The ND2 subunit is labeled by a photoaffinity analogue of asimicin, a potent complex I inhibitor

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.