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Crystal structures of the Asp96 to Asn mutant of the light-driven proton pump bacteriorhodopsin and its M photointermediate produced by illumination at ambient temperature have been determined to 1.8 and 2.0 angstroms resolution, respectively. The trapped photoproduct corresponds to the late M state in the transport cycle-that is, after proton transfer to Asp85 and release of a proton to the extracellular membrane surface, but before reprotonation of the deprotonated retinal Schiff base. Its density map describes displacements of side chains near the retinal induced by its photoisomerization to 13-cis,15-anti and an extensive rearrangement of the three-dimensional network of hydrogen-bonded residues and bound water that accounts for the changed pKa values (where Ka is the acid constant) of the Schiff base and Asp85. The structural changes detected suggest the means for conserving energy at the active site and for ensuring the directionality of proton translocation.

Photoisomerization of the retinal of bacteriorhodopsin initiates a cyclic reaction in which a proton is translocated across the membrane. Studies of this protein promise a better understanding of how ion pumps function. Together with a large amount of spectroscopic and mutational data, the atomic structure of bacteriorhodopsin, determined in the last decade at increasing resolutions, has suggested plausible but often contradictory mechanisms. X-ray diffraction of bacteriorhodopsin crystals grown in cubic lipid phase revealed unexpected two-fold symmetries that indicate merohedral twinning along the crystallographic c axis. The structure, refined to 2.3 angstroms taking this twinning into account, is different from earlier models, including that most recently reported. One of the carboxyl oxygen atoms of the proton acceptor Asp85 is connected to the proton donor, the retinal Schiff base, through a hydrogen-bonded water and forms a second hydrogen bond with another water. The other carboxyl oxygen atom of Asp85 accepts a hydrogen bond from Thr89. This structure forms the active site. The nearby Arg82 is the center of a network of numerous hydrogen-bonded residues and an ordered water molecule. This network defines the pathway of the proton from the buried Schiff base to the extracellular surface.

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Because asp-85 is the acceptor of the retinal Schiff base proton during light-driven proton transport by bacteriorhodopsin, modulation of its pKa in the photocycle is to be expected. The complex titration of asp-85 in the unphotolyzed protein was suggested [Balashov, S. P., Govindjee, R., Imasheva, E. S., Misra, S., Ebrey, T. G., Feng, Y., Crouch, R. K., & Menick, D. R (1995) Biochemistry 34, 8820-8834] to reflect the dependence of this residue on the protonation state of another, unidentified group. From the pH dependencies of the rate constant for the thermal equilibration of retinal isomeric states (dark adaptation) and the deprotonation kinetics of the Schiff base during the photocycle in the E204Q and E204D mutants, we identify the residue as glu-204. The nature of its interaction with asp-85 is that at neutral pH either residue can be anionic but not both. This is consistent with our recent finding that glu-204 is the origin of the proton released to the extracellular surface upon protonation of asp-85 during the transport. We propose, therefore, that the following series of events occur in the photocycle. Protonation of asp-85 in the proton equilibrium with the Schiff base of the photoisomerized retinal results in the dissociation of glu-204 and proton release to the extracellular surface. The deprotonation of glu-204, in turn, raises the pK(a) of asp-85, and the equilibrium with the Schiff base shifts toward complete proton transfer. This constitutes the first phase of the reprotonation switch because it excludes asp-85 as a donor in the reprotonation of the Schiff base that follows. The sequential structural changes of the protein that ensue, detected earlier by diffraction, are suggested to facilitate the change of the access of the Schiff base toward the cytoplasmic side as the second phase of the switch, and the lowering the pKa of asp-96, so as to make it a proton donor, as the third phase.

Glu-194 near the extracellular surface of bacteriorhodopsin is indispensable for proton release to the medium upon protonation of Asp-85 during light-driven transport. As for Glu-204, its replacement with glutamine (but not aspartate) abolishes both proton release and the anomalous titration of Asp-85 that originates from coupling between the pKa of this buried aspartate and those of the other acidic groups. Unlike the case of Glu-204, however, replacement of Glu-194 with aspartate raises the pKa for proton release. In Fourier transform infrared spectra of the E194D mutant a prominent positive band is observed at 1720 cm-1. It can be assigned from [4-13C]aspartate and D2O isotope shifts to the C&dbd;O stretch of protonated Asp-194. Its rise correlates with proton transfer from the retinal Schiff base to Asp-85. Its decay coincides with the appearance of a proton at the surface, detected under similar conditions with fluorescein covalently bound to Lys-129 and with pyranine. Its amplitude decreases with increasing pH, with a pKa of about 9. We show that this pKa is likely to be that of the internal proton donor to Asp-194, the Glu-204 site, before photoexcitation, while 13C NMR titration indicates that Asp-194 has an initial pKa of about 3. We propose that there is a chain of interacting residues between the retinal Schiff base and the extracellular surface. After photoisomerization of the retinal the pKa's change so as to allow (i) Asp-85 to become protonated by the Schiff base, (ii) the Glu-204 site to transfer its proton to Asp-194 in E194D, and therefore to Glu-194 in the wild type, and (iii) residue 194 to release the proton to the medium.

In the last step of the bacteriorhodopsin photocycle the initial state is regenerated from the O intermediate in an essentially unidirectional reaction. Comparison of the rate of this photocycle step and the rate of deprotonation of Asp-85 in pH jump experiments with various site-specific mutants indicates that recovery of the initial state is influenced by (1) residues such as Glu-204 that affect deprotonation of Asp-85 and (2) residues such as Leu-93 that contact the retinal and therefore must affect its thermal reisomerization from 13-cis to all-trans as suggested by Delaney, Schweiger, and Subramaniam (Proc. Natl. Acad. Sci. U.S.A. 92, 11120-11124, 1995). These results, together with FTIR spectra (Kandori, Hatanaka, Yamazaki, Needleman, Brown, Richter, Lanyi, & Maeda, manuscript in preparation) of the last intermediate in the photocycles of representatives of the two kinds of mutants, E204Q and L93M, suggest the following sequence of events: reisomerization of the retinal from 13-cis to an all-trans configuration that contains a twisted chain (with high amplitude hydrogen out-of-plane vibrational bands) triggers proton transfer from Asp-85 to Glu-204 or directly to the extracellular surface, and the proton transfer in turn triggers relaxation of the twist in the retinal. The involvement of the proton transfer in the kinetics of this sequence suggests the reason for the unidirectionality of the overall reaction: upon reisomerization of the retinal the very low pKa of Asp-85 in the unphotolyzed protein is reestablished and this residue thereby becomes a good proton donor.

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