Photocycle of the M-form in bacteriorhodopsin mutants without primary proton acceptor Asp-g5

The kinetics of photochemical transformation of a ``yellow{''} deprotonated form (M -> O transition) in bacteriorhodopsin mutants without primary proton acceptor D85 (D85N, D85N/D96N, D85T/E204Q) have been investigated in films at different humidities and in water suspension at different pi and temperatures. The rise of photoinduced O intermediate in double mutant D85N/D96N was slowed down comparing with D85N single mutant and was strongly pH sensitive. That was an-evidence for participation of Asp-96 in Schiff base protonation. The pH-dependencies of O decay in single mutant D85N and double mutant D85T/E204Q were remarkably different, manifesting that terminal group E204 is involved in proton release process in ``yellow{''} photocycle. All results support the idea that such photocycle is coupled with normal proton pumping in the same direction as in the wild type.