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

The kinetics of photochemical transformation of a "yellow" deprotonated for m (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 tempera tures. The rise of photoinduced O intermediate in double mutant D85N/D96N w as slowed down comparing with D85N single mutant and was strongly pH sensit ive. That was an-evidence for participation of Asp-96 in Schiff base proton ation. The pH-dependencies of O decay in single mutant D85N and double muta nt D85T/E204Q were remarkably different, manifesting that terminal group E2 04 is involved in proton release process in "yellow" photocycle. All result s support the idea that such photocycle is coupled with normal proton pumpi ng in the same direction as in the wild type.