chemical industry due to its ability to catalyze the formation of chiral epoxides. Phe103 and Phe186 located on the distal pocket have been proposed to guard the access of substrates to the ferryl oxygen of the heme center. The interactions of these two phenylalanine residues restricted the size of substrates and regulates CPO’s enantioselectivity. F186A- and F103A/F186A-CPO were generated and characterized where the rate of peroxidation and epoxidation were significantly enhanced at the expense of halogenation and dismutation activities. Our results demonstrated that Phe186 played a subtler role relative to Phe103 in terms of substrate specificity and product enantioselectivity of CPO.