Regeneration of bacteriorhodopsin (bR) from the M intermediate via a new intermediate P has been studied. In the purple sheets treated with 0.015% Triton X-100 (the P → bR transition is suppressed), the α-maximum of absorption of P is located at 560 ± 5 nm, the extinction coefficient being equal to 0.7 ± 0.1 of that of bR. Besides, there is a small but measurable absorbance increase at 330–350 nm, which seems to be due to a β-maximum of 13-cis-retinal residue in P. The similarity of the α-maximum of P and bR suggests the Schiff base nitrogen to be protonated at the M → P transition. The kinetics of P → bR transition measured at the 335 nm absorbance decrease coincides with that of proton uptake accompanying bR regeneration after flash. A scheme is proposed assuming that H+ absorption from the water phase is coupled to the 13-cis → all-trans isomerization of retinal residue in which the Schiff base is already protonated by a proton-donor group of the protein.
