Structural and mechanistic studies on a dimeric chlorite dismutase
Abstract
Chlorite dismutases (Clds) are able to efficiently decompose chlorite (ClO2-) into harmless chloride (Cl-) and dioxygen (O2) with chlorite being the sole source of dioxygen. Thereby, a covalent oxygen-oxygen bond is formed, a unique biochemical reaction that in addition is only catalyzed by the water-splitting manganese complex of photosystem II of oxygenic phototrophic organisms. The mechanism of cleavage of chlorite is still under heavy debate. Computational studies suggest homolytic cleavage, thereby producing chlorine monoxide (ClO●) and Compound II [Por…Fe(IV)=O] followed by a rebound step and release of chloride and dioxygen, whereas biochemical studies on pentameric (clade 1) Clds suggest heterolytic cleavage of chlorite thereby forming Compound I [Por+●…Fe(IV)=O] and hypochlorite (HOCl/-OCl). However, there is no experimental proof for the generation of Compound I nor is it known why these oxidoreductases have their pH optimum around pH 5 and are inactivated at alkaline pH. In order to elucidate structure-function relationships in Cld and understand the molecular basis of chlorite degradation, we have selected the dimeric Cld from Cyanothece sp. PCC7425 (CCld) as model enzyme for several reasons. CCld can easily be produced in E. coli, allows the generation of crystals of optimum size at all relevant pH values to be probed by both X-ray and neutron crystallography and for the first time showed distinct spectral features of typical heme b Compound II, which was formed immediately after mixing the ferric enzyme with chlorite. Moreover, CCld exhibits further enzymatic features that contradict the proposed heterolytic but favour the homolytic cleavage mechanism of chlorite. Thus it is the aim of this project to fully clarify the molecular mechanism of chlorite cleavage and O2 formation by using 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 (resonance Raman, UV-vis) and electrochemical techniques, (iii) analysis of all individual reaction steps of enzyme cycle by multi-mixing stopped-flow spectroscopy and freeze-quench electron paramagnetic resonance (EPR) spectroscopy; and (iv) elucidation of X-ray and neutron crystal structures in the pH range 5-10. The work will be performed in close cooperation with internationally well-known scientists from Austria (X-ray crystallography: Kristina Djinovic-Carugo), Italy (RR spectroscopy: Giulietta Smulevich), Belgium (EPR spectroscopy: Sabine Van Doorslaer) and USA (neutron crystallography: Leighton Coates). Understanding structure-function relationships of Cld will provide the basis for its application in chemical engineering and bioremediation.
molecular enzymology biomolecular spectroscopy heme protein chlorite dismutase structural biology
Publikationen
Crystal and Electronic Structure of Compound I in Dye-decolorizing Peroxidases
Autoren: Pfanzagl, V; Hofbauer, S; Beale, J; Mlynek, G; Michlits, H; Nys, K; Van Doorslaer, S; Djinovic-Carugo, K; Furtmüller, PG; Obinger, C Jahr: 2018
Conference & Workshop proceedings, paper, abstract
Kinetic and spectroscopic characterization of EfeB
Autoren: Michlits, H., Pfanzagl, V., Schwaiger, L., Furtmüller, P. G., Hofbauer, S., Obinger, C. Jahr: 2018
Conference & Workshop proceedings, paper, abstract
Arresting the Catalytic Arginine in Chlorite Dismutases: Impact on Heme Coordination, Thermal Stability, and Catalysis.
Autoren: Schmidt, D; Serra, I; Mlynek, G; Pfanzagl, V; Hofbauer, S; Furtmüller, PG; Djinović-Carugo, K; Van Doorslaer, S; Obinger, C; Jahr: 2021
Journal articles
Impact of the dynamics of the catalytic arginine on nitrite and chlorite binding by dimeric chlorite dismutase.
Autoren: Serra, I; Schmidt, D; Pfanzagl, V; Mlynek, G; Hofbauer, S; Djinović-Carugo, K; Furtmüller, PG; García-Rubio, I; Van Doorslaer, S; Obinger, C; Jahr: 2022
Journal articles
Project staff
Christian Obinger
Univ.Prof. Mag.rer.nat. Dr.rer.nat. Christian Obinger
christian.obinger@boku.ac.at
Tel: +43 1 47654-10011, 77273
Project Leader
01.01.2018 - 31.12.2020