Optical study of the light-induced protonation changes associated with the metarhodopson II intermediate in rod-outer-segment membranes.

The kinetics of the protonation changes and of the formation of the metarhodopsin II intermediate are measured in the presence of the pH indicator dye bromocresol purple after two consecutive flashes on the same suspension of right-side-out vesicles from cattle rod-outer-segment membranes. A rapid proton binding followed by partial proton release is observed. The formation of metarhodopsin II is found to be biphasic. The rapid phase of metarhodopsin II formation is slightly faster than the proton binding, and the kinetics of the slow phase are similar to that of the proton release. The protein groups which are protonated and deprotonated are shown to be situated on the outside of the vesicles. These results provide evidence that a structural change of the protein involving pK changes occurs without spectral modification before the formation of metarhodopsin III (half-time of the order of seconds).