Chloroperoxidase (CPO) from <i>Caldariomyces fumago</i> is a versatile heme enzyme with great potential for environmental and pharmaceutical applications. It catalyzes a plethora of reactions including halogenation, dismutation, epoxidation, and peroxidation. The diverse catalytic activities of CPO have long been attributed to the protein’s distinct active site structure, particularly the axial thiolate ligand. However, it is still poorly understood how CPO is able to perform such a broad range of activities, and no consensus has been achieved on the role of the thiolate ligand in CPO catalysis. To examine the role of the proximal thiolate ligand in defining the structural, physical, and biochemical properties of CPO, the Cys29 that coordinates to the heme has been replaced with a His using site-directed mutagenesis. To our surprise, the UV-Vis spectrum of the carbon monoxide complex of the ferrous C29H mutant remained essentially identical to that of wild type 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 is further confirmed by paramagnetic NMR spectroscopy. It is noted that the oxidative stability of the mutant protein is dramatically improved due to its remarkably decreased catalase activity. Contrary to a previous report, the specific chlorination activity of C29H is almost abolished but the epoxidation and peroxidation activities are significantly enhanced, especially in acidic conditions. These results demonstrate for the first time that the heme ligand, C29 in CPO is not a prerequisite for its unique P450-like spectroscopic signatures but is constitutive for the protein’s versatile catalytic activities.

Role of proximal thiolate ligand in chloroperoxidase catalysis

249th National Meeting (2015)

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