Exploring interactions between the 49 kDa and ND1 subunits in mitochondrial NADH-ubiquinone oxidoreductase (complex I) by photoaffinity labeling.

TitleExploring interactions between the 49 kDa and ND1 subunits in mitochondrial NADH-ubiquinone oxidoreductase (complex I) by photoaffinity labeling.
Publication TypeJournal Article
Year of Publication2011
AuthorsMurai, M, Mashimo, Y, Hirst, J, Miyoshi, H
JournalBiochemistry
Volume50
Issue32
Pagination6901-8
Date Published2011 Aug 16
ISSN1520-4995
KeywordsAnimals, Binding Sites, Cattle, Electron Transport Complex I, Electrophoresis, Polyacrylamide Gel, Hydrolysis, Mitochondria, Heart, Photoaffinity Labels, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Submitochondrial Particles
Abstract

Quinazolines are strong inhibitors of NADH-ubiquinone oxidoreductase (complex I) from bovine heart mitochondria. Using a photoreactive quinazoline, [(125)I]AzQ, and bovine heart submitochondrial particles (SMPs), we demonstrated previously that [(125)I]AzQ binds at the interface of the 49 kDa and ND1 subunits in complex I; it labeled a site in the N-terminal (Asp41-Arg63) region of the 49 kDa subunit, suggesting that this region contacts the ND1 subunit [Murai, M., et al. (2009) Biochemistry 48, 688-698]. The labeled region of ND1 could not be identified because it is highly hydrophobic, and the SMPs did not yield sufficient amounts of labeled protein. Here, we describe how photoaffinity labeling of isolated complex I by [(125)I]AzQ yielded sufficient material for identification of the labeled region of the ND1 subunit. The inhibition of the isolated enzyme by AzQ is comparable to that of SMPs. Our results reveal that the labeled site in ND1 is between Asp199 and Lys262, mostly likely in the third matrix loop that connects the fifth and sixth transmembrane helices. Thus, our results reveal new information about the interface between the hydrophilic and hydrophobic domains of complex I, a region that is thought to be important for ubiquinone reduction and energy transduction.

DOI10.1021/bi200883c
Alternate JournalBiochemistry
Citation Key10.1021/bi200883c
PubMed ID21721533
Grant ListMC_U105663141 / / Medical Research Council / United Kingdom