We had characterized the function of two distinct protein-associated ubisemiquinone molecules in the proton-pumping mechanism in complex I (NADH-UQ oxidoreductase).We constructed most of the frame work of our proton-pumping hypothesis, utilizing EPR techniques before the X-ray structures of bacterial and mitochondrial complex I were reported by Sazanov's group and Brandt and his collaborators, respectively. The fast relaxing semiquinone (SQ-Nf) signal is extremely sensitive to the proton motive force (ΔP) imposed to the energy transducing membrane, strongly indicating its direct involvement in proton-pumping mechanism. Slow relaxing semiquinone (SQ-Ns) is not sensitive to ΔP. Although they show identical piericidin A sensitivity, they differ in rotenone sensitivity considerably as well as their SQ binding subunits. These differences were exploited using tightly coupled bovine heart submitochondrial particles with a high respiratory control ratio (>8).We determined the center-to-center distance of 12 A between SQ-Nf and its direct electron donor, iron-sulfur cluster N2 based on their spin-spin interaction analysis. We have extended this work using reconstituted bovine heart complex I proteoliposomes which shows a respiratory control ratio >5. Our recent Q-band (33.9 GHz) EPR analysis of SQ-Nf and SQ-Ns spectra in the reconstituted proteoliposome system also supports our two-semiquinone model. We will compare our EPR-based model with the X-ray structure based proton-pumping models by Sazanov's group and Brandt with his collaborators.