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 sh ares with both of these families the seven helix structure. To generat e 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 rhodopsin s, and the proton pump bacteriorhodopsin. In MII, proton uptake from t he aqueous phase must be involved as well, since its formation increas es the pH of the aqueous medium and is accelerated under acidic condit ions. In the native membrane, the pH effect matches MII formation kine tically, suggesting that intramolecular and aqueous protonation change s contribute in concert to the protein transformation. We show here, h owever, that proton uptake, as indicated by bromocresol purple, and Sc hiff base deprotonation (380-nm absorption change) show different kine tics 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 linke d to Schiff base deprotonation; it is endothermic and depends on the h ydrophobic milieu around the protein. The second step is slightly exot hermic; 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 succ essively formed to generate full catalytic activity for nucleotide exc hange in the G protein.