EPR detection of two protein-associated ubiquinone components (SQNf and SQNs) in the membrane in situ and in proteoliposomes of isolated bovine heart complex I

► In many cases, two semiquinones are required to couple electron and proton transfer. ► In complex I, the fast relaxing semiquinone is sensitive to proton motive force. ► The semiquinone is spin-coupled with N2, and may function in “direct” proton pumping. ► In complex I, the slow relaxing semiquinone is insensitive to proton motive force. ► This semiquinone may transport scalar proton and trigger “indirect” proton transfer.