The influence of water on the photochemical reaction cycle of proteorhodopsin at low and high pH

Dried samples were prepared from suspension of proteorhodopsin. With HCl and NaOH the pH of the samples was adjusted below and above the pKa of the proton acceptor Asp-97, which was established earlier to be 7.1. Both types of samples were photoactive, and exhibited a truncated photocycle, compared to that measured in suspension. The photocycle of the low pH sample had a K like red shifted intermediate, decaying through an energized PR′ intermediate to the ground state protein. The high pH sample had a more complex photocycle in which beside of the red shifted intermediate an M like intermediate could be identified, having a deprotonated Schiff-base. This blue shifted intermediate decays through an intermediate we designated PR′, which is spectrally identical to the unphotolysed ground state. The humidity and temperature dependence of the photocycle in both cases was studied to understand the role of water in the function of the proteorhodopsin. The effects measured on proteorhodopsin were very similar to that measured earlier on bacteriorhodopsin.