Effects of the crystalline structure of purple membrane on the kinetics and energetics of the bacteriorhodopsin photocycle.
Time-resolved difference spectra were measured for Triton X-100 solubilized bacteriorhodopsin monomers between 100 ns and 100 ms after photoexcitation. The results are consistent with the general scheme K in equilibrium L in equilibrium M1 in equilibrium M2 in equilibrium N in equilibrium O----BR proposed previously for purple membranes [Váró, G., & Lanyi, J.K. (1990) Biochemistry 29, 2241-2250]. The rate constants which involve proton release or uptake, i.e., kLM1, kNO, and kON, were significantly higher in the monomeric protein than in purple membrane; the other steps were less affected. Analysis of the temperature dependencies of the rate constants between 5 and 30 degrees C yielded the enthalpies and entropies of activation for all steps except the two absent back-reactions. Comparison of these with data for purple membranes [Váró, G., & Lanyi, J.K. (1991) Biochemistry 30, 5016-5022] shows that the crystalline structure affects the energetics of the photocycle. In bacteriorhodopsin immobilized by the lattice of the purple membrane, the entropy changes leading to all transition states are more positive. Thus, the forward reactions proceed with less conformational hindrance. However, the thermal (enthalpic) barriers are higher. These effects are particularly pronounced for the M1----M2 and O----BR reactions. Large changes of the enthalpy and entropy levels of intermediates in the M2----BR reaction segment, but not in the K----M1 segment, upon solubilization of the protein are consistent with our earlier proposal that major protein conformational changes occur in the photocycle and they begin with the M1----M2 reaction.
Váró
G
G
Department of Physiology and Biophysics, University of California, Irvine 92717.
Lanyi
J K
JK
D001436:Bacteriorhodopsins / Q000737:chemistry*; D003460:Crystallization; D004734:Energy Metabolism; D007700:Kinetics; D010084:Oxidation-Reduction; D010777:Photochemistry; D010788:Photosynthesis; D013057:Spectrum Analysis; D013816:Thermodynamics
D001436:Bacteriorhodopsins