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EPR Characterization of Ubisemiquinones and Iron–Sulfur Cluster N2, Central Components of the Energy Coupling in the NADH-Ubiquinone Oxidoreductase (Complex I) In Situ

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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 \(\Delta \tilde \mu _{H^ + } \) 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 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 \(\Delta \tilde \mu _{H^ + } \) 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 SQNf and cluster N2. We discuss the implications of these findings in connection with the energy coupling mechanism in complex I.

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Authors and Affiliations

  1. Department of Biochemistry and Biophysics, Room 214A, Anatomy–Chemistry Building, Medical School, University of Pennsylvania, 36th and Hamilton Walk, Philadelphia, Pennsylvania, 19104-6059

    Sergey Magnitsky, Larisa Toulokhonova, Takahiro Yano, Vladimir D. Sled, Cecilia Hägerhäll, Andrei D. Vinogradov & Tomoko Ohnishi

  2. Department of Biochemistry, School of Biology, Moscow State University, Moscow, 119 899, Russia

    Vera G. Grivennikova

  3. Institute of Chemical Physics, Russian Academy of Sciences, Moscow, 117 977, Russia

    Doshimjan S. Burbaev

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  1. Sergey Magnitsky
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  2. Larisa Toulokhonova
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  3. Takahiro Yano
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  4. Vladimir D. Sled
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  5. Cecilia Hägerhäll
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  6. Vera G. Grivennikova
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  7. Doshimjan S. Burbaev
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  8. Andrei D. Vinogradov
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  9. Tomoko Ohnishi
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Magnitsky, S., Toulokhonova, L., Yano, T. et al. EPR Characterization of Ubisemiquinones and Iron–Sulfur Cluster N2, Central Components of the Energy Coupling in the NADH-Ubiquinone Oxidoreductase (Complex I) In Situ. J Bioenerg Biomembr 34, 193–208 (2002). https://doi.org/10.1023/A:1016083419979

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