Functional role of Coenzyme Q in the energy coupling of NADH-CoQ oxidoreductase (Complex I): Stabilization of the semiquinone state with the application of inside-positive membrane potential to proteoliposomes

Coenzyme Q(10) (which is also designated as CoQ(10), ubiquinone-10, UQ(10), CoQ, UQ or simply as Q) plays an important role in energy metabolism. For NADH-Q oxidoreductase (complex 1), Ohnishi and Salerno proposed a hypothesis that the proton pump is operated by the redox-driven conformational change of a Q-binding protein, and that the bound form of semiquinone (SQ) serves as its gate [FFBS Letters 579 (2005) 45-55]. This was based on the following experimental results: (i) EPR signals of the fast-relaxing SQ anion (designated as Q(Nf)(center dot-)) are observable only in the presence of the proton electrochemical potential (Delta mu(+)(H)); (ii) iron-sulfur cluster N2 and Q(Nf)(center dot-) are directly spin-coupled; and (iii) their center-to-center distance was calculated as 12 angstrom, but Q(Nf)(center dot-) is only 5 angstrom deeper than N2 perpendicularly to the membrane. After the priming reduction of Q to Q(Nf)(center dot-), the proton pump operates only in the steps between the semiquinone anion (Q(Nf)(center dot-)) and fully reduced quinone (QH(2)). Thus, by cycling twice for one NADH molecule, the pump transports 4H(+) per 2e(-). This hypothesis predicts the following phenomena: (a) Coupled with the piericidin A sensitive NADH-DBQ or Q(1)reductase reaction, Delta mu(+)(H) would be established; (b) Delta mu(+)(H) would enhance the SQ EPR signals; and (c) the dissipation of Delta mu(+)(H) with the addition of an uncoupler would increase the rate of NADH oxidation and decrease the SQ signals. We reconstituted bovine heart complex 1, which was prepared at Yoshikawa's laboratory, into proteoliposomes. Using this system, we succeeded in demonstrating that all of these phenomena actually took place. We believe that these results strongly support our hypothesis.