Alternative translocation of protons and halide ions by bacteriorhodopsin.

Abstract

however, the transport rate decreases by a factor of -2 in bRHFr compared with bRHCI'. The data indicate that in the acid purple form bR transports the halide anions instead of protons. We present a testable model for the transport mechanism, which should also be applicable to halorhodopsin.Halobacterium halobium contains two light energytransducing pigments: bacteriorhodopsin, with an absorption maximum at 568 nm (bR5M), which transports H+ out of the cell; and halorhodopsin (hR), which transports Cl-into the cell. In the cell membrane and in the purified state, bR occurs in the form of small membrane patches, the purple membrane, which are two-dimensional crystals ofbR and lipid (for review, see refs. 1-4). Both pigments are intrinsic membrane proteins, contain nearly identical all-trans-retinylidene chromophores, and show extensive sequence homologies (for review, see ref. 5).The absorption maximum of bR is red-shifted from 568 to 605 nm at low pH, forming blue bR, or bRF d, and transport activity ceases (6-8). The same effect is obtained by removing residual metal cations with an ion-exchange resin because the high negative surface charge of the purple membrane then drops the surface pH below 1.0 (9, 10). The addition of Clreverses the red shift and restores charge transport in this "acid purple" form (bR5HC) (11). The absorption changes accompanying charge transport ofbR568 are changed in bR5'and resemble those of hR (12). The question arises, which ion is translocated by the bR54 form?Direct measurements of the expected changes in Cl-or H+ concentration under the required low pH conditions are difficult and so far have not succeeded. However, substantial information on the ion transport in bR and hR has been obtained by measuring the kinetics of the absorption changes and charge shifts that accompany ion transport, and these should be sensitive to the properties of the transported ion. We have, therefore, measured current and absorption kinetics ofpurple membrane after substituting 2H' for H+ and Brfor Cl-. Techniques and results of photoelectric measurements have recently been reviewed comprehensively (13) and will not be described here. MATERIALS AND METHODSThe bR-containing purple membrane fragments were prepared from H. halobium strain JW-3 (ET 1001) according to the standard procedure (14). After orientation in an electric field, they were immobilized in a polyacrylamide gel (15), and slabs of 10 x 5 x 2 mm were placed in a cuvette. The gel samples were preequilibrated in the desired solution overnight, and the pH was monitored with an OP-0808 electrode (Radelkis, Budapest) and adjusted by adding appropriate amounts ofconcentrated HCI or HBr solutions (Aldrich). The measured p2H values were corrected by subtracting 0.41 pH unit.Optical and electrical signals were measured after laser pulse excitation at 532 nm from a frequency-doubled neodymium-yttrium/aluminum garnet (Nd-YAG) laser with 8-ns pulse length and 1.5-mJ pulse energy. To record photosteadystate currents, we used 0.8-to 4-s-long illuminatio...

Anion sensitivity and spectral tuning of cone visual pigments in situ.

“…It seems likely, therefore, that the Schiff base counterion in hR consists of D238, R108, and an exchangeable (and transportable) Cl-ion, which takes the place of the lost carboxylate and hydrogen bonds to T111. Der et al (32) have offered a similar argument to explain light-driven Cl-pumping by hR at normal pH and by bR at acid pH. Interestingly, in the bovine rhodopsin mutant E113Q, external Cl-also appears to be able to substitute for a carboxylate as the Schiff base counterion (33)(34)(35).…”

Residue replacements of buried aspartyl and related residues in sensory rhodopsin I: D201N produces inverted phototaxis signals.

“…The D201N results, therefore, genetically separate the attractant and repellent signal generation processes, which were previously concluded to be distinct based on photophysiological measurements (13 (6). A key residue involved in mediating proton pumping in BR is the Schiff base proton acceptor, Asp-85, which together with Asp-212 and Arg-82 forms a counterion for the protonated Schiff base (19)(20)(21)(22)(23). When Asp-85 is protonated by lowering the pH or substituted with a neutral residue, the absorption maximum of BR shifts from 568 to -590 nm and the pumping activity is eliminated (24).…”

Specificity of Anion Binding in the Substrate Pocket of Bacteriorhodopsin

“…In addition, a form of wild-type bacteriorhodopsin known as the acid-blue state, which occurs at sufficiently low pH to protonate residue 85, has also been shown to bind and pump both chloride and bromide (1,4,5). Thus, the possibility exists that simply neutralizing a single amino-acid can convert bacteriorhodopsin from an outward-directed proton pump to an inward-directed anion pump.…”