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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

Magnitsky, Sergey; Toulokhonova, Larisa; Yano, Takahiro; Sled, Vladimir D; Hägerhäll, Cecilia LU ; Grivennikova, Vera G; Burbaev, Doshimjan S; Vinigradov, Andrei D and Ohnishi, Tomoko (2002) In Journal of Bioenergetics and Biomembranes 34(3). p.193-208
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 (Q_1) reactions in the tightly coupled, activated bovine heart submitochondrial particles, and were named SQ_Nf (fast-relaxing component), SQ_Ns (slow-relaxing), and SQ_Nx (very slow... (More)
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_Ns (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_Nf, because of its high sensitivities to Delta ilde{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 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_vertical = 2.05 signal of the cluster N2 and the quenching of the SQ_Nf signal, using two different experimental approaches: (1) changing the Delta ilde{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 SQ_Nf and cluster N2. We discuss the implications of these findings in connection with the energy coupling mechanism in complex I. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
NADH-ubiquinone oxidoreductase, complex I, EPR, ubisemiquinone, iron-sulfur cluster N2, rotenone, piericidin A, energy-transduction
in
Journal of Bioenergetics and Biomembranes
volume
34
issue
3
pages
193 - 208
publisher
Kluwer
external identifiers
  • scopus:0036321770
ISSN
1573-6881
DOI
10.1023/A:1016083419979
language
English
LU publication?
yes
id
4f6fbfe7-13db-4257-affa-62de495fb64c (old id 124847)
date added to LUP
2007-07-06 10:56:47
date last changed
2017-01-01 04:57:18
@article{4f6fbfe7-13db-4257-affa-62de495fb64c,
  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 (Q_1) reactions in the tightly coupled, activated bovine heart submitochondrial particles, and were named SQ_Nf (fast-relaxing component), SQ_Ns (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_Nf, because of its high sensitivities to Delta ilde{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 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_vertical = 2.05 signal of the cluster N2 and the quenching of the SQ_Nf signal, using two different experimental approaches: (1) changing the Delta ilde{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 SQ_Nf and cluster N2. We discuss the implications of these findings in connection with the energy coupling mechanism in complex I.},
  author       = {Magnitsky, Sergey and Toulokhonova, Larisa and Yano, Takahiro and Sled, Vladimir D and Hägerhäll, Cecilia and Grivennikova, Vera G and Burbaev, Doshimjan S and Vinigradov, Andrei D and Ohnishi, Tomoko},
  issn         = {1573-6881},
  keyword      = {NADH-ubiquinone oxidoreductase,complex I,EPR,ubisemiquinone,iron-sulfur cluster N2,rotenone,piericidin A,energy-transduction},
  language     = {eng},
  number       = {3},
  pages        = {193--208},
  publisher    = {Kluwer},
  series       = {Journal of Bioenergetics and Biomembranes},
  title        = {EPR Characterization of Ubisemiquinones and Iron-Sulfur Cluster N2, Central Components of the Energy Coupling in the NADH-Ubiquinone Oxidoreductase (Complex I) In Situ},
  url          = {http://dx.doi.org/10.1023/A:1016083419979},
  volume       = {34},
  year         = {2002},
}