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Abstract

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 (Q1) reactions in the tightly coupled, activated bovine heart submitochondrial particles, and were named SQNf (fast-relaxing component), SQNS (slow-relaxing), and SQNx (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 SQNf, because of its high sensitivities to DeltamicroH+ 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 SQNf signal at a much lower concentration than that required to quench the slower relaxing components (SQNs and SQNx). A close correlation was shown between the line shape alteration of the g// = 2.05 signal of the cluster N2 and the quenching of the SQNf signal, using two different experimental approaches: (1) changing the DeltamicroH+ 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 SQNf and cluster N2. We discuss the implications of these findings in connection with the energy coupling mechanism in complex .

Evidence for Two Sites of Superoxide Production by Mitochondrial NADH-Ubiquinone Oxidoreductase (Complex I)

“…Possible candidates for this second superoxide-producing site include an unstable semiquinone, such as the ⌬p-sensitive species reported in Ref. 21, or alternatively this superoxide may come from the interaction of this semiquinone and the terminal Fe-S center N2 or, as suggested in Ref. 3, from N2 itself.…”

Inhibitors of the Quinone-binding Site Allow Rapid Superoxide Production from Mitochondrial NADH:Ubiquinone Oxidoreductase (Complex I)

“…EPR studies have detected at least three ubisemiquinone species within complex I, termed SQ Nf , SQ Ns , and SQ Nx for fast relaxing, slow relaxing, and very slow relaxing semiquinone (31)(32)(33). If there is a Q cycle mechanism in complex I, then as well as the canonical quinone reducing site there must be at least one additional quinone reduction and one quinol oxidation site.…”

The Role of External and Matrix pH in Mitochondrial Reactive Oxygen Species Generation

“…Among all possible radicals arising as intermediates of one-electron transport, the semiquinones, emerging in several places of electron transport sequence, are considered the most capable to reduce molecular oxygen producing superoxide anion (14). The semiquinone anion radical (SQ Ϫ ) appears in respiratory complex I and complex III as a product of either oneelectron ubiquinone reduction or one-electron ubiquinol oxidation followed by two protons released into the medium surrounding the inner mitochondrial membrane (15)(16)(17). The elementary reactions resulting in production of SQ Ϫ are presented by Reactions 1 and 2.…”