Light-induced changes in H+ binding to the purple membrane. Effect of pH, light, temperature, and ionic strength.

Abstract

Under continuous illumination, isolated planar sheets of purple membrane from Halobacterium halobium acidify the surroundings at alkaline pH. This light-induced change in H+ binding to the purple membrane (delta h) was studied by differential titration under varying conditions of pH, temperature, ionic strength, salt composition, light intensity, and wavelength. A maximum acidification was found between pH 9 and 10, with delta h less at neutral or more alkaline pH, consistent with a previously proposed three-state model. The light intensity and wavelength dependence also support this model. The temperature dependence of delta h, interpreted in terms of the three-state model, is anomalous. The apparent enthalpy of proton dissociation (delta H0) is -6 kcal/mol, a value of opposite sign to the expected delta H0 for a group of pK = 10. The apparent activation energy (Ea) for proton uptake is 14 kcal/mol in 15 mM NaCl and 18 kcal/mol in 3 M KCl, 5 to 10 times too large for a diffusion-limited proton transfer reaction from water. However, both delta H0 and Ea are consistent with conformational changes linked to light-independent proton dissociation and pump-dependent proton uptake. An increase in ionic strength increases delta h. This effect is shown to be quantitatively explained by a high negative electrostatic surface potential, which accumulates protons in a diffuse electrical double layer.