2 DIFFERENT FORMS OF METARHODOPSIN-II - SCHIFF-BASE DEPROTONATION PRECEDES PROTON UPTAKE AND SIGNALING STATE

Rhodopsin is a retinal protein and a G-protein-coupled receptor; it shares with both of these families the seven helix structure. To generate the G-interacting helix-loop conformation, generally identified with the 380-nm absorbing metarhodopsin II (MII) photoproduct, the retinal Schiff base bond to the apoprotein must be deprotonated. This occurs as a key event also in the related retinal proteins, sensory rhodopsins, and the proton pump bacteriorhodopsin. In MII, proton uptake from the aqueous phase must be involved as well, since its formation increases the pH of the aqueous medium and is accelerated under acidic conditions. In the native membrane, the pH effect matches MII formation kinetically, suggesting that intramolecular and aqueous protonation changes contribute in concert to the protein transformation. We show here, however, that proton uptake, as indicated by bromocresol purple, and Schiff base deprotonation (380-nm absorption change) show different kinetics when the protein is solubilized in suitable detergents. Our data are consistent with a two-step reaction: MI half arrow right over half arrow left MII(a) half arrow right over half arrow left + nH+ MII(b). The first step, with an activation energy E(A) = 160 kJ/mol, is linked to Schiff base deprotonation; it is endothermic and depends on the hydrophobic milieu around the protein. The second step is slightly exothermic; E(A) = 60 kj/mol and n = 2. The transformation of the protein determines the apparent pK(a) of 6.75. From the known pH dependence of G-protein activation, we conclude that MII(a) and MII(b) must be successively formed to generate full catalytic activity for nucleotide exchange in the G protein.