Characterization of the inhibitor binding site in mitochondrial NADH-ubiquinone oxidoreductase by photoaffinity labeling using a quinazoline-type inhibitor

Masatoshi Murai, Koji Sekiguchi, Takaaki Nishioka, Hideto Miyoshi

研究成果: Article査読

62 被引用数 (Scopus)

抄録

The diverse inhibitors of bovine heart mitochondrial complex I (NADH-ubiquinone oxidoreductase) are believed to share a common large binding domain with partially overlapping sites, though it remains unclear how these binding sites relate to each other. To obtain new insight into the inhibitor binding domain in complex I, we synthesized a photoreactive azidoquinazoline {[125I]-6-azido-4-(4-iodophenethylamino)quinazoline, [ 125I]AzQ}, in which a photolabile azido group was introduced into the toxophoric quinazoline ring to allow specific cross-linking, and carried out a photoaffinity labeling study using bovine heart submitochondrial particles. Analysis of the photo-cross-linked proteins by peptide mass fingerprinting and immunoblotting revealed that [125I]AzQ specifically binds to the 49 kDa and ND1 subunits with a frequency of ∼4:1. The cross-linking was completely blocked by excess amounts of other inhibitors such as acetogenin and fenpyroximate. Considerable cross-linking was also detected in the ADP/ATP carrier and 3-hydroxybutyrate dehydrogenase, though it was not associated with dysfunction of the two proteins. The partial proteolysis of the [ 125I]AzQ-labeled 49 kDa subunit by V8-protease and N-terminal sequencing of the resulting peptides revealed that the amino acid residue cross-linked by [125I]AzQ is within the sequence region Thr25-Glu143 (118 amino acids). Furthermore, examination of fragment patterns generated by exhaustive digestion of the [125I]AzQ-labeled 49 kDa subunit by V8-protease, lysylendopeptidase, or trypsin strongly suggested that the cross-linked residue is located within the region Asp41-Arg63 (23 amino acids). The present study has revealed, for the first time, the inhibitor binding site in complex I at the sub-subunit level.

本文言語English
ページ(範囲)688-698
ページ数11
ジャーナルBiochemistry
48
4
DOI
出版ステータスPublished - 2009 2 3

ASJC Scopus subject areas

  • 生化学

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