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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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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
Springer
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
2016-04-01 12:13:37
date last changed
2022-02-03 19:17:52
@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}},
  keywords     = {{NADH-ubiquinone oxidoreductase; complex I; EPR; ubisemiquinone; iron-sulfur cluster N2; rotenone; piericidin A; energy-transduction}},
  language     = {{eng}},
  number       = {{3}},
  pages        = {{193--208}},
  publisher    = {{Springer}},
  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}},
  doi          = {{10.1023/A:1016083419979}},
  volume       = {{34}},
  year         = {{2002}},
}