Characterization of the Inhibitor Binding Site in Mitochondrial NADH−Ubiquinone Oxidoreductase by Photoaffinity Labeling Using a Quinazoline-Type Inhibitor

Abstract

The diverse inhibitors of bovine heart mitochondrial complex I (NADH-ubiquinone oxidoreductase) are believed to share a common large binding domain with partially overlapping sites, though it remains unclear how these binding sites relate to each other. To obtain new insight into the inhibitor binding domain in complex I, we synthesized a photoreactive azidoquinazoline {[(125)I]-6-azido-4-(4-iodophenethylamino)quinazoline, [(125)I]AzQ}, in which a photolabile azido group was introduced into the toxophoric quinazoline ring to allow specific cross-linking, and carried out a photoaffinity labeling study using bovine heart submitochondrial particles. Analysis of the photo-cross-linked proteins by peptide mass fingerprinting and immunoblotting revealed that [(125)I]AzQ specifically binds to the 49 kDa and ND1 subunits with a frequency of approximately 4:1. The cross-linking was completely blocked by excess amounts of other inhibitors such as acetogenin and fenpyroximate. Considerable cross-linking was also detected in the ADP/ATP carrier and 3-hydroxybutyrate dehydrogenase, though it was not associated with dysfunction of the two proteins. The partial proteolysis of the [(125)I]AzQ-labeled 49 kDa subunit by V8-protease and N-terminal sequencing of the resulting peptides revealed that the amino acid residue cross-linked by [(125)I]AzQ is within the sequence region Thr25-Glu143 (118 amino acids). Furthermore, examination of fragment patterns generated by exhaustive digestion of the [(125)I]AzQ-labeled 49 kDa subunit by V8-protease, lysylendopeptidase, or trypsin strongly suggested that the cross-linked residue is located within the region Asp41-Arg63 (23 amino acids). The present study has revealed, for the first time, the inhibitor binding site in complex I at the sub-subunit level.

Identification of the binding sites for ubiquinone and inhibitors in the Na+-pumping NADH-ubiquinone oxidoreductase from Vibrio cholerae by photoaffinity labeling

“…The gels were stained with CBB, dried, exposed to an imaging plate (BAS-MS2040, Fuji Film) for 12-24 h, and visualized with the FLA-5100 Bio-Imaging analyzer. The radioactivity of each band was quantified from the digitized data using MultiGauge (Fuji Film) or directly from the gel slices using a ␥-counting system (COBLA II, Packard), as described elsewhere (32).…”

“…To partially digest the NqrA subunit labeled by PUQ-3, the CBB-stained NqrA band was treated with V8-protease (Roche Applied Science, Penzberg, Germany) in a 15% Tris-EDTA mapping gel using according to the procedures described previously (32)(33)(34). The partial digests were characterized by mass spectrometry or N-terminal sequence analysis.…”

The Membrane Subunit NuoL(ND5) Is Involved in the Indirect Proton Pumping Mechanism of Escherichia coli Complex I

“…In the direct coupling mechanism, quinone-binding site(s) and proton-translocation machinery need to be located close to the peripheral arm with its redox centers to allow direct interaction. The x-ray crystallographic structure (10), photoaffinity labeling/cross-linking studies (24,25), extensive mutagenesis studies of subunits 49 kDa and PSST in Y. lipolytica complex I (26,27) and the NuoH subunit (a ND1 homologue in Escherichia coli complex I) (28) support that the primary Q-binding site is in the pocket surrounded by the 49 kDa, PSST, and ND1 subunits, and that H ϩ translocation is directly coupled to electron transfer from cluster N2 in PSST to UQ in membrane close to the interface region. In the indirect coupling mechanism, energy transduction takes place through long-range conformational changes, thus the electron transfer module can be away from the H ϩ -pumping module.…”

Metabolic enzymes of helminth parasites: potential as drug targets

“…E. multilocularis complex I is inhibited by quinazoline (Scheme 6) and this compound kills the parasite [205]. This compound, and its derivatives, also inhibit mammalian mitochondrial complex I [206]. So, the challenge would be to identify derivatives which selectively inhibit the complex from helminths.…”