ENDOR spectroscopic studies of stable semiquinone radicals bound to the Escherichia coli cytochrome bo(3) quinol oxidase

The putative oxidation of ubiquinol by the cytochrome bo(3) terminal oxidas e of Escherichia coli in sequential one-electron steps requires stabilizati on of the semiquinone. ENDOR spectroscopy has recently been used to study t he native ubisemiquinone radical formed in the cytochrome bo(3) quinone-bin ding site [Veselov, A.V., Osborne, J.P., Gennis, R.B. & Scholes, C.P. (2000 ) Biochemistry 39, 3169-3175]. Comparison of these spectra with those from the decyl-ubisemiquinone radical in vitro indicated that the protein induce d large changes in the electronic structure of the ubisemiquinone radical. We have used quinone-substitution experiments to obtain ENDOR spectra of ub isemiquinone, phyllosemiquinone and plastosemiquinone anion radicals bound at the cytochrome bo(3) quinone-binding site. Large changes in the electron ic structures of these semiquinone anion radicals are induced on binding to the cytochrome bo(3) oxidase. The changes in electronic structure are, how ever, independent of the electronic structures of these semiquinones in vit ro. Thus it is shown to be the structure of this binding site in the protei n, not the covalent structure of the bound quinone, that determines the ele ctronic structure of the protein-bound semiquinone.