Characterization of the ubiquinone binding site in the alternative NADH-quinone oxidoreductase of Saccharomyces cerevisiae by photoaffinity labeling.
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 approximately 8 and approximately 4 kDa peptides, respectively. The approximately 8 kDa V8 digest was identified as the Thr329-Glu399 region (71 amino acids) by an N-terminal sequence analysis. Although the approximately 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.
Murai
Masatoshi
M
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Yamashita
Tetsuo
T
Senoh
Mai
M
Mashimo
Yuko
Y
Kataoka
Michihiko
M
Kosaka
Hiroaki
H
Matsuno-Yagi
Akemi
A
Yagi
Takao
T
Miyoshi
Hideto
H
D001665:Binding Sites; D042967:Electron Transport Complex I / Q000737:chemistry* / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000737:chemistry; D010449:Peptide Mapping / Q000379:methods; D019920:Photoaffinity Labels / Q000138:chemical synthesis / Q000737:chemistry*; D011487:Protein Conformation; D011808:Quinone Reductases / Q000737:chemistry* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism; D014451:Ubiquinone / Q000378:metabolism*
D024101:Mitochondrial Proteins; C493237:Ndi1 protein, S cerevisiae; D019920:Photoaffinity Labels; D029701:Saccharomyces cerevisiae Proteins; D014451:Ubiquinone; C041831:NADH dehydrogenase (quinone); D011808:Quinone Reductases; D042967:Electron Transport Complex I