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

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

doi:10.1021/acscatal.7b01749

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

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

STS Project Technical Systems

Research output: Contribution to journal › Article › peer-review

28 Scopus citations

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.

Original language	English
Pages (from-to)	7962-7976
Number of pages	15
Journal	ACS Catalysis
Volume	7
Issue number	11
DOIs	https://doi.org/10.1021/acscatal.7b01749
State	Published - Nov 3 2017

Funding

This project was supported by the Austrian Science Fund, FWF [Doctoral program BioToPMolecular Technology of Proteins (W1224) and the projects P25270 and P22276] and the Federal Ministry of Economy, Family and Youth through the initiative “Laura Bassi Centres of Expertise”, funding the Center of Optimized Structural Studies, No. 253275. Research at the Spallation Neutron Source (SNS) at ORNL was sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy. We thank Elisabeth Lobner, Christa Jakopitsch, and Rupert Tscheließnig for lab assistance, discussions and assistance in data analysis.

Funders	Funder number
Office of Basic Energy Sciences
Scientific User Facilities Division
U.S. Department of Energy
Austrian Federal Ministry of Economy, Family and Youth	253275
Austrian Science Fund	P25270, P22276, W1224

Keywords

O generation
X-ray diffraction
chlorite dismutase
heme enzyme
neutron diffraction
resonance Raman spectroscopy
spectroelectrochemistry
stopped-flow spectroscopy

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10.1021/acscatal.7b01749

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Schaffner, I., Mlynek, G., Flego, N., Pühringer, D., Libiseller-Egger, J., Coates, L., Hofbauer, S., Bellei, M., Furtmüller, P. G., Battistuzzi, G., Smulevich, G., Djinović-Carugo, K., & Obinger, C. (2017). Molecular Mechanism of Enzymatic Chlorite Detoxification: Insights from Structural and Kinetic Studies. ACS Catalysis, 7(11), 7962-7976. https://doi.org/10.1021/acscatal.7b01749

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title = "Molecular Mechanism of Enzymatic Chlorite Detoxification: Insights from Structural and Kinetic Studies",

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.",

keywords = "O generation, X-ray diffraction, chlorite dismutase, heme enzyme, neutron diffraction, resonance Raman spectroscopy, spectroelectrochemistry, stopped-flow spectroscopy",

author = "Irene Schaffner and Georg Mlynek and Nicola Flego and Dominic P{\"u}hringer and Julian Libiseller-Egger and Leighton Coates and Stefan Hofbauer and Marzia Bellei and Furtm{\"u}ller, {Paul G.} and Gianantonio Battistuzzi and Giulietta Smulevich and Kristina Djinovi{\'c}-Carugo and Christian Obinger",

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doi = "10.1021/acscatal.7b01749",

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Schaffner, I, Mlynek, G, Flego, N, Pühringer, D, Libiseller-Egger, J, Coates, L, Hofbauer, S, Bellei, M, Furtmüller, PG, Battistuzzi, G, Smulevich, G, Djinović-Carugo, K & Obinger, C 2017, 'Molecular Mechanism of Enzymatic Chlorite Detoxification: Insights from Structural and Kinetic Studies', ACS Catalysis, vol. 7, no. 11, pp. 7962-7976. https://doi.org/10.1021/acscatal.7b01749

TY - JOUR

T1 - Molecular Mechanism of Enzymatic Chlorite Detoxification

T2 - Insights from Structural and Kinetic Studies

AU - Schaffner, Irene

AU - Mlynek, Georg

AU - Flego, Nicola

AU - Pühringer, Dominic

AU - Libiseller-Egger, Julian

AU - Coates, Leighton

AU - Hofbauer, Stefan

AU - Bellei, Marzia

AU - Furtmüller, Paul G.

AU - Battistuzzi, Gianantonio

AU - Smulevich, Giulietta

AU - Djinović-Carugo, Kristina

AU - Obinger, Christian

PY - 2017/11/3

Y1 - 2017/11/3

N2 - 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.

AB - 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.

KW - O generation

KW - X-ray diffraction

KW - chlorite dismutase

KW - heme enzyme

KW - neutron diffraction

KW - resonance Raman spectroscopy

KW - spectroelectrochemistry

KW - stopped-flow spectroscopy

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U2 - 10.1021/acscatal.7b01749

DO - 10.1021/acscatal.7b01749

M3 - Article

AN - SCOPUS:85032933754

SN - 2155-5435

VL - 7

SP - 7962

EP - 7976

JO - ACS Catalysis

JF - ACS Catalysis

IS - 11

ER -

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

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