Comparative studies on chlorite dismutases

Some microorganisms can reduce perchlorate to chlorate and chlorate to chlorite via perchlorate reductase. The key enzyme in these (per)chlorate-reducing bacteria is the heme b oxidoreductase chlorite dismutase (Cld) that finally detoxifies chlorite by its conversion into chloride and dioxygen. Until recently, all isolated Clds were thought to come from facultatively anaerobic heterotrophs from different subclasses of Proteobacteria, but sequence and phylogenetic analyses now demonstrate that many bacterial and archaebacterial genomes seem to encode Cld-like proteins. This now offers the opportunity to perform a comprehensive and comparative biochemical and biophysical investigation of structure-function relationships of these enzymes. Based on our successful recombinant production and elucidation of the X-ray structures of Clds from Nitrospira defluvii and Nitrobacter winogradskyi, that have comparable enzymatic activity but show significant differences in subunit size and oligomerization, we aim at investigation of these two enzymes together with Clds from Bradyrhizobium japonicum and from the cyanobacterium Cyanothece sp. PCC7425. These four oxidoreductases from lineages I & II will be recombinantly produced and compared by using a broad set of methods including UV-Vis, electronic circular dichroism and resonance Raman spectroscopy, multi-mixing stopped-flow spectroscopy, electronic paramagnetic resonance spectroscopy, spectroelectrochemistry, and X-ray crystallography. Together with site-directed mutagenesis these biophysical techniques will help to find a general reaction mechanism of chlorite degradation that might include chlorite-mediated formation of a ferryl-porphyryl radical intermediate that recombines with transiently produced hypochlorite forming a novel O-O-bond and chloride. We intend to understand ligand and substrate access and binding to the distal heme site, the role of the protein environment in redox regulation as well as the actual electronic structure, reactivity and kinetics of interconversion of intermediates and internal electron transfer pathway(s). Moreover, since the selected model proteins differ in subunit size and oligomerization, we want to probe the impact of these structural differences on conformational and thermal stability and enzymatic activity. This project will open new perspectives for future research on biocatalysis of heme enzymes as well as on bioremediation since rising concentrations of harmful anthropogenic chlorite has been detected in groundwater, drinking waters, and soils. The work will be performed in close cooperation with internationally well-known scientists, namely Prof. Smulevich in Florence (resonance Raman spectroscopy), Prof. Battistuzzi in Modena (spectroelectrochemistry) and Prof. Djinovic-Carugo in Vienna (X-ray crystallography).

Some microorganisms can reduce perchlorate to chlorate and chlorate to chlorite via perchlorate reductase. The key enzyme in these (per)chlorate-reducing bacteria is the heme b oxidoreductase chlorite dismutase (Cld) that finally detoxifies chlorite by its conversion into chloride and dioxygen. Until recently, all isolated Clds were thought to come from facultatively anaerobic heterotrophs from different subclasses of Proteobacteria, but sequence and phylogenetic analyses now demonstrate that many bacterial and archaebacterial genomes seem to encode Cld-like proteins, too. Based on our successful recombinant production and elucidation of the X-ray structures of Clds from Nitrospira defluvii, Nitrobacter winogradskyi, and Cyanothece sp. PCC7425 and the chlorite dismutase like protein from Listeria monocytogenes we could investigate these oxidoreductases by using a broad set of biochemical and biophysical methods. In the current project we could figure out the interaction of chlorite with heme enzymes and reveal the biological role, phylogeny and structure of functional chlorite dismutases with differences in overall structure and subunit architecture. With experimental and computational studies on chlorite degradation, we were able to present a general reaction mechanism that includes binding of chlorite to ferric chlorite dismutase, followed by subsequent heterolytic O-Cl bond cleavage leading to the formation of Compound I and hypochlorite, which finally recombine for production of chloride and O2. The role of the Cld-typical distal arginine in catalysis could be also analysed together with the pH dependence of the reaction as the role of transiently produced hypochlorite in irreversible inactivation of the enzyme. Based on our analysis and currently available data, chlorite dismutase is suggested for future application in biotechnology and bioremediation since rising concentrations of harmful anthropogenic chlorite has been detected in groundwater, drinking waters, and soils.