ENERGY-DEPENDENT COMPLEX I-ASSOCIATED UBISEMIQUINONES IN SUBMITOCHONDRIAL PARTICLES

Two distinct species of Complex I-associated ubisemiquinones (SQ(Nf) and SQ(NS) were detected by cryogenic EPR analysis of tightly coupled submitochondrial particles oxidizing NADH or succinate under steady-state conditions, The g = 2.00 signals from both fast-relaxing SQ(NF) (P-1/2 = 170 mW at 40 K) and slow-relaxing SQ(NS) (P-1/2 = 0.7 mW) are sensitive to uncouplers, rotenone and thermally induced deactivation of Complex I, At higher temperatures the SQ(Nf) signal is broadened and only the SQ(NS) signal is seen (P-1/2 = 7 mW at 105 K). The spin-spin interaction between SQ(Nf) and the iron-sulfur cluster N2 was detected as split peaks of the g(parallel to) 2.05 signal with a coupling constant of 1.65 mT, revealing their mutual distance of 8-11 Angstrom. The data obtained are consistent with a model in which N2 and two interacting bound ubisemiquinone species are spatially arranged within the hydrophobic domain of Complex I, participating in the vectorial proton translocation.