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...

Photocycle and flash-induced proton release and uptake were investigated for bacteriorhodopsin mutants in which Asp-85 was replaced by Ala, Asn, or Glu; Asp-212 was replaced by Asn or Glu; Asp-115 was replaced by Ala, Asn, or Glu; Asp-96 was replaced by Ala, Asn, or Glu; and Arg-82 was replaced by Ala or Gln in dimyristoylphosphatidylcholine/3- [(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate miceiles at pH 7.3. In the Asp-85 -Ala and Asp-85 --Asn mutants, the absence of the charged carboxyl group leads to a blue chromophore at 600 and 595 nm, respectively, and lowers the pK of the Schiff base deprotonation to 8.2 and 7, respectively, suggesting a role for Asp-85 as counterion to the Schiff base. The early part of the photocycles of the Asp-85 -* Ala and Asp-85 -* Asn mutants is strongly perturbed; the formation of a weak M-like intermediate is slowed down about 100-fold over wild type. In both mutants, proton release is also slower but dearly precedes the rise of M. The amplitude of the early (<0.2 ps) reversed photovoltage component in the Asp-85 -* Asn mutant is very large, and the net charge displacement is close to zero, indicating proton release and uptake on the cytoplasmic side of the membrane. The data suggest an obligatory role for Asp-85 in the efficient deprotonation of the Schiff base and in the proton release phase, probably as proton acceptor. In the Asp-212 -* Asn mutant, the rise of the absorbance change at 410 nm is slowed down to 220 Ls, its amplitude is small, and the release of protons is delayed to 1.9 ms. The absorbance changes at 650 nm indicate perturbations in the early time range with a slow K intermediate. Thus Asp-212 also participates in the early events of charge translocation and deprotonation of the Schiff base. In the Arg-82 -Gln mutant, no net transient proton release was observed, whereas, in the Arg-82 Ala mutant,, uptake and release were reversed. The pK shift of the purple-to-blue transition in the Asp-85 --Glu, Arg-82-Ala, and Arg-82 --Gin mutants and the similarity in the photocycle and photoelectrical signals of the Asp-85 -Ala, Asp-85 --Asn, and Asp-212 --Asn mutants suggest the interaction between Asp-85, Arg-82, Asp-212, and the Schiff base as essential for proton release.Site-directed mutagenesis has shown and Asp-212 to be essential for proton translocation by bacteriorhodopsin (bR) (1). The very low activity in mutants Asp-96 -+ Ala (D96A) and Asp-96 -* Asn (D96N) at pH 7 is due to a markedly slowed-down decay of the photocycle intermediate M and the associated charge movement (2-5). was concluded to be the internal proton donor for the reprotonation of the Schiff base (SB) leading to the decay of M (3, 4). Fourier-transform infrared spectroscopy has revealed the protonation states of Asp-85, Asp-96, Asp-115, and Asp-212 in the K, L, and M intermediates and provided clues to the time course of proton transfer (6, 7). The data suggest that Asp-85 and Asp-212 are deprotonated in the ground state and become protonated in the L -* M transition (6). A...

The photocycle, electrical charge translocation, and release and uptake of protons from the aqueous phase were investigated for bacteriorhodopsin mutants with aspartic acid-96 replaced by asparagine or glutamic acid. At

The flash-induced charge movements during the photocycle of light-adapted bacteriorhodopsin in purple membranes attached to a black lipid membrane were investigated under voltage clamp and current clamp conditions. Signal registration ranged from 200 ns to 30 s after flash excitation using a logarithmic clock, allowing the equally weighted measurement of the electrical phenomena over eight decades of time. The active pumping signals were separated from the passive system discharge on the basis of an equivalent circuit analysis. Both measuring methods were shown to yield equivalent results, but the charge translocation could be accurately monitored over the whole time range only under current clamp conditions. To describe the time course of the photovoltage signals a model based on distributed kinetics was found to be more appropriate than discrete first order processes suggesting the existence of conformational substates with distributed activation energies. The time course of the active charge displacement is characterised by a continuous relaxation time spectrum with three broad peaks plus an unresolved fast transient (<0.3 mus) of opposite polarity. The time constants and relative amplitudes (in brackets) derived from the peak rate constants and relative areas of the three bands are: tau(1) = 32 mus (20%), tau(2) = 0.89 ms (15%) and tau(3) = 18 ms (65%) at 25 degrees C in 150 mM KCl at pH7. The Arrhenius plots of the peak rate constants were linear yielding activation energies of E(A1) = 57 kJ/mol, E(A2) = 52 kJ/mol, and E(A3) = 44 kJ/mol. The electrical signal at 890 mus has no counterpart in the photocycle of bacteriorhodopsin suspensions. Fits with a sum of exponentials required 5 to 6 components and were not reproducible. Analysis of photoelectrical signals with continuous relaxation time spectra gave equally good fits with fewer parameters and were well reproducible.

Using three selectively deuterated retinals, the in‐plane structure of the chromophore of bacteriorhodopsin was determined by neutron diffraction. The time‐resolved photovoltage of bacteriorhodopsin was analysed on the basis of distributed kinetics indicating the presence of three major phases in the charge displacement with time constants of 32 μs, 890 μs and 18 ms at 25°C, pH 7. Sixty to seventy percent of the relative charge displacement occurred in the slowest step. The current‐voltage characteristic of bacteriorhodopsin incorporated in planar lipid bilayers was measured and found to be distinctly non‐linear. Between −80 mV and +180 mV it can be approximated by a single exponential plus a constant. This behaviour can be explained in terms of a barrier model for the pump with the slowest step dominating the photocurrent and its voltage dependence. Combining the information from the current‐voltage dependence with the kinetic information, a stoichiometry of one proton pumped per bR cycling is obtained. There is no evidence for a reversal potential down to −160 mV.