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Exploring the binding site of delta(lac)-acetogenin in bovine heart mitochondrial NADH-ubiquinone oxidoreductase.
- Source :
-
Biochemistry [Biochemistry] 2010 Jun 15; Vol. 49 (23), pp. 4794-803. - Publication Year :
- 2010
-
Abstract
- 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.
- Subjects :
- Acetogenins chemistry
Amino Acid Sequence
Animals
Binding Sites
Binding, Competitive
Cattle
Diazomethane chemistry
Diazomethane metabolism
Electron Transport Complex I antagonists & inhibitors
Electron Transport Complex I chemistry
Furans chemistry
Furans metabolism
Hydrophobic and Hydrophilic Interactions
Iodine Radioisotopes metabolism
Membrane Proteins chemistry
Membrane Proteins metabolism
Mitochondria, Heart enzymology
Molecular Sequence Data
NADH Dehydrogenase antagonists & inhibitors
NADH Dehydrogenase chemistry
NADH Dehydrogenase metabolism
Photoaffinity Labels metabolism
Protein Structure, Tertiary
Submitochondrial Particles enzymology
Submitochondrial Particles metabolism
Acetogenins metabolism
Electron Transport Complex I metabolism
Mitochondria, Heart metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1520-4995
- Volume :
- 49
- Issue :
- 23
- Database :
- MEDLINE
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 20459120
- Full Text :
- https://doi.org/10.1021/bi100454b