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 (SQNf) species associated with NADH−ubiquinone oxidoreductase (complex I) detected in tightly coupled submitochondrial particles (SMP). The signals of SQNf are observed only in the presence of ΔμH + , whereas other slowly relaxing SQ species, SQNs and SQNx, are not sensitive to ΔμH + . In this study, we resolved the EPR spectrum of the ΔμH + -sensitive SQNf, which was trapped during the steady-state NADH−Q1 oxidoreductase reaction, as the difference between coupled and uncoupled SMP. Thorough analyses of the temperature profile of the resolved SQNf signals have revealed previously unrecognized spectra from ΔμH + -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 (ΔB) 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 Å. This finding delineates an important functional role for this coupled [(N2)red−SQNf] structure in complex I, which is discussed in connection with electron transfer and energy coupling.