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

• Published in: Journal of bioenergetics and biomembranes Vol. 34; no. 3; pp. 193 - 208
• Main Authors: Magnitsky, Sergey, Toulokhonova, Larisa, Yano, Takahiro, Sled, Vladimir D, Hägerhäll, Cecilia, Grivennikova, Vera G, Burbaev, Doshimjan S, Vinogradov, Andrei D, Ohnishi, Tomoko
• Format: Journal Article
• Language: English
• Published: United States Springer Nature B.V 01.06.2002
• Subjects: Animals; Binding Sites; Biologi; Biological Sciences; Cattle; Coenzymes; complex I; Electron Spin Resonance Spectroscopy; Electron Transport - drug effects; Electron Transport Complex I; energy-transduction; EPR; iron-sulfur cluster N2; Iron-Sulfur Proteins - chemistry; Mitochondria, Heart - chemistry; NADH, NADPH Oxidoreductases - antagonists & inhibitors; NADH, NADPH Oxidoreductases - chemistry; NADH-ubiquinone oxidoreductase; Natural Sciences; Naturvetenskap; piericidin A; rotenone; Sulfur; Ubiquinone - analogs & derivatives; Ubiquinone - chemistry; ubisemiquinone; Uncoupling Agents - pharmacology
• ISSN: 0145-479X; 1573-6881; 1573-6881
• DOI: 10.1023/a:1016083419979

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 .