Spin labeling of the Escherichia coli NADH ubiquinone oxidoreductase (complex I).
Biochim Biophys Acta
; 1797(12): 1894-900, 2010 Dec.
Article
em En
| MEDLINE
| ID: mdl-20959113
ABSTRACT
The proton-pumping NADHubiquinone oxidoreductase, the respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. Electron microscopy revealed the two-part structure of the complex with a peripheral arm involved in electron transfer and a membrane arm most likely involved in proton translocation. It was proposed that the quinone binding site is located at the joint of the two arms. Most likely, proton translocation in the membrane arm is enabled by the energy of the electron transfer reaction in the peripheral arm transmitted by conformational changes. For the detection of the conformational changes and the localization of the quinone binding site, we set up a combination of site-directed spin labeling and EPR spectroscopy. Cysteine residues were introduced to the surface of the Escherichia coli complex I. The spin label (1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl)-methanethiosulfonate (MTSL) was exclusively bound to the engineered positions. Neither the mutation nor the labeling had an effect on the NADHdecyl-ubiquinone oxidoreductase activity. The characteristic signals of the spin label were detected by EPR spectroscopy, which did not change by reducing the preparation with NADH. A decyl-ubiquinone derivative with the spin label covalently attached to the alkyl chain was synthesized in order to localize the quinone binding site. The distance between a MTSL labeled complex I variant and the bound quinone was determined by continuous-wave (cw) EPR allowing an inference on the location of the quinone binding site. The distances between the labeled quinone and other complex I variants will be determined in future experiments to receive further geometry information by triangulation.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Espectroscopia de Ressonância de Spin Eletrônica
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Proteínas de Escherichia coli
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Complexo I de Transporte de Elétrons
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Escherichia coli
Tipo de estudo:
Prognostic_studies
Idioma:
En
Ano de publicação:
2010
Tipo de documento:
Article