Probing the ubiquinone reduction site of mitochondrial complex I using novel cationic inhibitors.
A wide variety of N-methylpyridinium and quinolinium cationic inhibitors of mitochondrial complex I was synthesized to develop potent and specific inhibitors acting selectively at one of the two proposed ubiquinone binding sites of this enzyme (Gluck, M. R., Krueger, M. J., Ramsay, R. R., Sablin, S. O., Singer, T. P., and Nicklas, W. J. (1994) J. Biol. Chem. 269, 3167-3174). N-Methyl-2-n-dodecyl-3-methylquinolinium (MQ18) inhibited electron transfer of complex I at under microM order regardless of whether exogenous or endogenous ubiquinone was used as an electron acceptor. The presence of tetraphenylboron (TPB-) potentiated the inhibition by MQ18 in a different way depending upon the molar ratio of TPB- to MQ18. In the presence of a catalytic amount of TPB-, the inhibitory potency of MQ18 was remarkably enhanced, and the extent of inhibition was almost complete. The presence of equimolar TPB- partially reactivated the enzyme activity, and the inhibition was saturated at an incomplete level (approximately 50%). These results are explained by the proposed dual binding sites model for ubiquinone (cited above). The inhibition behavior of MQ18 for proton pumping activity was similar to that for electron transfer activity. The good correlation of the inhibition behavior for the two activities indicates that both ubiquinone binding sites contribute to redox-driven proton pumping. On the other hand, N-methyl-4-[2-methyl-3-(p-tert-butylphenyl)]propylpyridinium (MP6) without TPB- brought about approximately 50% inhibition at 5 microM, but the inhibition reached a plateau at this level over a wide range of concentrations. Almost complete inhibition was readily obtained at low concentrations of MP6 in the presence of TPB-. Thus MP6 appears to be a selective inhibitor of one of the two ubiquinone binding sites. With a combined use of MP6 and 2,3-diethoxy-5-methyl-6-geranyl-1,4-benzoquinone, we also provided kinetic evidence for the existence of two ubiquinone binding sites.
Miyoshi
H
H
Department of Agricultural Chemistry, Kyoto University, Sakyo-ku, Kyoto 606, Japan. miyoshi@kais.kyoto-u.ac.jp
Inoue
M
M
Okamoto
S
S
Ohshima
M
M
Sakamoto
K
K
Iwamura
H
H
D015655:1-Methyl-4-phenylpyridinium / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D042967:Electron Transport Complex I; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors*; D010084:Oxidation-Reduction; D017494:Proton Pumps / Q000187:drug effects; D013775:Tetraphenylborate / Q000494:pharmacology; D014451:Ubiquinone / Q000378:metabolism*
D017494:Proton Pumps; D009243:NAD; D014451:Ubiquinone; D013775:Tetraphenylborate; D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I; D015655:1-Methyl-4-phenylpyridinium