Distortion of the L→M transition in the photocycle of the bacteriorhodopsin mutant D96N:: a time-resolved step-scan FTIR investigation

The D96N mutant of bacteriorhodopsin has often been taken as a model system to study the M intermediate of the wild type photocycle due to the long life time of the corresponding intermediate of the mutant, Using time-resolved step-scan FTIR spectroscopy in combination with a sample changing wheel we investigated the photocycle of the mutant with microsecond time resolution. Already after several microseconds an intermediate similar to the M-N state is observed, which contrasts with the nil state of the wild type protein. At reduced hydration M and N intermediates similar to those of wild type BR can he detected, These results have a hearing on the interpretation of the photocycle of this mutant, A mechanism is suggested for the fast rise of MN which provides some insight into the molecular events involved in triggering the opening of the cytosolic channel also of the wild type protein. (C) 1999 Federation of European Biochemical Societies.