The ND5 subunit was labeled by a photoaffinity analogue of fenpyroximate in bovine mitochondrial complex I.
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.
Nakamaru-Ogiso
Eiko
E
Division of Biochemistry, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.
Sakamoto
Kimitoshi
K
Matsuno-Yagi
Akemi
A
Miyoshi
Hideto
H
Yagi
Takao
T
D000595:Amino Acid Sequence; D000818:Animals; D001565:Benzoates / Q000378:metabolism*; D002417:Cattle; D008929:Mitochondria, Heart / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009243:NAD / Q000737:chemistry; D009249:NADP / Q000737:chemistry; D019920:Photoaffinity Labels / Q000378:metabolism*; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011720:Pyrazoles / Q000378:metabolism*; D011808:Quinone Reductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors; D013367:Submitochondrial Particles / Q000378:metabolism
C472745:(trifluoromethyl)phenyldiazirinylfenpyroximate; D001565:Benzoates; D019920:Photoaffinity Labels; D021122:Protein Subunits; D011720:Pyrazoles; D017923:Sodium-Hydrogen Exchangers; D009243:NAD; D009249:NADP; C415144:fenpyroximate; C041831:NADH dehydrogenase (quinone); D011808:Quinone Reductases