Asp76 Is the Schiff Base Counterion and Proton Acceptor in the Proton-Translocating Form of Sensory Rhodopsin I

Abstract

Both sensory rhodopsin I, a phototaxis receptor, and bacteriorhodopsin, a light-driven proton pump, have homologous residues which have been identified as critical for bacteriorhodopsin functioning. This includes Asp76, which in the case of bacteriorhodopsin (Asp85) functions as both the Schiff base counterion and the proton acceptor. Sensory rhodopsin I exists in a pH dependent equilibrium between two different forms in the absence of its transducer protein HtrI. At pH below 7, it exists primarily in a blue form (lambda max = 587 nm) which functions as a phototaxis signal transducer when complexed to HtrI, while at higher pH, it converts to a purple proton-transporting form similar to bacteriorhodopsin (lambda max = 550 nm). We report ATR-FTIR difference spectra obtained from both low- and high-pH forms of purified sensory rhodopsin I reconstituted into lipid vesicles. The low-pH species has an ethylenic C = C stretch mode at 1520 cm-1 which shifts to 1526 cm-1 in the high-pH form. No frequency shift was found for the mutant D76N, in agreement with visible absorption measurements. Weak negative/positive bands at 1763/1751 cm-1 previously assigned to a perturbation of the C = O stretch mode of Asp76 during S373 formation in the low-pH form are replaced by a single intense positive band near 1749 cm-1 in the high-pH form. These results along with the effects of H/D exchange show that Asp76 is protonated in the signal-transducing form of sensory rhodopsin I and is ionized and functions as the counterion and Schiff base proton acceptor in the proton-transporting high-pH form of sensory rhodopsin I similar to bacteriorhodopsin.

A Schiff base connectivity switch in sensory rhodopsin signaling

“…1 A Inset), whereas the slow negative current is pH-independent in the range examined. Mutation to Asn of Asp-76, the homolog to the Schiff base proton acceptor in BR and SRII, previously shown to be a Schiff base proton acceptor in purified SRI at alkaline pH (27), eliminates the fast positive photocurrent (Fig. 1 A).…”

Primary reactions of sensory rhodopsins

“…A second peak is seen at 400 nm. The absorption spectrum is characteristic for the ''blue form'' of SRI expected for the ambient pH value (6.0) of the sample (22,28). The red shift of the main absorption band relative to BR indicates the protonation of Asp-76 (28), which corresponds to Asp-85 in BR, located in the vicinity of the Schiff base.…”

Proton Transfers in a Channelrhodopsin-1 Studied by Fourier Transform Infrared (FTIR) Difference Spectroscopy and Site-directed Mutagenesis

“…For example, neutralization of Asp-85 in BR at low pH accounts for its observed red shift (37). The red-shifted max of sensory rhodopsin I at pH below 7 is also accounted for by neutralization of the Asp-76 SB proton counterion (38). However, other factors such the existence of an internal water molecule(s) positioned near the SB as in the case of W402 in BR (17) are also likely to play a role in the determination of the wavelength of visible absorption.…”