University of Illinois Urbana-Champaign

EPR and solid-state NMR studies on the mechanism of cytochrome bo3 ubiquinol oxidase from escherichia coli

Lin, Myat T.

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Lin_Myat.pdf

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https://hdl.handle.net/2142/15565

Description

Title
EPR and solid-state NMR studies on the mechanism of cytochrome bo3 ubiquinol oxidase from escherichia coli
Author(s)
Lin, Myat T.
Issue Date
2010-05-14T20:50:36Z
Director of Research (if dissertation) or Advisor (if thesis)
Gennis, Robert B.
Doctoral Committee Chair(s)
Gennis, Robert B.
Committee Member(s)
  • Crofts, Antony R.
  • Gruebele, Martin
  • Nair, Satish K.
Department of Study
School of Molecular & Cell Bio
Discipline
Biophysics & Computnl Biology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Date of Ingest
2010-05-14T20:50:36Z
Keyword(s)
  • cytochrome bo3
  • ubiquinol oxidase
  • Electron paramagnetic resonance (EPR)
  • Solid-state NMR
  • selective isotope labeling
  • auxotrophs
  • quinone binding site
  • Nuclear magnetic resonance (NMR)
Abstract
Cytochrome bo3 ubiquinol oxidase from E. coli is a member of heme-copper oxidase superfamily. This trans-membrane enzyme complex catalyzes two-electron oxidation of ubiquinol and reduction of molecular oxygen to water. During the process, the protons from ubiquinol are released to the periplasmic side of the membrane, whereas the protons used in the reduction of O2 are taken from the cytoplasmic side. In addition to the protons involved in the redox reaction, the enzyme also translocates four protons from the cytoplasmic side to the periplasmic side for each molecule of oxygen reduced. Thus, cytochrome bo3 contributes to the electrochemical potential difference across the membrane. During turnover, electrons from the ubiquinol pool are first transferred to a ubiquinone cofactor bound at the high affinity binding site known as the QH-site, from which electrons are moved one at a time sequentially to the low-spin heme b and the CuB-heme o3 catalytic site. In this study, E. coli C43(DE3) auxotroph strains were constructed with highly efficient λ-Red recombination system. Wild-type and D75H mutant cytochrome bo3 samples with selectively isotope labeled Arg, Gln or His were prepared from these C43(DE3) auxotroph strains, and the semiquinone radicals at the QH-site of these samples were analyzed by pulsed EPR spectroscopy. Selective 15N labeling has revealed the Nε of R71 in the wild type cytochrome bo3 and the Nε of H75 in the D75H mutant cytochrome bo3 as the nitrogen atoms that are strongly coupled with the carbonyl oxygen-1 of the semiquinone and produce 14N ESEEM features observed in previous studies. Pulsed EPR experiments performed on these selectively 15N labeled samples also enabled the investigation of nitrogen nuclei weakly coupled to the semiquinone. In addition, three amino-acid pair-wise isotope labeled cytochrome bo3 samples were prepared from C43(DE3) auxotrophs for solid-state NMR (SSNMR) experiments. The initial SSNMR spectra proved that clean isotope labeling at the selected amino acid types was achieved through the use of auxotroph strains. This selective labeling approach dramatically simplifies the SSNMR signals and presents a great possibility to accomplish the chemical shift assignments of critical residues at the active site of such a large membrane protein complex. Furthermore, several cytochrome bo3 samples with selectively isotope labeled ubiquinone cofactors were successfully assembled. The procedure described in this study establishes a major step towards structural and mechanistic studies on the ubiquinone binding site of cytochrome bo3 by EPR and SSNMR techniques.
Graduation Semester
2010-5
Permalink
http://hdl.handle.net/2142/15565
Copyright and License Information
Copyright 2010 Myat T. Lin

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