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 bo(3) 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 bos 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 Q(H)(.-) 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.