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

Abstract

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 I) detected in tightly coupled submitochondrial particles (SMP). The signals of SQ_Nf are observed only in the presence of Δμ_H⁺, whereas other slowly relaxing SQ species, SQ_Ns and SQ_Nx, are not sensitive to Δμ_H⁺. In this study, we resolved the EPR spectrum of the Δμ_H⁺-sensitive SQ_Nf, 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 SQ_Nf signals have revealed previously unrecognized spectra from Δμ_H⁺-sensitive SQ_Nf species. This newly detected SQ_Nf 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 SQ_Nf 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−SQ_Nf] structure in complex I, which is discussed in connection with electron transfer and energy coupling.