Structural and functional characterisation of the chlorite dismutase from the nitrite-oxidizing bacterium "Candidatus Nitrospira defluvii": identification of a catalytically important amino acid residue.
Chlorite dismutase (Cld) is a unique heme enzyme which transforms chlorite to chloride and molecular oxygen (reaction: ClO(2)(-)→Cl(-)+O(2)). Since bacteria with Cld play significant roles in the bioremediation of industrially contaminated sites and also in wastewater treatment, it is of high interest to understand the molecular mechanism of chlorite detoxification. Here we investigate a highly active Cld from Candidatus Nitrospira defluvii (NdCld), a key nitrifier in biological wastewater treatment, using a comprehensive structural, biochemical and bioinformatics approach. We determined the crystal structure of Cld from Candidatus Nitrospira defluvii and showed that functional NdCld is a homopentamer possessing a fold found in other Clds and Cld-like enzymes. To investigate the Cld function in more detail, site-directed mutagenesis of a catalytically important residue (Arg173) was performed and two enzyme mutants were structurally and biochemically characterized. Arginine 173 is demonstrated to play a key role in (i) controlling of ligand and substrate access and binding and (ii) in chlorite dismutation reaction. The flexible residue modulates the electrostatic potential and size of the active site entrance and might be involved in keeping transiently formed hypochlorite in place for final molecular oxygen and chloride formation. Furthermore, using a structure-based sequence alignment, we show that the residue corresponding to Arg173 is conserved in all known active forms of Cld and propose it as a marker for Cld activity in yet uncharacterized Cld-like proteins. Finally, our analysis indicates that all Clds and Cld-like enzymes employ a non-covalently bound heme as a cofactor.