Characterization of the Ubiquinone Binding Site in the Alternative NADH-Quinone Oxidoreductase of Saccharomyces cerevisiae by Photoaffinity Labeling

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• 作者：Masatoshi Murai ; Tetsuo Yamashita ; Mai Senoh ; Yuko Mashimo ; Michihiko Kataoka ; Hiroaki Kosaka ; Akemi Matsuno-Yagi ; Takao Yagi ; Hideto Miyoshi
• 刊名：Biochemistry
• 出版年：2010
• 出版时间：April 6, 2010
• 年：2010
• 卷：49
• 期：13
• 页码：2973-2980
• 全文大小：845K
• 年卷期：v.49,no.13(April 6, 2010)
• ISSN：1520-4995

文摘

The Ndi1 enzyme found in the mitochondrial membrane of Saccharomyces cerevisiae is an NDH-2-type alternative NADH-quinone oxidoreductase. As Ndi1 is expected to be a possible remedy for complex I defects of mammalian mitochondria, a detailed biochemical characterization of the enzyme is needed. To identify the ubiquinone (UQ) binding site in Ndi1, we conducted photoaffinity labeling using a photoreactive biotinylated UQ mimic (compound 2) synthesized following a concept of the least possible modification of the substituents on the quinone ring. Cleavage with CNBr of Ndi1 cross-linked by 2 revealed the UQ ring of 2 to be specifically cross-linked to the Phe281−Met410 region (130 amino acids). Digestion of the CNBr fragment with V8 protease and lysylendopeptidase (Lys-C) gave 8 and 4 kDa peptides, respectively. The 8 kDa V8 digest was identified as the Thr329−Glu399 region (71 amino acids) by an N-terminal sequence analysis. Although the 4 kDa Lys-C digest could not be identified by N-terminal sequence analysis, the band was thought to cover the Gly374−Lys405 region (32 amino acids). Taken together, the binding site of the Q ring of 2 must be located in a common region of the V8 protease, and Lys-C digests Gly374−Glu399 (26 amino acids). Superimposition of the Ndi1 sequence onto a three-dimensional structural model of NDH-2 from Escherichia coli suggested that the C-terminal portion of this region is close to the isoalloxazine ring of FAD.