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

  • Author / Creator
    Zhang, Yanfei
  • 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.

  • Subjects / Keywords
  • Graduation date
    2015-11
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/R3ZW18Z0R
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
    English
  • Institution
    University of Alberta
  • Degree level
    Doctoral
  • Department
    • Department of Biochemistry
  • Supervisor / co-supervisor and their department(s)
    • Weiner, Joel H. (Biochemistry)
  • Examining committee members and their departments
    • Weiner, Joel H. (Biochemistry)
    • Raivio, Tracy (Biological Sciences)
    • Lemieux, Joanne (Biochemistry)
    • Turner, Raymond (Biological Sciences)
    • Glover, Mark (Biochemistry)