The Influence of the Proximal Thiolate Ligand and Hydrogen Bond Network of the Proximal Helix on the Structural and Biochemical Properties of Chloroperoxidase

• Main Author: Shersher, Elena
• Format: Others
• Published: FIU Digital Commons 2016
• Subjects: Chloroperoxidase; Peroxygenase; Heme Protein; Catalysis; Proximal Thiolate; Hydrogen Bond Network; Biochemistry; Biotechnology
• Online Access: http://digitalcommons.fiu.edu/etd/2483; http://digitalcommons.fiu.edu/cgi/viewcontent.cgi?article=3643&context=etd

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.