Hydrogen-Bonding Effect on ¹³C and Proton Hyperfine Couplings of [4-¹³C]-Labeled Ubisemiquinones in Vitro

EPR spectra of ubiquinone-0 (UQ 0 ) and UQ 10 anion radicals selectively labeled with 13 C at the 4-C O position are reported. The environmental effect on the 4- 13 C 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- 13 C 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 13 C-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.