VOLUME AND ENTHALPY CHANGES IN THE EARLY STEPS OF BACTERIORHODOPSIN PHOTOCYCLE STUDIED BY TIME-RESOLVED PHOTOACOUSTICS

We have studied the photoinduced volume changes, energetics, and kinet ics in the early steps of the bacteriorhodopsin (BR) photocycle with p ulsed, time-resolved photoacoustics. Our data show that there are two volume changes. The fast volume change (less than or equal to 200 ns) is an expansion (2.5 +/- 0.3 Angstrom(3)/molecule) and is observed exc lusively in the purple membrane (PM), vanishing in the 3-[(3-cholamido propyl)-dimethylammonio]- BR sample; the slow change (similar to 1 mu S) is a volume contraction (-3.7 +/- 0.3 Angstrom(3)/molecule). The fa st expansion is assigned to the restructuring of the aggregated BR in the PM, and the 1-mu s contraction to the change in hydrogen bonding o f water at Asp 212 (Kandori et al. 1995. J. Am. Chem. Sec. 117:2118-21 19). The formation of the K intermediate releases most of the absorbed energy as heal, with Delta H-K = -36 +/- 8 kJ/mol. The activation ene rgy of the K --> L step is 49 +/- 6 kJ/mol, but the enthalpy change is small, -4 +/- 10 kJ/mol. On the time scale we studied, the primary ph otochemical kinetics, enthalpy, and volume changes are not affected by substituting the solvent D2O for H2O. Comparing data on monomeric and aggregated BR, we conclude that the functional unit for the photocycl e is the BR monomer, because both the kinetics (rate constant and acti vation energy) and the enthalpy changes are independent of its aggrega tion state.