1. Identification of the binding sites for ubiquinone and inhibitors in the Na+-pumping NADH-ubiquinone oxidoreductase from Vibrio cholerae by photoaffinity labeling
- Author
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Ito, Takeshi, Murai, Masatoshi, Ninokura, Satoshi, Kitazumi, Yuki, Mezic, Katherine G, Cress, Brady F, Koffas, Mattheos AG, Morgan, Joel E, Barquera, Blanca, and Miyoshi, Hideto
- Subjects
Biological Sciences ,Medical Physiology ,Biomedical and Clinical Sciences ,Rare Diseases ,Digestive Diseases ,Bacterial Proteins ,Binding Sites ,Catalysis ,Computer Simulation ,Crystallography ,X-Ray ,Electron Transport ,Electron Transport Complex I ,Enzyme Inhibitors ,Fatty Acids ,Unsaturated ,Lactones ,Mass Spectrometry ,Molecular Structure ,Mutation ,Photoaffinity Labels ,Protein Binding ,Pseudoalteromonas ,Quinolones ,Quinone Reductases ,Sodium ,Ubiquinone ,Vibrio cholerae ,Na+-pumping NADH-ubiquinone oxidoreductase ,aurachin ,chemical biology ,enzyme inhibitor ,photoaffinity labeling ,respiratory chain ,ubiquinone ,Chemical Sciences ,Medical and Health Sciences ,Biochemistry & Molecular Biology ,Biological sciences ,Biomedical and clinical sciences ,Chemical sciences - Abstract
The Na+-pumping NADH-quinone oxidoreductase (Na+-NQR) is the first enzyme of the respiratory chain and the main ion transporter in many marine and pathogenic bacteria, including Vibrio cholerae The V. cholerae Na+-NQR has been extensively studied, but its binding sites for ubiquinone and inhibitors remain controversial. Here, using a photoreactive ubiquinone PUQ-3 as well as two aurachin-type inhibitors [125I]PAD-1 and [125I]PAD-2 and photoaffinity labeling experiments on the isolated enzyme, we demonstrate that the ubiquinone ring binds to the NqrA subunit in the regions Leu-32-Met-39 and Phe-131-Lys-138, encompassing the rear wall of a predicted ubiquinone-binding cavity. The quinolone ring and alkyl side chain of aurachin bound to the NqrB subunit in the regions Arg-43-Lys-54 and Trp-23-Gly-89, respectively. These results indicate that the binding sites for ubiquinone and aurachin-type inhibitors are in close proximity but do not overlap one another. Unexpectedly, although the inhibitory effects of PAD-1 and PAD-2 were almost completely abolished by certain mutations in NqrB (i.e. G140A and E144C), the binding reactivities of [125I]PAD-1 and [125I]PAD-2 to the mutated enzymes were unchanged compared with those of the wild-type enzyme. We also found that photoaffinity labeling by [125I]PAD-1 and [125I]PAD-2, rather than being competitively suppressed in the presence of other inhibitors, is enhanced under some experimental conditions. To explain these apparently paradoxical results, we propose models for the catalytic reaction of Na+-NQR and its interactions with inhibitors on the basis of the biochemical and biophysical results reported here and in previous work.
- Published
- 2017