Structural and mechanistic studies on a dimeric chlorite dismutase

Rising concentrations of chlorite (ClO2-) have been detected in groundwater, drinking waters, and soils. Due to its oxidative nature, chlorite reacts easily with organic material and thus has toxic effects on living cells. Numerous bacterial phyla have evolved an enzyme called chlorite dismutase, which efficiently decomposes chlorite into harmless chloride (Cl-) and dioxygen (O2). Thereby, a covalent oxygen-oxygen bond is formed, a unique biochemical reaction so far found only in phototrophic organisms like plants. Due to its enzymatic properties chlorite dismutase could be used for bioremediation of chlorite but also as dioxygen generator in future biotechnological applications. For this reason it is very important to understand the relation between structure and function of this enzyme. In this project we want to understand how this enzyme works and degrades chlorite. Based on the successful recombinant production and elucidation of the structure of this enzyme at atomic resolution, we aim at understanding all individual steps and intermediates during enzyme turnover. The mechanism of cleavage of chlorite and O2 formation is still under heavy debate. So far it is not clear whether chlorine monoxide or hypochlorite is transiently produced or why and how the enzyme is irreversibly inhibited at alkaline pH values. Such a project in the field of molecular enzymology needs the comprehensive application of a broad set of biochemical and biophysical methods: (i) the recombinant production and purification of the wild-type protein and selected single mutants; (ii) characterization of these iron-proteins by various spectroscopic and electrochemical techniques, which provide information of the active site structure in solution; (iii) analysis of individual reaction steps by stopped-flow spectroscopy and freeze-quench electron paramagnetic resonance spectroscopy, which gives information about the electronic structure of the redox intermediates relevant for catalysis; and (iv) elucidation of X-ray and neutron crystal structures. The work will be performed in close cooperation with internationally well-known scientists from Austria, Italy, Belgium and USA. The established knowledge of structure and function of chlorite dismutase will provide the basis for a better understanding of the functional and physiological role of chlorite dismutases in prokaryotic organisms but also for its application in the field of chemical engineering and bioremediation.

Chlorite contamination of drinking water, groundwater and soils is becoming an ever-increasing environmental problem because chlorite is toxic to living cells due to its oxidative properties. Interestingly, however, many species of bacteria can degrade chlorite into harmless chloride and oxygen with the help of an enzyme called chlorite dismutase. In the process, an O-O bond is formed, a reaction previously observed only in phototrophic and oxygen-releasing organisms (e.g., plants). Due to these unique properties, chlorite dismutase could be used in the future in the degradation of chlorite in drinking water or as a generator of oxygen in a wide variety of biotechnological applications. However, this required a better understanding of the structure-function relationships in this biological catalyst. This was realized in this project. Therefore, the aim of this project was to find out the exact mechanism of chlorite degradation by chlorite dismutase. To this end, the iron enzyme was recombinantly produced functionally in large quantities and the three-dimensional structure of the wild-type protein and many mutants was elucidated at atomic resolution. Fourteen X-ray structures of chlorite dismutases with and without bound ligands could be elucidated. Furthermore, this project clarified the role of the dynamics of the catalytic arginine on the distal hydrophobic heme side in the individual reaction steps. In addition, the electronic and spectroscopic properties of the redox intermediates involved were elucidated by the multimixing UV-vis-stopped-flow technique and by means of electron spin resonance spectroscopy, and a reaction mechanism was proposed that also involves the irreversible inactivation of the enzyme observed at higher pH values. It was shown that the stability of the key redox intermediate Compound I decreases with increasing pH and that the addition of single electron donors such as serotonin can partially stop the inhibition. This work was carried out in close collaboration with nationally and internationally recognized specialists from Austria and Belgium. The knowledge gained about the structure and function of chlorite dismutase provides the basis both for understanding the physiological role of these enzymes in bacteria and for potential biotechnological applications such as the decontamination of contaminated drinking water.