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Biochim Biophys Acta. 2010 Dec;1797(12):1894-900. doi: 10.1016/j.bbabio.2010.10.013. Epub 2010 Oct 16.

Spin labeling of the Escherichia coli NADH ubiquinone oxidoreductase (complex I).

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  • 1Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität Freiburg, Albertstr. 21, 79104 Freiburg, Germany.

Abstract

The proton-pumping NADH:ubiquinone 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 NADH:decyl-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.

Copyright © 2010 Elsevier B.V. All rights reserved.

PMID:
20959113
[PubMed - indexed for MEDLINE]
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