Molecular Mechanism of Enzymatic Chlorite Detoxification: Insights from Structural and Kinetic Studies.

Irene Schaffner,Marzia Bellei,Christian Obinger,Kristina Djinović-Carugo,Dominic Pühringer,Stefan Hofbauer,Julian Libiseller-Egger,Leighton Coates,Giulietta Smulevich,Nicola Flego,Gianantonio Battistuzzi,Paul G Furtmüller,Georg Mlynek

doi:10.1021/acscatal.7b01749

Abstract

The heme enzyme chlorite dismutase (Cld) catalyzes the degradation of chlorite to chloride and dioxygen. Although structure and steady-state kinetics of Clds have been elucidated, many questions remain (e.g., the mechanism of chlorite cleavage and the pH dependence of the reaction). Here, we present high-resolution X-ray crystal structures of a dimeric Cld at pH 6.5 and 8.5, its fluoride and isothiocyanate complexes and the neutron structure at pH 9.0 together with the pH dependence of the Fe(III)/Fe(II) couple, and the UV–vis and resonance Raman spectral features. We demonstrate that the distal Arg127 cannot act as proton acceptor and is fully ionized even at pH 9.0 ruling out its proposed role in dictating the pH dependence of chlorite degradation. Stopped-flow studies show that (i) Compound I and hypochlorite do not recombine and (ii) Compound II is the immediately formed redox intermediate that dominates during turnover. Homolytic cleavage of chlorite is proposed.

Highlights

Chlorite dismutases (Clds) are heme b-containing oxidoreductases (EC 1.13.11.49) found in prokaryotic organisms.[1]
PCC7425 is a dimeric representative of clade 2 functional Clds and represents the first protein of this clade which has been comprehensively characterized regarding structure−function relationships
Similar to its characterized homologues (e.g., NdCld, DaCld, Cld from GR-1, KpCld), it is capable of efficiently converting chlorite into chloride and molecular oxygen, the reaction being strongly dictated by pH

Summary

INTRODUCTION

Chlorite dismutases (Clds) are heme b-containing oxidoreductases (EC 1.13.11.49) found in prokaryotic organisms.[1]. Computational studies suggest homolytic cleavage of OClO− thereby producing chlorine monoxide (ClO) and Compound II [Por···Fe(IV) O], followed by a rebinding step and production of Fe(III)−peroxyhypochlorite that releases chloride and dioxygen (Reactions I and II).[7] Binding of the anionic substrate chlorite (pKa = 1.728) to ferric Cld is exergonic[7] and independent of the Cld-typical arginine.[9]. Our data demonstrate that the distal Arg is protonated at pH 9.0 and indicate that ClO is the product of chlorite cleavage, at least in the O2 generative part of the reaction This contradicts the hypothesis that the protonation state of the catalytic arginine modulates chlorite degradation. On the basis of our data, we discuss the postulated mechanism(s) of chlorite conversion and provide a more detailed view of the mode of action including potential side reactions catalyzed by the enzyme

RESULTS

DISCUSSION

Impact of Ligand Binding on the Structure of the

■ ACKNOWLEDGMENTS

■ REFERENCES