Identification of Glutamic Acid 113 as the Schiff Base Proton Acceptor in the Metarhodopsin II Photointermediate of Rhodopsin

Abstract

In order to investigate the molecular mechanism of rhodopsin photoactivation, site-directed mutants of bovine rhodopsin were studied by Fourier-transform infrared (FTIR) difference spectroscopy. Rhodopsin mutants E113D and E113A were prepared in which the retinylidene Schiff base counterion, Glu113, was replaced by Asp and Ala, respectively. FTIR difference spectra were recorded and compared with spectra of recombinant native rhodopsin. Both mutant pigments formed photoproducts at 0 degrees C with vibrational absorption bands typical of the metarhodopsin II (MII) state of rhodopsin. The FTIR difference spectrum of E113D was nearly identical to that of rhodopsin. A positive band at 1712 cm-1 caused by the protonation of an internal carboxylic acid in rhodopsin was shifted slightly to 1709 cm-1 in mutant E113D. E113A was studied at acidic pH in the presence of chloride as an inorganic counterion to the protonated Schiff base. The 1712-cm-1 (1709-cm-1) band was absent in the FTIR difference spectrum of mutant E113A. Therefore, we have assigned the 1712-cm-1 absorbance band to the C = O stretching vibration of protonated Glu113 in MII of rhodopsin. These results show that the Schiff base counterion of rhodopsin, the carboxylate side chain of Glu113, becomes protonated during MII formation.

Crystal structure of the ligand-free G-protein-coupled receptor opsin

“…Light induced retinal cis→trans isomerization in rhodopsin causes rearrangements in the retinal binding site, leading to the disrupture of the Schiff base/ Glu-113 salt bridge by proton transfer to Glu-113 [161]. Glu-113 was suggested as Schiff base counterion and predicted to be the cause of energy increase and spectral red shift in the primary photon-induced event [58].…”

G Protein-Coupled Receptor Rhodopsin: A Prospectus

“…The consequence of this movement is the breakage of a salt bridge between Glu 113 and the protonated Schiff base between Lys 296 and the retinal (108). This involves movement of a proton from the donor to the acceptor (142) in conjunction with the motion of the transmembrane helices (136,143). The movement of helix VII away from helix I by 2-4 Å and the pushing of helix VI (64) ∼3 Å by the photolyzed chromophore from the first cytoplasmic loop lead to removal of Glu 247 from the ionic interaction in the DRY region ( Figure 6) [summarized in (136)].…”

How a small change in retinal leads to G‐protein activation: Initial events suggested by molecular dynamics calculations

“…11,32,[54][55][56][57][58][59][60][61][62][63][64][65] This would involve a change in protonation states during the photocycle 33 and experimental work has confirmed that Glu 113 is the dark-state counterion and been suggestive, but not conclusive, that Glu 181 is active during the light-activated stages. 66,67 The simulation results are intriguing in suggesting that this counterion switch mechanism could be present as part of the relaxation mechanism of the protein to the retinal conformational change.…”