The G-protein-coupled receptor rhodopsin is activated by photoconversion of its covalently bound ligand 11-cis-retinal to the agonist all-trans-retinal. After lightinduced isomerization and early photointermediates, the receptor reaches a G-protein-dependent equilibrium between active and inactive conformations distinguished by the protonation of key opsin residues. In this report, we study the role of the 9-methyl group of retinal, one of the crucial steric determinants of light activation. We find that when this group is removed, the protonation equilibrium is strongly shifted to the inactive conformation. The residually formed active species is very similar to the active form of normal rhodopsin, metarhodopsin II. It has a deprotonated Schiff base, binds to the retinal G-protein transducin, and is favored at acidic pH. Our data show that the normal proton transfer reactions are inhibited in 9-demethyl rhodopsin but are still mandatory for receptor activation. We propose that retinal and its 9-methyl group act as a scaffold for opsin to adjust key proton donor and acceptor side chains for the proton transfer reactions that stabilize the active conformation. The mechanism may also be applicable to related receptors and may thus explain the partial agonism of certain ligands.The retinal photoreceptor rhodopsin is one of the archetypes of the G-protein-coupled receptor superfamily. It is composed of the apoprotein opsin comprising seven transmembrane helices (TMs) 1 and the chromophore 11-cis-retinal, which is covalently bound to Lys 296 in TM7 via a protonated Schiff base (SB), keeps the receptor in the inactive conformation, and acts as a highly effective inverse agonist. Following the absorption of a photon, the retinal isomerizes to a strained all-trans-conformation (1), which induces a series of conformational rearrangements of the opsin moiety. The final product of this reaction sequence is the active conformation (R*) that contains all-trans-retinal as a covalently bound agonist and is capable of catalyzing the activation of the retinal G-protein transducin (G t ) (for reviews see Refs. 2-4).These events can be summarized as shown in Fig. 1A). After cis-trans-isomerization and initial, short lived intermediates, illuminated rhodopsin progresses from the lumirhodopsin form to the first long lived state, metarhodopsin I (meta I). Up to and including meta I, the SB of the pigment remains protonated. Deprotonation of the SB and protonation of its counterion Glu 113 mark the transition from the meta I ( max ϭ 478 nm) to the metarhodopsin II (meta II) conformation (5), which is characterized by a strongly blue-shifted absorbance maximum ( max ϭ 380 nm). SB deprotonation is known to be necessary for interaction with G t (6 -8) and is accompanied by the uptake of a proton from the aqueous solution (9 -11). pH rate profiles of G t activation and proton uptake measurements have shown the opsin residue Glu 134 to be an important mediator of the uptake or even the acceptor of this proton (12, 13). Based on computer ...