The Photochemical Reaction Cycle of Proteorhodopsin at Low pH

Abstract

The proton acceptor group in the recently described retinal protein, proteorhodopsin has an unusually high pK(a) of 7.1. It was shown that at pH above this pK(a), illumination initiates a photocycle similar to that of bacteriorhodopsin, and the protein transports proton across the cell membrane. Recently it was reported that proteorhodopsin, unlike bacteriorhodopsin, transports protons at pH below the pK(a) of the proton acceptor, and this transport is in the reverse direction. We have investigated the photocycle of proteorhodopsin at such low pH. At pH 5, three spectrally distinct intermediates K, L, and N, and another spectrally silent one, PR', could be identified, but a deprotonated Schiff base containing M-like intermediate, characteristic for proton pumping activity, does not accumulate. All the reactions between the intermediates are close to equilibrium, except the last transition from PR' to PR, when the protein returns to its initial unexcited state in a quasiunidirectional reaction. The electric signal measurements indicate that although charge motions are detected inside the protein, their net dislocation is zero, indicating that contrary to the earlier reported, at low pH no charged particle is transported across the membrane.

Lipid Bilayer Composition Can Influence the Orientation of Proteorhodopsin in Artificial Membranes

“…pR has seven transmembrane a-helices that enclose a retinal chromophore. On light absorption and under conditions of neutral pH, the protein undergoes a series of conformational shifts that translocate one proton across the membrane from the C-terminal face of pR to its N-terminal face (23). Interestingly, insertion into the lipid bilayer is required for pR to be functional.…”

Microbial Rhodopsins: Phylogenetic and Functional Diversity

“…The new pigment, designated GPR (λ max = 525 nm), exhibited a photochemical reaction cycle with intermediates and kinetics characteristic of archaeal proton-pumping rhodopsins. Its transport, spectroscopic, and photochemical reactions have now been characterized by a number of laboratories in Escherichia coli-expressed forms (Beja et al, 2000;Beja et al, 2001;Dioumaev et al, 2002;Dioumaev et al, 2003;Friedrich et al, 2002;Krebs et al, 2002;Lakatos et al, 2003;Man et al, 2003). The efficient proton pumping and rapid photocycle (15 ms halftime) of the new pigment strongly suggested that proteorhodopsin functions as a proton pump in its natural environment.…”

Voltage- and pH-Dependent Changes in Vectoriality of Photocurrents Mediated by Wild-type and Mutant Proteorhodopsins upon Expression in Xenopus Oocytes

“…Thus, the structurally and functionally important amino acids are highly conserved, with the notable exception of Glu-108 (Asp-96 in BR) and the carboxylic acid residues of the extracellular proton release cluster, which are not present in PR. The spectral properties and photocycle characteristics of PR have been thoroughly analyzed by UV-Vis and Fourier-transformed infrared spectroscopy , 1,[17][18][19][20][21][22][23][24][25][26] and also proton pumping has been investigated in reconstituted and cellular expression systems. 17 Typical for light-driven proton pumps, the PR photocycle at alkaline pH consists of the archetypical intermediates M and O and has a short turnover time of~20 ms, with decay of an O-like state as rate-limiting step.…”

“…These include, first, the typical absorption changes at around 400 nm, indicative of formation and decay of an M-like state with a deprotonated SB, which could not be observed within the time resolution of spectroscopic experiments at ambient temperatures. 1,[17][18][19][20][21][22][23] Second, the direction of proton pumping was found to reverse in a pH-and potential-dependent manner. 17 Although the inversion of pumping direction was shown in two independent functional assays, this property has been particularly challenged by other groups.…”

“…17 Although the inversion of pumping direction was shown in two independent functional assays, this property has been particularly challenged by other groups. 19,22 In this study, we used the Xenopus oocyte expression system to analyze the voltage dependence of stationary or transient photocurrents of wild-type (WT) and mutant PRs under a variety of extracellular and intracellular pH conditions. The appearance and lifetime of an M-like intermediate was probed by measuring the time course of blue and green laser flash-induced transient currents of PR during and after continuous illumination with green light, in each case using BR as a reference.…”