The N intermediate of bacteriorhodopsin at low temperatures: Stabilization and photoconversion

In aqueous suspensions of purple membranes (pH 10.2, 0.4 M KCl) an intermediate having an absorption maximum at 570–575 nm (at −196°C) was produced by first heating the M intermediate up to −30°C and then stabilizing it by subsequent cooling to −60°C. We suggest that this species is the intermediate N (or P or R) found and characterized earlier near room temperature. Upon illumination at −196°C N is transformed into a bathochromically absorbing species K n which has an absorption maximum near 605 nm and an extinction 1.35 times that of N. This light reaction is photoreversible. The quantum yield ratio for the forward and back reaction is 0.18 ± 0.02. The maximum photo steady state concentration of K n is about 0.24. The N intermediate was also trapped in water suspensions of purple membranes at neutral pH and low salt concentration by illumination at λ > 620 nm during cooling. In addition to N another intermediate absorbing in the red (maximum at 610–620 nm) was accumulated in smaller amounts. It is not photoactive at 196°C and apparently is the O intermediate or a photoproduct of N.