Q-band ENDOR (Electron Nuclear Double Resonance) of the high-affinity ubisemiquinone center in cytochrome bo₃ from Escherichia coli

Electron nuclear double resonance (ENDOR) was performed on the protein- bound, stabilized, high-affinity ubisemiquinone radical, Q(H)·^-, of b(O₃) quinol oxidase to determine its electronic spin distribution and to probe its interaction with its surroundings. Until this present work, such ENDOR studies of protein-stabilized ubisemiquinone centers have only been done on photosynthetic reaction centers whose function is to reduce a ubiquinol pool. In contrast, Q(H)·^-, serves to oxidize a ubiquinol pool in the course of electron transfer from the ubiquinol pool to the oxygen-consuming center of terminal b(O₃) oxidase. As documented by large hyperfine couplings (> 10 MHz) to nonexchangeable protons on the Q(H)·^- ubisemiquinone ring, we provide evidence for an electronic distribution on Q(H)·^- that is different from that of the semiquinones of reaction centers. Since the ubisemiquinone itself is physically nearly identical in both QH·^- and the bacterial photosynthetic reaction centers, this electronic difference is evidently a function of the local protein environment. Interaction of Q(H)·^- with this local protein environment was explicitly shown by exchangeable deuteron ENDOR that implied hydrogen bonding to the quinone and by weak proton hyperfine couplings to the local protein matrix.