| | The ND2 subunit is labeled by a photoaffinity analogue of asimicin, a potent complex I inhibitorEdited by Peter Brzezinski Received 13 December 2009; received in revised form 7 January 2010; accepted 7 January 2010. published online 13 January 2010. Abstract NADH:ubiquinone oxidoreductase (complex I) is the entry enzyme of mitochondrial oxidative phosphorylation. To obtain the structural information on inhibitor/quinone binding sites, we synthesized [3H]benzophenone-asimicin ([3H]BPA), a photoaffinity analogue of asimicin, which belongs to the acetogenin family known as the most potent complex I inhibitor. We found that [3H]BPA was photo-crosslinked to ND2, ND1 and ND5 subunits, by the three dimensional separation (blue-native/doubled SDS–PAGE) of [3H]BPA-treated bovine heart submitochondrial particles. The cross-linking was blocked by rotenone. This is the first finding that ND2 was photo-crosslinked with a potent complex I inhibitor, suggesting its involvement in the inhibitor/quinone-binding. Abbreviations: BN–PAGE, blue native-PAGE, BPA, benzophenone-asimicin, 1-D, one-dimensional, 2-D, two-dimensional, 3-D, three-dimensional, IC50, the molar concentration of substance that provides 50% inhibition of the control NADH oxidase activity, Q, quinone, SMP, submitochondrial particles, SQ, semiquinone, [125I]TDA, [125I](trifluoromethyl)phenyldiazirinylacetogenin a Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA b Johnson Research Foundation, Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA c Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA  Corresponding author. Address: Rm 214 Anatomy/Chemistry Building, 36th & Hamilton Walk, Philadelphia, PA 19104, USA. Fax: +1 215 898 2939.
PII: S0014-5793(10)00021-9 doi:10.1016/j.febslet.2010.01.004 © 2010 Federation of European Biochemical Societies | |
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ABSTRACT
