EPR characterization of ubisemiquinones and iron-sulfur cluster N2, central components of the energy coupling in the NADH-ubiquinone oxidoreductase (complex I) in situ.
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 .
Magnitsky
Sergey
S
Department of Biochemistry and Biophysics, Medical School, University of Pennsylvania, Philadelphia 19104-6059, USA.
Toulokhonova
Larisa
L
Yano
Takahiro
T
Sled
Vladimir D
VD
Hägerhäll
Cecilia
C
Grivennikova
Vera G
VG
Burbaev
Doshimjan S
DS
Vinogradov
Andrei D
AD
Ohnishi
Tomoko
T
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003067:Coenzymes; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport / Q000187:drug effects; D042967:Electron Transport Complex I; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D008929:Mitochondria, Heart / Q000737:chemistry; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000737:chemistry*; D014475:Uncoupling Agents / Q000494:pharmacology
D003067:Coenzymes; D007506:Iron-Sulfur Proteins; D014475:Uncoupling Agents; D014451:Ubiquinone; D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I; C024989:coenzyme Q10