The photocycle and the proton pumping kinetics of bacteriorhodopsin, as well as the transfer rate of protons from the membrane surface into the aqueous bulk phase were examined for purple membranes in water and in ice. In water, the optical pH indicator pyranine residing in the aqueous bulk phase monitors the H+-release later than the pH indicator fluorescein covalently linked to the extracellular surface of BR. In the frozen state, however, pyranine responds to the ejected H+ as fast as fluorescein attached to BR, demonstrating that the surface/bulk transfer is in ice no longer rate limiting. The pumped H+ appears at the extracellular surface during the transition of the photocycle intermediate L550 to the intermediate M412. The Arrhenius plot of the M formation rate suggests that the proton is translocated through the protein via an ice-like structure.
