Dynamics of the proton transfer reaction on the cytoplasmic surface of bacteriorhodopsin

The cytoplasmic surface of bacteriorhodopsin is characterized by a group of carboxylates that function as a proton attractive domain [Checover, S., Na chliel, E., Dencher, N. A., and Gutman, M. (1997) Biochemistry 36, 13919-13 928]. To identify these carboxylates, we selectively mutated them into cyst eine residues and monitored the effects of the dynamics of proton transfer between the bulk and the surface of the protein. The measurements were carr ied out without attachment of a pH-sensor to the cysteine residue, thus avo iding any structural perturbation and change in the surface charge caused b y the attachment of a reporter group, and the protein was in its BR state. The purple membranes were suspended in an unbuffered solution of pyranine ( 8-hydroxypyrene-1,3,6-trisulfonate) and exposed to a train of 1000 laser pu lses (2.1 mJ/pulse, lambda = 355 nm, at 10 Hz). The excitation of the dye e jected the hydroxyl's proton, and a few nanoseconds later, a pair of free p rotons and ground-state pyranine anion was formed. The experimental observa tion was the dynamics of the relaxation of the system;co the prepulse state . The observed signals were reconstructed by a numeric method that replicat es the chemical reactions proceeding in the perturbed space. The detailed r econstruction of the measured signal assigned the various proton-binding si tes with rate constants for proton binding and proton exchange and the pK v alues. Comparison of the results obtained by the various mutants indicates that the dominant proton-binding cluster of the wild-type protein consists of D104, E161, and E234. The replacement of D104 or E161 with cysteine lowe red the proton binding capacity of the cluster to similar to 60% of that of the native protein. The replacement of E234 with cysteine disrupted the st ructure of the cluster, causing the two remaining carboxylates to function as isolated residues that do not interact with each other. The possibility of proton transfer between monomers is discussed.