Identification of two different Q-binding sites in QH2-cytochrome c oxidoreductase, using the Q analogue n-heptadecylmercapto-6-hydroxy-5,8-quinolinequinone.
The pK and mid-point redox potential of the Q-analogue 7-(n-heptadecyl)mercapto-6-hydroxy-5,8-quinolinequinone (HMHQQ) in aqueous medium are so low that under the experimental conditions used for studying the inhibition of electron transfer in submitochondrial particles only the oxidized, anionic form is present. The KD of the analogue, determined by comparing its inhibitory effect with that of n-heptyl-4-hydroxyquinoline N-oxide, is (0.003 + 0.24 x mg protein/ml) microM. The inhibition of succinate oxidation is pH dependent, due to a pH-dependent change in the overcapacity of the QH2-oxidizing system above the Q-reducing system. If the terminal part of the respiratory chain is reduced with ascorbate, the analogue inhibits the reduction of cytochrome b by substrate in the presence of antimycin with a similar KD value. In the absence of ascorbate the KD value is 100-times higher. The reduction of cytochrome b by substrate in particles treated with 2,3-dimercaptopropanol (BAL) + O2 is also sensitive to HMHQQ, with a KD value in between the two values given above. It is concluded that the QH2 oxidase system contains two different sites for interaction with ubiquinone. The site responsible for the inhibition of steady-state electron transfer is near the Fe-S cluster, as is shown by the sensitivity to the redox state of this cluster and by the effect of HMHQQ on the EPR signal of the reduced cluster. The second site, which is similar to the antimycin-binding site, is occupied only at higher concentrations of inhibitor. The affinity of HMHQQ for this site is not affected by the redox state of the Fe-S cluster.
D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D003573:Cytochrome b Group / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D014450:Electron Transport Complex III; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D011804:Quinolines / Q000494:pharmacology*; D015363:Quinolones; D011808:Quinone Reductases / Q000378:metabolism*; D013367:Submitochondrial Particles / Q000201:enzymology*
D003573:Cytochrome b Group; D009097:Multienzyme Complexes; D011804:Quinolines; D015363:Quinolones; C036314:n-heptadecylmercapto-6-hydroxy-5,8-quinolinequinone; D000968:Antimycin A; D009247:NADH, NADPH Oxidoreductases; D011808:Quinone Reductases; D014450:Electron Transport Complex III