Exploring the binding site of acetogenin in the ND1 subunit of bovine mitochondrial complex I.
125I-labeled (trifluoromethyl)phenyldiazirinyl acetogenin, [125I]TDA, a photoaffinity labeling probe of acetogenin, photo-cross-links to the ND1 subunit of bovine heart mitochondrial NADH-ubiquinone oxidoreductase (complex I) with high specificity [M. Murai, A. Ishihara, T. Nishioka, T. Yagi, and H. Miyoshi, (2007) The ND1 subunit constructs the inhibitor binding domain in bovine heart mitochondrial complex I, Biochemistry 46 6409-6416.]. To identify the binding site of [125I]TDA in the ND1 subunit, we carried out limited proteolysis of the subunit cross-linked by [125I]TDA using various proteases and carefully analyzed the fragmentation patterns. Our results revealed that the cross-linked residue is located within the region of the 4th to 5th transmembrane helices (Val144-Glu192) of the subunit. It is worth noting that an excess amount of short-chain ubiquinones such as ubiquinone-2 (Q2) and 2-azido-Q2 suppressed the cross-linking by [125I]TDA in a concentration-dependent way. Although the question of whether the binding sites for ubiquinone and different inhibitors in complex I are identical remains to be answered, the present study provided, for the first time, direct evidence that an inhibitor (acetogenin) and ubiquinone competitively bind to the enzyme. Considering the present results along with earlier photoaffinity labeling studies, we propose that not all inhibitors acting at the terminal electron transfer step of complex I necessarily bind to the ubiquinone binding site itself.
Sekiguchi
Koji
K
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Murai
Masatoshi
M
Miyoshi
Hideto
H
D054378:Acetogenins / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D042967:Electron Transport Complex I / Q000737:chemistry* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008929:Mitochondria, Heart; D008954:Models, Biological; D011485:Protein Binding
D054378:Acetogenins; D042967:Electron Transport Complex I