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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.

Authors
  • Magnitsky, Sergey
  • Toulokhonova, Larisa
  • Yano, Takahiro
  • Sled, Vladimir D
  • Hägerhäll, Cecilia
  • Grivennikova, Vera G
  • Burbaev, Doshimjan S
  • Vinogradov, Andrei D
  • Ohnishi, Tomoko
Type
Published Article
Journal
Journal of bioenergetics and biomembranes
Publication Date
Jun 01, 2002
Volume
34
Issue
3
Pages
193–208
Identifiers
PMID: 12171069
Source
Medline
License
Unknown

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 .

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