EPR characterization of ubisemiquinones and iron-sulfur cluster N2, central components of the energy coupling in the NADH-ubiquinone oxidoreductase (complex I) in situ

The proton-translocating NADH-ubiquinone oxidoreductase (complex I) is the largest and least understood respiratory complex. The intrinsic redox components (FMN and iron-sulfur clusters) reside in the promontory part of the complex. Ubiquinone is the most possible key player in proton-pumping reactions in the membrane part. Here we report the presence of three distinct semiquinone species in complex I in situ, showing widely different spin relaxation profiles. As our first approach, the semiquinone forms were trapped during the steady state NADH-ubiquinone-1 (Q(1)) reactions in the tightly coupled, activated bovine heart submitochondrial particles, and were named SQ(Nf) (fast-relaxing component), SQ(N)s (slow-relaxing), and SQ(Nx) (very slow relaxing). This indicates the presence of at least three different quinone-binding sites in complex I. In the current study, special attention was placed on the SQ(N)f, because of its high sensitivities to Delta(mu) over tilde (H+) and to specific complex I inhibitors (rotenone and piericidin A) in a unique manner. Rotenone inhibits the forward electron transfer reaction more strongly than the reverse reaction, while piericidine A inhibits both reactions with a similar potency. Rotenone quenched the SQ(Nf) signal at a much lower concentration than that required to quench the slower relaxing components (SQ(Ns) and SQ(Nx)). A close correlation was shown between the line shape alteration of the g(parallel to) = 2.05 signal of the cluster N2 and the quenching of the SQ(N)f signal, using two different experimental approaches: (1) changing the Delta(mu) over tilde (H+) poise by the oligomycin titration which decreases proton leak across the SMP membrane; (2) inhibiting the reverse electron transfer with different concentrations of rotenone. These new experimental results further strengthen our earlier proposal that a direct spin-coupling occurs between SQ(Nf) and cluster N2. We discuss the implications of these findings in connection with the energy coupling mechanism in complex I.