Transmembrane location of retinal in bacteriorhodopsin by neutron diffraction

We report that the replacement of Leu-93 in bacteriorhodopsin by Ala (L93A) or Thr (L93T) slows down the photocycle by -100-fold relative to wild-type bacteriorhodopsin. Time-resolved visible absorption spectroscopy and resonance Raman experimnents, respectively, show the presence of long-lived O4ike and N-like intermediates in the photocycles of the above mutants. We infer the existence of an equilibrium between the N and 0 intermediates in the photocycles of these mutants. The L93A and L93T mutants exhibit normal proton pumping under continuous illumination, suggesting that the decay of the N and/or 0 intermediate, and consequently, proton translocation, can be accelerated by the absorption of a second photon. Since the 13-cis --all-trans reisomerization of retinal is completed during the decay of the N and 0 intermediates, we conclude that the interaction of Leu-93 with retinal is important in this phase of the photocycle. This conclusion is supported by a recent structural model of bacteriorhodopsin that suggests that Leu-93 is near the C-13 methyl group of retinal. intermediate (17). Both isomerization steps are generally believed to be associated with changes in protein conformation. To understand how changes in retinal geometry are coupled to conformational changes in the protein, we are carrying out systematic replacements of amino acids that line the retinal binding pocket. We report here that the interaction of Leu-93, a residue in helix C (Fig. 1), with retinal has an important role in the photocycle and in light/dark adaptation. Time-resolved visible spectroscopic studies show that the half-time for the decay of the 0 intermediate of the photocycle is increased to -800 ms in the mutants where Leu-93 was replaced by Ala (L93A) or Thr (L93T) as compared to a rate of <5 ms for wild-type bR. Resonance Raman (RR) experiments provide evidence for the presence ofa long-lived N intermediate in the photocycles of these mutants. We conclude that Leu-93 in bR is involved in coupling protein conformational changes that occur during the decay of the N and 0 intermediates of the photocycle to 13-cis -+ all-trans reisomerization of the chromophore.

Section: Discussionmentioning

confidence: 67%

Replacement of leucine-93 by alanine or threonine slows down the decay of the N and O intermediates in the photocycle of bacteriorhodopsin: implications for proton uptake and 13-cis-retinal----all-trans-retinal reisomerization.

Subramaniam

Greenhalgh

Rath

et al. 1991

“…Fourier transform infrared measurements indicate that in the photocycle Asp-96 remains protonated until the decay of M (6), consistent with its postulated deprotonation in the M --N transition. The SB is located in the middle of the membrane (22), and Asp-96 is located approximately halfway between the SB and the cytoplasmic end of helix C (R. Henderson, personal communication). Thus, Asp-96 is in a proper position to serve as an internal donor in the proton-uptake pathway.…”

Section: Resultsmentioning

confidence: 99%

Aspartic acid-96 is the internal proton donor in the reprotonation of the Schiff base of bacteriorhodopsin.

Otto

Marti

Holz

et al. 1989

346

351

Above pH 8 the decay of the photocycle intermediate M of bacteriorhodopsin splits into two components: the usual millisecond pH-independent component and an additional slower component with a rate constant proportional to the molar concentration of HI, 1H+]. In parallel, the charge translocation signal associated with the reprotonation of the Schiff base develops a similar slow component. These observations are explained by a two-step reprotonation mechanism. An internal donor ru-st reprotonates the Schiffbase in the decay of M to N and is then reprotonated from the cytoplasm in the N -O 0 transition. The decay rate of N is proportional to [HI].By postulating a back reaction from N to M, the M decay splits up into two components, with the slower one having the same pH dependence as the decay of N. Photocycle, photovoltage, and pH-indicator experiments with mutants in which aspartic acid-96 is replaced by asparagine or alanine, which we call D96N and D96A, suggest that Asp-96 is the internal proton donor involved in the re-uptake pathway. In both mutants the stoichiometry of proton pumping is the same as in wild type. However, the M decay is monophasic, with the logarithm of the decay time [log (7)] linearly dependent on pH, suggesting that the internal donor is absent and that the Schiff base is directly reprotonated from the cytoplasm. Like HI, azide increases the M decay rate in D96N. The rate constant is proportional to the azide concentration and can become >100 times greater than in wild type. Thus, azide functions as a mobile proton donor directly reprotonating the Schiffbase in a bimolecular reaction. Both the proton and azide effects, which are absent in wild type, indicate that the internal donor is removed and that the reprotonation pathway is different from wild type in these mutants.Bacteriorhodopsin (bR) is a light-driven proton pump from Halobacterium halobium that transports H+ ions from the cytoplasm to the extracellular space with a stoichiometry of one proton per cycle (1). The transmembrane H+ translocation involves distinct electrogenic steps associated with H+ ejection from the protein interior into the periplasm and the subsequent H+ rebinding from the cytoplasmic side of the membrane. The proton uptake occurs on the millisecond time scale and is apparently coupled to the reprotonation of the Schiff base (SB) and the decay of the M intermediate of the photochemical cycle. Fourier transform infrared (FTIR) spectroscopy first indicated that several aspartate carboxyl groups undergo protonation changes during the photocycle (2, 3). Site-directed mutagenesis of bR showed that substitution of aspartate residues at positions 85, 96, and 212 by asparagine reduced the proton pumping activity to a few percent (4) and revealed, in combination with FTIR measurements, the protonation states of specific aspartate residues in various photocycle intermediates (5, 6). In the mutant D96N, the kinetics of proton uptake is affected (7-9). We recently showed that the low steady-state proton pumping act...

“…The Schiff base is located near the center of the membrane (25), but the rapid exchange of the Schiff base proton in the dark and the effect of azide in a bR mutant indicate that diffusion pathways to and from the membrane surfaces exist (26,27). We postulate that an increased positive electrical potential near the Schiff base in the L state provides the driving force for H' ejection.…”

Section: Biophysics: Der Et Amentioning

confidence: 85%

Alternative translocation of protons and halide ions by bacteriorhodopsin.

Dér

Száraz

Tóth-Boconádi

et al. 1991

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...