Exploring the binding site of delta(lac)-acetogenin in bovine heart mitochondrial NADH-ubiquinone oxidoreductase.
Biochemical characterization of the inhibition mechanism of Deltalac-acetogenins synthesized in our laboratory indicated that they are a new type of inhibitor of bovine heart mitochondrial NADH-ubiquinone oxidoreductase (complex I) [Murai, M., et al. (2006) Biochemistry 45, 9778-9787]. To identify the binding site of Deltalac-acetogenins with a photoaffinity labeling technique, we synthesized a photoreactive Deltalac-acetogenin ([(125)I]diazinylated Deltalac-acetogenin, [(125)I]DAA) which has a small photoreactive diazirine group attached to a pharmacophore, the bis-THF ring moiety. Characterization of the inhibitory effects of DAA on bovine complex I revealed unique features specific to, though not completely the same as those of, the original Deltalac-acetogenin. Using [(125)I]DAA, we carried out photoaffinity labeling with bovine heart submitochondrial particles. Analysis of the photo-cross-linked protein by Western blotting and immunoprecipitation revealed that [(125)I]DAA binds to the membrane subunit ND1 with high specificity. The photo-cross-linking to the ND1 subunit was suppressed by an exogenous short-chain ubiquinone (Q(2)) in a concentration-dependent manner. Careful examination of the fragmentation patterns of the cross-linked ND1 generated by limited proteolysis using lysylendopeptidase, endoprotease Asp-N, or trypsin and their changes in the presence of the original Deltalac-acetogenin strongly suggested that the cross-linked residues are located at two different sites in the third matrix-side loop connecting the fifth and sixth transmembrane helices.
Kakutani
Nobuyuki
N
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Murai
Masatoshi
M
Sakiyama
Naoto
N
Miyoshi
Hideto
H
D054378:Acetogenins / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D003978:Diazomethane / Q000737:chemistry / Q000378:metabolism; D042967:Electron Transport Complex I / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D005663:Furans / Q000737:chemistry / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D007457:Iodine Radioisotopes / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008929:Mitochondria, Heart / Q000201:enzymology / Q000378:metabolism*; D008969:Molecular Sequence Data; D009245:NADH Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D019920:Photoaffinity Labels / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013367:Submitochondrial Particles / Q000201:enzymology / Q000378:metabolism
D054378:Acetogenins; D005663:Furans; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D019920:Photoaffinity Labels; C061022:bullatacin; C018674:tetrahydrofuran; D003978:Diazomethane; D009245:NADH Dehydrogenase; C515075:NADH dehydrogenase subunit 1, human; D042967:Electron Transport Complex I