Fenpyroximate Binds to the Interface between PSST and 49 kDa Subunits in Mitochondrial NADH-Ubiquinone Oxidoreductase

Abstract

Using a photoaffinity labeling technique, Nakamaru-Ogiso et al. demonstrated that fenpyroximate, a strong inhibitor of bovine heart mitochondrial NADH-ubiquinone oxidoreductase (complex I), binds to the ND5 subunit [Nakamaru-Ogiso, E., et al. (2003) Biochemistry 42, 746-754]. Considering that the main body of the ND5 subunit composed of transmembrane helixes 1-15 is located at the distal end of the membrane domain [Efremov, R. G., et al. (2010) Nature 465, 441-445], however, their result may be questionable. Because establishing the number and location of inhibitors and/or quinone binding sites in the membrane domain is necessary to elucidate the function of the enzyme, it is critical to clarify whether there is an additional inhibitor and/or quinone binding site besides the interface between the hydrophilic and membrane domains. We therefore performed photoaffinity labeling experiments using two newly synthesized fenpyroximate derivatives [[(125)I]-4-azidophenyl fenpyroximate ([(125)I]APF) and [(125)I]-3-azido-5-iodobenzyl fenpyroximate ([(125)I]AIF)] possessing a photoreactive azido group at and far from the pharmacophoric core moiety, respectively. Doubled sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that [(125)I]APF and [(125)I]AIF bind to the PSST and 49 kDa subunits, respectively. Careful examination of the fragmentation patterns of the labeled PSST and 49 kDa subunits generated by limited proteolysis indicated that the residues labeled by [(125)I]APF and [(125)I]AIF are located in the Ser43-Arg66 (PSST) and Asp160-Arg174 (49 kDa) regions, respectively, which face the supposed quinone-binding pocket formed at the interface of the PSST, 49 kDa, and ND1 subunits. We conclude that fenpyroximate does not bind to the distal end of the membrane domain but rather resides at the interface between the two domains in a manner such that the pharmacophoric pyrazole ring and side chain of the inhibitor orient toward the PSST and 49 kDa subunits, respectively. This study answers a critical question relating to complex I.

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

“…The tryptic digests were desalted with ZipTip (Merck-Millipore) and spotted onto the target plate using ␣-cyano-4-hydroxycinnamic acid as a matrix (36). A mass spectrometric analysis was conducted using a Bruker Autoflex III Smartbeam MALDI-TOF/TOF instrument (Bruker Daltonics).…”

“…A mass spectrometric analysis was conducted using a Bruker Autoflex III Smartbeam MALDI-TOF/TOF instrument (Bruker Daltonics). The mass spectra obtained were analyzed according to previously published procedures (36). N-terminal amino acid residues were examined with a Procise 494 HT protein sequencing system (Applied Life Sciences, Foster City, CA) at the APRO Life Science Institute, Inc. (Tokushima, Japan).…”

Production of new amilorides as potent inhibitors of mitochondrial respiratory complex I

“…3) We also confirmed that the IC 50 values of these amilorides determined with the NADH-Q 1 oxidoreductase assay matched those determined with the NADH oxidase assay, indicating that the inhibition of other respiratory complexes is negligible. Since the IC 50 values of the photoreactive inhibitors used in earlier photoaffinity labeling studies with complex I, such as quinazoline, 8) fenpyroximate, 9) and acetogenins, 17) were all at one digit nm levels, the commercially available amilorides are not potent enough to be used as templates for designing photoreactive amilorides. Therefore, we needed to produce more potent amiloride derivatives (Scheme 1).…”

“…Photoaffinity labeling is a powerful technique to identify the binding site of amilorides in bovine complex I. 8,9) In the photoaffinity labeling technique, less nonspecific binding occurs at higher binding affinities of photoreactive ligands; therefore, using a ligand with high binding affinity as possible allows for the accurate interpretation of data obtained in photoaffinity labeling experiments. Commercially available amilorides are not suitable as design templates for the synthesis of photoreactive amilorides possessing high binding affinities to complex I because of their low binding affinities to bovine complex I.…”

Roles of Subunit NuoK (ND4L) in the Energy-transducing Mechanism of Escherichia coli NDH-1 (NADH:Quinone Oxidoreductase)

“…The transfer of electron from NADH starts at the tip of the peripheral arm with FMN being the primary electron acceptor and then through a chain of seven conserved Fe/S clusters to the likely quinone-binding site expected to be present at the interface with the membrane domain (11)(12)(13)(14). The hydrophobic arm is embedded in the inner mitochondrial/cytoplasmic membrane and is believed to participate in the proton translocation and in the binding of quinone and inhibitors (9,(15)(16)(17)(18).…”