Exploring by pulsed EPR the electronic structure of ubisemiquinone bound at the QH site of cytochrome bo3 from Escherichia coli with in vivo 13C-labeled methyl and methoxy substituents

J Biol Chem. 2011 Mar 25;286(12):10105-14. doi: 10.1074/jbc.M110.206821. Epub 2011 Jan 19.


The cytochrome bo(3) ubiquinol oxidase from Escherichia coli resides in the bacterial cytoplasmic membrane and catalyzes the two-electron oxidation of ubiquinol-8 and four-electron reduction of O(2) to water. The one-electron reduced semiquinone forms transiently during the reaction, and the enzyme has been demonstrated to stabilize the semiquinone. The semiquinone is also formed in the D75E mutant, where the mutation has little influence on the catalytic activity, and in the D75H mutant, which is virtually inactive. In this work, wild-type cytochrome bo(3) as well as the D75E and D75H mutant proteins were prepared with ubiquinone-8 (13)C-labeled selectively at the methyl and two methoxy groups. This was accomplished by expressing the proteins in a methionine auxotroph in the presence of l-methionine with the side chain methyl group (13)C-labeled. The (13)C-labeled quinone isolated from cytochrome bo(3) was also used for the generation of model anion radicals in alcohol. Two-dimensional pulsed EPR and ENDOR were used for the study of the (13)C methyl and methoxy hyperfine couplings in the semiquinone generated in the three proteins indicated above and in the model system. The data were used to characterize the transferred unpaired spin densities on the methyl and methoxy substituents and the conformations of the methoxy groups. In the wild type and D75E mutant, the constraints on the configurations of the methoxy side chains are similar, but the D75H mutant appears to have altered methoxy configurations, which could be related to the perturbed electron distribution in the semiquinone and the loss of enzymatic activity.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Substitution
  • Carbon Isotopes
  • Cytochrome b Group
  • Cytochromes
  • Electron Spin Resonance Spectroscopy
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins
  • Isotope Labeling
  • Mutation, Missense
  • Oxidation-Reduction
  • Ubiquinone / analogs & derivatives*
  • Ubiquinone / chemistry
  • Ubiquinone / metabolism


  • Carbon Isotopes
  • Cytochrome b Group
  • Cytochromes
  • Escherichia coli Proteins
  • cytochrome bo3, E coli
  • Ubiquinone
  • coenzyme Q10