Hydration effects on the photocycle of bacteriorhodopsin in thin layers of purple membrane

Abstract

THE purple membrane of Halobacterium halobium acts as a light-driven proton pump, producing a transmembrane proton gradient which is coupled to ATP synthesis¹, and to phototaxis² in the intact bacteria. It contains a single type of protein, bacteriorhodopsin (BR) which spans a 45-Å membrane. The isolated purple membranes are flat oval sheets with an average diameter of 0.5 µm (refs 3, 4). Bacteriorhodopsin contains a retinal molecule (all-trans and 13-cis)⁵ which is covalently bound via a protonated Schiff base to a lysine residue. It undergoes a photocycle described by the following scheme^6–8: where proton ejection to the bulk solution occurs on the route ‘550’ → ‘412’ (refs 9,10), whereas protonation of the bacteriorhodopsin takes place parallel to the , process¹¹. It has been reported that the reconstituted undergoes a cycle which involves the ‘X’ and the ‘610’ intermediates¹². It was demonstrated that proton transfer is a vectorial process where the proton is ejected from one side of the purple membrane and reprotonation takes place on the other side¹³. We present here results on the effects of the specific hydration of the purple membrane on the relaxation times of ‘412’ and on the formation of the ‘660’ and ‘610’ intermediates. The results demonstrate that the full photocycle of bacteriorhodopsin can be observed in thin purple membrane layers even at the lowest hydration state and that the amount of absorbed water is rate limiting for the molecular process of the cycle.