title: Exploring interactions between the 49 kDa and ND1 subunits in mitochondrial NADH-ubiquinone oxidoreductase (complex I) by photoaffinity labeling.
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.
authors: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
authors: Masatoshi
authors: M
authors: Murai
authors: Yuko
authors: Y
authors: Mashimo
authors: Judy
authors: J
authors: Hirst
authors: Hideto
authors: H
authors: Miyoshi
mesh_terms: D000818:Animals; D001665:Binding Sites; D002417:Cattle; D042967:Electron Transport Complex I / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008929:Mitochondria, Heart / Q000201:enzymology; D019920:Photoaffinity Labels; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013367:Submitochondrial Particles / Q000378:metabolism
chemical_list: D019920:Photoaffinity Labels; D042967:Electron Transport Complex I