The pH dependencies of the rate constants in the photocycles of recombinant D96N and D115N/D96N bacteriorhodopsins were determined from time-resolved difference spectra between 70 ns and 420 ms after photoexcitation. The results were consistent with the model suggested earlier for proteins containing D96N substitution: BR hv----K----L----M1----M2----BR. Only the M2----M1 back-reaction was pH-dependent: its rate increased with increasing [H+] between pH 5 and 8. We conclude from quantitative analysis of this pH dependency that its reverse, the M1----M2 reaction, is linked to the release of a proton from a group with a pKa = 5.8. This suggests a model for wild-type bacteriorhodopsin in which at pH greater than 5.8 the transported proton is released on the extracellular side from this as yet unknown group and on the 100-microseconds time scale, but at pH less than 5.8, the proton release occurs from another residue and later in the photocycle most likely directly from D85 during the O----BR reaction. We postulate, on the other hand, that proton uptake on the cytoplasmic side will be by D96 and during the N----O reaction regardless of pH. The proton kinetics as measured with indicator dyes confirmed the unique prediction of this model: at pH greater than 6, proton release preceded proton uptake, but at pH less than 6, the release was delayed until after the uptake. The results indicated further that the overall M1----M2 reaction includes a second kinetic step in addition to proton release; this is probably the earlier postulated extracellular-to-cytoplasmic reorientation switch in the proton pump.

During the M in equilibrium with N----BR reaction sequence in the bacteriorhodopsin photocycle, proton is exchanged between D96 and the Schiff base, and D96 is reprotonated from the cytoplasmic surface. We probed these and the other photocycle reactions with osmotically active solutes and perturbants and found that the M in equilibrium with N reaction is specifically inhibited by withdrawing water from the protein. The N----BR reaction in the wild-type protein and the direct reprotonation of the Schiff base from the cytoplasmic surface in the site-specific mutant D96N are much less affected. Thus, it appears that water is required inside the protein for reactions where a proton is separated from a buried electronegative group, but not for those where the rate-limiting step is the capture of a proton at the protein surface. In the wild type, the largest part of the barrier to Schiff base reprotonation is the enthalpy of separating the proton from D96, which amounts to about 40 kJ/mol. We suggest that in spite of this D96 confers an overall kinetic advantage because when this residue becomes anionic in the N state its electric field near the cytoplasmic surface lowers the free energy barrier of the capture of a proton in the next step. In the D96N protein, the barrier to the M----BR reaction is 20 kJ/mol higher than what would be expected from the rates of the M----N and N----BR partial reactions in the wild type, presumably because this mechanism is not available.

X-ray diffraction experiments revealed the structure of the N photointermediate of bacteriorhodopsin. Since the retinal Schiff base is reprotonated from Asp-96 during the M to N transition in the photocycle, and Asp-96 is reprotonated during the lifetime of the N intermediate, or immediately after, N is a key intermediate for understanding the light-driven proton pump. The N intermediate accumulates in large amounts during continuous illumination of the F171C mutant at pH 7 and 5°C. Small but significant changes of the structure were detected in the x-ray diffraction profile under these conditions. The changes were reversible and reproducible. The difference Fourier map indicates that the major change occurs near helix F. The observed diffraction changes between N and the original state were essentially identical to the diffraction changes reported for the M intermediate of the D96N mutant of bacteriorhodopsin. Thus, we find that the protein conformations of the M and N intermediates of the photocycle are essentially the same, in spite of the fact that in M the Schiff base is unprotonated and in N it is protonated. The observed structural change near helix F will increase access of the Schiff base and Asp-96 to the cytoplasmic surface and facilitate the proton transfer events that begin with the decay of the M state.The active site of an ion pump must communicate alternately with the two opposite sides of the membrane. Change of the protein conformation, linked to this switch, is therefore expected to be an essential step in the reaction cycle. In the light-driven proton pump bacteriorhodopsin (bR), the switch must occur after proton transfer toward the extracellular side but before proton transfer from the cytoplasmic side-i.e., while the proton binding site, the retinal Schiff base, is unprotonated. Thus, the switch reaction is expected to take place during the lifetime of the M intermediate in what was termed the Ml to M2 reaction (1-5). Structural changes are indeed revealed by neutron, x-ray, and electron diffraction when photostationary states are created where the M intermediate accumulates (6-9). We have shown that this structural change is closely related to the deprotonation of Schiff base; in the original structure (conformation E) the proton channel is open to the extracellular side, and when an M-type conformation is assumed (conformation C) it is open to the cytoplasmic side (10). Thus, the proton transport mechanism is elegantly explained by the alternating protein conformation model (11, 12). Light energy is utilized to cause the initial proton transfer that triggers the structural transition from conformation E to conformation C, and that results in the change of access of the retinal Schiff base as well as further changes in proton affinity (pK) of donor and acceptor groups so as to drive proton transfers at strategic locations in the protein.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" i...

The pH dependence of the absorption spectra of bacteriorhodopsin and its photocycle intermediates was studied in the pH range 4.5−9. The spectra of the intermediates were determined from difference spectra taken during the photocycle with an optical multichannel analyzer. The data analysis was based on various criteria concerning the shape of the spectra, but no assumption was made about the kinetic model that describes the photocycle. The strategy for calculation of the spectra was one described earlier, as well as a newly introduced algorithm based on the Monte Carlo method. The search methods used gave very similar results. Like the absorption spectrum of bacteriorhodopsin in the above-mentioned pH range, the spectra of all the intermediates were found to be almost unchanged. The spectrum of intermediate M displayed a 2 nm, and that of intermediate L a 1 nm, red shift with rising pH, but this latter shift was within the overall error of the measurements. The similarity of all the intermediate spectra calculated with different procedures at all pH values points to the reliability of the method and the validity of the spectra. Averaging of the calculated spectra over the whole pH range furnished a well-determined set of intermediate spectra, suitable for further kinetic and spectroscopic studies of the photocycle.