Unraveling the function in chlorite dismutases and related peroxidases by EPR

The heme b containing enzymes chlorite dismutase and dye-decolorizing peroxidase belong to the same protein superfamily. Despite their high structural similarity, their enzymatic activity is very different. Chlorite dismutase is capable of degrading the strong and toxic oxidant chlorite to chloride and dioxygen. In contrast, dye-decolorizing peroxidases are able to degrade a large variety of molecules, including textile dyes and lignin. This project aims at understanding which local structural elements in the heme environment govern the observed high variations in enzymatic functions. Two representative chlorite dismutases and two dye- decolorizing peroxidases will be recombinantly produced in E. coli and investigated by a broad set of biochemical and biophysical techniques available in the Belgian and Austrian laboratories. Importantly, advanced electron paramagnetic resonance spectroscopy in combination with protein engineering will be used as major tools to determine the local geometric and electronic structure of the heme site during the different steps in protein turnover. The same biophysical spectroscopic method will also be used to investigate the interaction of the enzymes with different heme-ligating molecules, such as substrates, ligands and inhibitors. Insight into the mechanistic working of the enzymes is essential in view of their potential technological applications, such as in bioremediation of the environmental pollutant chlorite or degradation of lignin in the transformation of biomass to biofuel.

Heme enzymes are found in all kingdoms of life. They catalyse a broad array of reactions and include oxygen transport proteins like haemoglobin, electron transfer proteins like cytochromes, catalases, peroxidases, monooxygenases, biosensors and many more. In the recent years many new so far unknown heme enzymes have been detected including chlorite dismutases (Clds) and dye-decolorizing peroxidases (DyPs) which have been investigated in this project. Bacterial Clds and DyPs share the same overall structure and active site architecture but catalyse completely different reactions. Both enzyme families contain heme b as redox active prosthetic group in their active site which is coordinated by a proximal histidine. In this project we elucidated the role of the catalytically relevant redox intermediates (i.e. Compound I and Compound II) of these iron proteins and of the conserved distal amino acids that participate in the enzymatic reactions. We could demonstrate the impact of distinct residues on conversion of the environmental pollutant chlorite to chloride and dioxygen in Clds as well as on peroxidase activity in case of DyPs. We succeeded in unravelling (i) the crystal structures of wild-type and mutant Clds and DyPs, (ii) the electronic configuration of the redox intermediates by electron paramagnetic resonance (EPR) spectroscopy, and, finally, (iii) the reaction mechanism of degradation of toxic chlorite to harmless chloride and dioxygen as well as of Compound I formation in DyPs. Further obtained data on conformational and thermal stability, pH dependence of enzymatic activity and self-inhibition provide additional important information for using engineered Clds in bioremediation, e.g. future application in purification of polluted drinking water or generation of dioxygen. The studies on DyPs showed that this novel peroxidase family exhibits a broad variability in catalysis. We demonstrate that class B DyPs do not to act as classical heme peroxidases because of extremely poor reactivity of the redox intermediate Compound I and suggest a role in redox- based controlling of distinct membrane channels in bacteria. The project benefited from the close cooperation and complementary expertise between the Austrian group and the Belgian partner, which is an expert in EPR spectroscopy.