Hydrogen-bonding effect on C-13 and proton hyperfine couplings of [4-C-13]-labeled ubisemiquinones in vitro

EPR spectra of ubiquinone-0 (UQ(0)) and UQ(10) anion radicals selectively labeled with C-13 at the 4-C=O position are reported. The environmental effect on the 4-C-13 splitting constant was studied in mixed solvents in which the molar fraction of the protic component was changed over a wide range. The quinones were electrochemically reduced, with cyclic voltammetry and concomitant optical spectroscopy being performed as controls. The value and the sign of the 4-C-13 hyperfine splitting constant strongly depend on the H-bonding properties (proticity) of the solvent, due to the formation of mono- and disolvates through hydrogen bonding of the carbonyl oxygens in protic solvents, The formation of disolvates is less favorable by a factor of 80 than formation of the monosolvated anion. The results are discussed in light of recent EPR, FTIR, and NMR experiments on the primary C-13-labeled acceptor quinone Q(A) in bacterial photosynthetic reaction centers. We propose that in the neutral state both carbonyls of Q(A) are not or only weakly hydrogen bonded to the protein and that formation of anionic Q(A)(.-) results in a much stronger H-bond for the 4-carbonyl only. The large shift of the 4-C=O IR signal reported for UQ in vivo is in our view mostly due to a change in bond order induced by binding of UQ to the protein, perhaps through a change in sp(2) hybridization at the 4-C position.