Proton Transfers in the Photochemical Reaction Cycle of Proteorhodopsin

Abstract

The spectral and photochemical properties of proteorhodopsin (PR) were determined to compare its proton transport steps to those of bacteriorhodopsin (BR). Static and time-resolved measurements on wild-type PR and several mutants were done in the visible and infrared (FTIR and FT-Raman). Assignment of the observed C=O stretch bands indicated that Asp-97 and Glu-108 serve as the proton acceptor and donor, respectively, to the retinal Schiff base, as do the residues at corresponding positions in BR, but there are numerous spectral and kinetic differences between the two proteins. There is no detectable dark-adaptation in PR, and the chromophore contains nearly entirely all-trans retinal. Because the pK(a) of Asp-97 is relatively high (7.1), the proton-transporting photocycle is produced only at alkaline pH. It contains at least seven transient states with decay times in the range from 10 micros to 200 ms, but the analysis reveals only three distinct spectral forms. The first is a red-shifted K-like state. Proton release does not occur during the very slow (several milliseconds) rise of the second, M-like, intermediate, consistent with lack of the residues facilitating extracellular proton release in BR. Proton uptake from the bulk, presumably on the cytoplasmic side, takes place prior to release (tau approximately 2 ms), and coincident with reprotonation of the retinal Schiff base. The intermediate produced by this process contains 13-cis retinal as does the N state of BR, but its absorption maximum is red-shifted relative to PR (like the O state of BR). The decay of this N-like state is coupled to reisomerization of the retinal to all-trans, and produces a state that is O-like in its C-C stretch bands, but has an absorption maximum apparently close to that of unphotolyzed PR.

Spectroscopic and Photochemical Characterization of a Deep Ocean Proteorhodopsin

“…In GPR, Asp-97 is the proton acceptor from the Schiff base in the corresponding photocycle transition and M formation in GPR_D97N is essentially completely blocked (6). BPR_D97N shows a 7-fold reduced accumulation of M, confirming a similar role of Asp-97 as a proton acceptor in BPR (Fig.…”

Photochemical reaction cycle transitions during anion channelrhodopsin gating

“…7A, red symbols and line), as expected from its predicted position close to the Schiff base (3). The pH titration of the S97E mutant resembled that of the protonated Schiff base counterions in many other microbial retinal proteins, i.e., BR (17) and proteorhodopsins (18,19). Introduction of this Glu results in appearance of a fast phase in M formation in the S97E mutant, which was absent in the WT.…”

Expression of holo-proteorhodopsin in Synechocystis sp. PCC 6803

“…The small contribution of PR to the energy metabolism in Synechocystis observed here will in part be due to its relatively slow turnover (i.e. 'pumping') rate, which may be close to only 10 protons per second (79)(80)(81). Taking the latter value as a starting point one can calculate that in Synechocystis, growing with a doubling time of about 8 h and carrying out predominantly linear electron flow, the retinal-based pump, when expressed at levels of up to 10 5 molecules per cell, contributes to proton motive force generation (and hence ATP production) less than 1 % (this is assuming that one CO 2 fixed requires the pumping of 10 protons).…”