Role of bulk water in the hydrolysis of rhodopsin’s chromophore

Rhodopsin (Rho) is a prototypical G proteincoupled receptor (GPCR) that changes from an inactive conformational state to the G protein-activating state as a consequence of its retinal chromophore isomerization, 11-cisretinal → all-trans-retinal. The photoisomerized chromophore covalently linked to Lys by a Schiff base is subsequently hydrolyzed but little is known about this reaction. Recent research indicates a significant role for tightly bound, transmembrane water molecules in the Rho activation process. Atomic structures of Rho and hydroxyl radical footprinting reveal ordered waters within Rho transmembrane helices which are located close to highly conserved and functionally important receptor residues, forming a hydrogen bond network. Using O labeled H2O, we now report that water from bulk solvent, but not tightly-bound water, is involved in the hydrolytic release of chromophore upon Rho activation by light. Moreover, small molecules, and presumably water, enter the Rho structure from the cytoplasmic side of the membrane. Thus, this work indicates two distinct origins of water vital for Rho function. Rhodopsin (Rho), the visual pigment in retinal rod photoreceptors, is a G protein-coupled receptor (GPCR) responsible for dim light vision (1,2). Rho is composed of its apo-protein, opsin, and a retinylidene chromophore in an 11-cis conformation covalently linked by a Schiff base to Lys of the opsin (Fig. 1A). Photoactivation of Rho is initiated by isomerization of the retinylidene ligand to its all-trans-configuration (3), allowing the photoreceptor after deprotonation of the Schiff base to adopt the active Meta II state required for G protein (transducin) activation. The atomic structure of Rho led to the identification of crystallographically ordered water molecules adjacent to functionally important conserved protein residues (4) (Fig. 1A), supporting the conclusion that these waters are essential, not only for structural stabilization of the receptor but also for the activation process. One of these