Characterization of the ΔμH+-sensitive ubisemiquinone species (SQNf) and the interaction with cluster N2:: New insight into the energy-coupled electron transfer in complex I

In this report, we describe the electron paramagnetic resonance (EPR) spectroscopic characterizations of the fast-relaxing ubisemiquinone (SQ(Nf)) species associated with NADH-ubiquinone oxidoreductase (complex 1) detected in tightly coupled submitochondrial particles (SMP). The signals of SQ(Nf) are observed only in the presence of Deltamu(H)+, whereas other slowly relaxing SQ species, SQ(Ns) and SQ(Nx), are not sensitive to Deltamu(H)+. In this study, we resolved the EPR spectrum of the Deltamu(H)+-sensitive SQ(Nf), which was trapped during the steady-state NADH-Q(1) oxidoreductase reaction, as the difference between coupled and uncoupled SMP. Thorough analyses of the temperature profile of the resolved SQ(Nf) signals have revealed previously unrecognized spectra from Deltamu(H)+-sensitive SQNf species. This newly detected SQNf signals are observable only below 25 K, similar to the cluster N2 signals, and exhibit a doublet signal with a peak-to-peak separation (AB) of 56 G. In this work, we identify the partner to the interacting cluster N2. We have analyzed the g = 2.00 and g = 2.05 splittings using a computer simulation program that includes both exchange and dipolar interactions as well as the g-strain effect. Computer simulation of these interaction spectra showed that cluster N2 and fast-relaxing SQNf species undergo a spin-spin interaction, which contains both exchange (55 MHz) and dipolar interaction (16 MHz) with an estimated center-to-center distance of 12 A. This finding delineates an important functional role for this coupled [(N2)(red)-SQ(Nf)] structure in complex I, which is discussed in connection with electron transfer and energy coupling.