Abstract
The thermal retinal isomerization from all-trans, 15-anti to 13-cis, 15-syn of bacteriorhodopsin in purple membrane in H <sub>2</sub> O and D <sub>2</sub> O during dark adaptation was investigated at 30-55 C at neutral pH. In this temperature range, phase transition of purple membrane and destruction of the tertiary structure of bacteriorhodopsin did not take place. We found that the solvent isotope effect is inverted below about 45 C; i.e., k <sub>f</sub> (D <sub>2</sub> O)/k <sub>f</sub> (H <sub>2</sub> O) > 1. Applying the transition state theory, the changes in enthalpy from the initial state to the transition state along the thermal trans-to-cis forward reaction coordinate, ΔHf <sub>*</sub> , were determined to be 24.7 ± 1.2 and 20.1 ± 0.4 kcal mol <sup>-1</sup> in H <sub>2</sub> O and D <sub>2</sub> O, respectively. The relative entropic change of the transition state in H <sub>2</sub> O and D <sub>2</sub> O, ΔΔSf <sub>*</sub> = ΔSf <sub>*</sub> (D <sub>2</sub> O) - ΔSf <sup>*</sup> (H <sub>2</sub> O), was -14.4 ± 3.9 cal mol <sup>-1</sup> K <sup>-1</sup> . In addition, the Gibbs free energy of trans-to-cis thermal isomerization reaction in D <sub>2</sub> O is 0.4-0.7 kcal mol <sup>-1</sup> lower than that in H <sub>2</sub> O. It is the first time the entropy and enthalpy of the transition state have been quantified to elucidate the solvent isotope effect in the retinal thermal isomerization of bacteriorhodopsin during dark adaptation. The solvent isotope effect on the thermodynamics properties and kinetics implied that the hydrogen bonding in the transition state during the dark adaptation of bR is stronger than that in the initial state. © 2014 American Chemical Society.