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Characterization, Structure and Mechanism of Sulfide:quinone oxidoreductase (SQR) from Acidithiobacillus ferrooxidans Open Access


Other title
Flavin adenine dinucleotide (FAD)
Flavin radical
Quinone binding site
Sulfide-FAD-quinone redox reaction mechanisms
sulfide:quinone oxidoreductase (SQR)
X-ray crystallography
Acidithiobacillus ferrooxidans
Electron Paramagnetic Resonance (EPR)
Type of item
Degree grantor
University of Alberta
Author or creator
Zhang, Yanfei
Supervisor and department
Weiner, Joel H. (Biochemistry)
Examining committee member and department
Weiner, Joel H. (Biochemistry)
Glover, Mark (Biochemistry)
Turner, Raymond (Biological Sciences)
Lemieux, Joanne (Biochemistry)
Raivio, Tracy (Biological Sciences)
Department of Biochemistry

Date accepted
Graduation date
Doctor of Philosophy
Degree level
A key enzyme in maintaining sulfide homeostasis is the membrane-associated flavoenzyme sulfide:quinone oxidoreductase (SQR) found in nearly all domains of life (except plants). SQR maintains a critical equilibrium between sulfide (H2S, HS- and S2-) and elemental sulfur (S0), coupling the oxidation of sulfide to the reduction of ubiquinone via a non-covalent FAD cofactor. SQR interacts with the membrane via two amphipathic helices and the Q-pool through a conserved hydrophobic domain. The active site of SQR includes three cysteines (Cys160, Cys356 and Cys128) and the FAD in close juxtaposition to the ubiquinone binding site. To understand the sulfide oxidation mechanism, we expressed the wild-type sqr gene from Acidithiobacillus ferrooxidans, as well as several variants of conserved, catalytically important amino acids in E. coli BL21(DE3) and purified soluble, active, His-tagged SQR. The purified wild-type SQR and the variants were subjected to extensive kinetic (pre-steady state and steady state) and structural analysis (X-ray crystallography). We also monitored SQR activity in vivo by detecting the H2S produced by growing E. coli transformed with wild-type and variant SQR. Our catalytic activity analysis and structural determination led us to propose two alternative mechanisms: (1) A nucleophilic attack mechanism that involves Cys356–S–S− as a nucleophile which attacks the C4A atom of FAD; or (2) A radical mechanism of direct electron transfer from Cys356 disulfide to FAD. The growing polysulfide is held between Cys160 and Cys356. The role of Cys128 (most likely in the form of a disulfide) is confined to the release of the polysulfur product. We further investigated the role of the FAD and the conserved Cys and His residues using a combination of kinetics and EPR spectroscopy. Using steady state kinetics of Na2S-dependent decylubiquinone (DUQ) reduction we measured a kcat of 6.5 s-1 and a Km (Na2S) of 3.0 M and a Km (DUQ) of 3.4 M. Variants of Cys160, Cys356 and His198 had greatly diminished DUQ reduction activity whereas variants of Cys128 and His132 were less affected. A neutral flavin semiquinone was observed in the EPR spectrum of SQR reduced with Na2S which was enhanced in the Cys160Ala variant suggesting the presence of a Cys356-Sγ-S-C4A-FAD adduct. Potentiometric titrations of the FAD semiquinone revealed an midpoint potential (Em) of -139 ± 4 mV at pH 7.0 in wild-type SQR. The Em of the FAD in SQRCys160Ala (Em= -135 ± 5 mV) is similar to that in wild-type SQR. We also combined computational docking and kinetic approaches to analyze quinone binding. SQR can reduce both benzoquinones and naphthoquinones. The alkyl side chain of ubiquinone derivatives enhances binding to SQR but limits the enzyme turnover. Pentachlorophenol and 2-n-heptyl-4-hydroxyquinoline-N-oxide are potent inhibitors of SQR with apparent inhibition constants (Ki) of 0.46 µM and 0.58 µM, respectively. The highly conserved amino acids surrounding the quinone binding site play an important role in quinone reduction. The phenyl sidechains of Phe357 and Phe391 sandwich the benzoquinone head group and are critical for quinone binding. Importantly, conserved amino acids that define the ubiquinone-binding site also play an important role in flavin reduction.
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
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