Inhibition of the M1-->M2 (M(closed) --> M(open)) transition in the D96N mutant photocycle and its relation to the corresponding transition in wild-type bacteriorhodopsin.

Glutaraldehyde, lutetium ions and glycerol inhibit the blue shift of the difference spectra maximum of the M intermediate in the D96N mutant. The M formed has a spectrum indistinguishable from the M intermediate in wild-type bacteriorhodopsin. It has been concluded that the M(open) form previously described by us is identical to the M2 and Mn intermediates postulated by Zimanyi et al. (Photochem. Photobiol. (1992) 56, 1049-1055) and Sasaki et al. (J. Biol. Chem. (1992) 267, 20782-20786), respectively. It is supposed that its formation is accompanied by the appearance of the cytoplasmic proton half-channel. M(open) in the wild-type protein is present in a very low amount due to the shift of the M(closed) <--> M(open) equilibrium towards the M(closed). The inhibitors used do not prevent the multiphase pattern of the M formation in either mutant or wild-type proteins.