Energy-dependent Complex I-associated ubisemiquinones in submitochondrial particles.

Two distinct species of Complex I-associated ubisemiquinones (SQNf and SQNs) 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 SQNf (P1/2 = 170 mW at 40 K) and slow-relaxing SQNs (P1/2 = 0.7 mW) are sensitive to uncouplers, rotenone and thermally induced deactivation of Complex I. At higher temperatures the SQNf signal is broadened and only the SQNs signal is seen (P1/2 = 7 mW at 105 K). The spin-spin interaction between SQNf and the iron-sulfur cluster N2 was detected as split peaks of the g parallel 2.5 signal with a coupling constant of 1.65 mT, revealing their mutual distance of 8-11 A. 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.