Journal
BIOCHEMISTRY
Volume 49, Issue 13, Pages 2973-2980Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi100005j
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Funding
- Scientific Research [20380068]
- Japan Society for the Promotion of Science [21880024]
- National Institutes of Health [R01GM033712]
- Grants-in-Aid for Scientific Research [21880024] Funding Source: KAKEN
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The Ndil enzyme found in the mitochondrial membrane of Saccharomyces cerevisiae is an NDH-2-type alternative NADH-quinone oxidoreductase. As Ndil 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 Ndil, 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 Ndil 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 similar to 8 and similar to 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 similar to 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-G1u399 (26 amino acids). Superimposition of the Ndil 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.
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