Murai M, et al. (2010) Characterization of the ubiquinone binding site in the alternative NADH-quinone oxidoreductase of Saccharomyces cerevisiae by photoaffinity labeling. Biochemistry 49(13):2973-80
Abstract: The Ndi1 enzyme found in the mitochondrial membrane of Saccharomyces cerevisiae is an NDH-2-type alternative NADH-quinone (Q) 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 carried out photoaffinity labeling using a photoreactive biotinylated UQ mimic (compound 2) synthesized on the basis of a rational design concept. Cleavage with CNBr of Ndi1 cross-linked by 2 revealed the UQ-ring of 2 to be specifically cross-linked to the region Phe281-Met410 (130 amino acids). Digestion of the CNBr fragment with V8 protease and lysylendopeptidase (Lys C) gave ~8 kDa and ~4 kDa peptides, respectively. The ~8 kDa V8 digest was identified as Thr329-Glu399 (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 region Gly374-Lys405 (32 amino acids). Taken together, the binding site of the Q-ring of 2 must be located in a common region of the V8 and the Lys C digests Gly374-Glu399 (26 amino acids). Superimposition of the Ndi1 sequence onto a 3D-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.
|Status: Published||Type: Journal Article||PubMed ID: 20192260|
Topics addressed in this paper
- To go to the Locus page for a gene, click on the gene name.
|Protein Physical Properties|
|Protein/Nucleic Acid Structure|