Fenpyroximate is a potent inhibitor of the mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I). We synthesized its photoaffinity analogue [(3)H](trifluoromethyl)phenyldiazirinylfenpyroximate ([(3)H]TDF). When bovine heart submitochondrial particles (SMP) were illuminated with UV light in the presence of [(3)H]TDF, radioactivity was mostly incorporated into a 50 kDa band. There was a good correlation between radioactivity labeling of the 50 kDa band and inhibition of the NADH oxidase activity, indicating that a 50 kDa protein is responsible for the inactivation of complex I. Blue native gel electrophoresis of the [(3)H]TDF-labeled SMP revealed that the majority of radioactivity was found in complex I. Analysis of the complex I band on an SDS gel showed a major peak of radioactivity at approximately 50 kDa. There are three subunits in complex I that migrate in this region: FP51K, IP49K, and ND5. Further analysis using the 2D gel electrophoresis implied that the labeled protein was the ND5 subunit. Labeling of the ND5 subunit was stimulated by NADH/NADPH but was prevented by various complex I inhibitors. Amiloride derivatives that are known to be inhibitors of Na(+)/H(+) antiporters also diminished the labeling. In agreement with the protective effect, we observed that the amiloride derivatives inhibited NADH-ubiquinone-1 reductase activity but not NADH-K(3)Fe(CN)(6) reductase activity in bovine SMP. These results suggest that the ND5 subunit is involved in construction of the inhibitor- and quinone-binding site(s). Furthermore, it seems likely that the ND5 subunit may participate in H(+)(Na(+)) translocation in coupling site 1.