Much progress has been made in recent years in describing how protons are transported in the light driven pump, bacteriorhodopsin. The structure of the protein in the non-illuminated, but light-adapted, BR-state is available to 1.4–1.6 Å resolution1, 2
, and the participation of the retinal Schiff base, acidic side-chains, and numerous bound water molecules, in the internal proton transfers and in proton release and uptake at the two membrane surfaces are now understood. High-resolution x-ray structural models of nearly all of the intermediate states of the transport cycle1, 3–15
give a step by step atomic level “movie” containing details of the structural rearrangements in the protein up to the M and N states. Large-scale conformational changes in the second half of the photocycle have been described, in turn, by cryo-electron microscopy16, 17
, low-resolution x-ray and neutron diffraction projection maps18–22
, EPR spectroscopy with site-specific spin-labels23–27
and cysteine reactivity changes28
at the protein surface. The changes in the strength of the retinal Schiff base counter-ion, inter-atomic distances at functionally important locations, and dihedral angles in the retinal have been determined29–33
with solid-state NMR. Rotation of retinal bonds, hydrogen-out-of-plane (HOOP) motions, protonation changes in the protein, and perturbations of polar groups and the peptide backbone as well as water and a hydrogen-bonded aqueous continuum, are described34–38
by vibrational spectroscopy. Combined quantum-mechanics/molecular dynamics calculations39, 40
of the barriers to changes in local geometry, and their comparison with the measured thermodynamics of the photocycle reactions, have produced a suggested reaction mechanism for the early events in the cycle.
According to the crystal structures,1, 41
it is because the binding site does not initially accommodate the changed shape of the polyene that photoisomerization produces a twisted 13-cis,15-anti retinal. Relaxation of this high-energy state is what drives both the critical deprotonation of the Schiff base and the ensuing stepwise conformational changes of the protein that propagate toward the two membrane surfaces. There is an emerging consensus from both crystal structures and spectroscopic information on how these conformational changes allow conduction of a proton from the cytoplasmic aqueous interface to the retinal and from the retinal to the extracellular interface. However, important elements of the transport mechanism have remained controversial because there is disagreement about the structure of the L intermediate. The protonation of the anionic Asp85 by NZ+
-H of Lys216 (the Schiff base connection of Lys216 to the retinal) is the crucial step in the transport, and it occurs in the L to M reaction. Its mechanism is determined by the pKa
difference that develops between the proton donor to its acceptor, and by the path taken by the transferred proton. Alternatively, protonation of Asp85 might be by a water molecule that dissociates and leaves behind a hydroxyl ion, and in this case the proton pump was proposed30, 31, 33, 42–45
to be, in fact, a hydroxyl ion pump. Thus, the central issues in the transport depend on how the geometry of this region is changed from the equilibrium that exists in the BR-state to the unstable arrangement in the L state.
In the BR state wat402 receives a hydrogen-bond from the Schiff base of the all-trans retinal and donates hydrogen-bonds to the carboxylates of Asp85 and Asp212. This stable structure will be perturbed upon photoisomerization of the retinal to 13-cis,15-anti. The three crystal structures reported1, 8, 15
for the K state are in agreement with the conclusions from changes in the C=N stretch frequency that had suggested46, 47
that the Schiff base loses its hydrogen-bond. There is no such consensus for the L state. Although produced in the crystals by similar illumination protocols, for this intermediate three very different crystallographic models have been reported. They suggest different means for energy conservation in the cycle and different paths for transferring the Schiff base proton to Asp85. The crystallographic considerations in evaluating these models48
have been reviewed. In model a), from data to 2.1 Å resolution8
and a claimed 70% occupancy for L (although there is a differing opinion49
), the Lys216 NZ+
-H vector has turned away from wat402 that had connected it to Asp85 in the BR state, and now points into the cytoplasmic, hydrophobic region. Wat402 is missing from the model, but bending of helix C, with Asp85, toward the Schiff base site (although without a decrease in the Asp85 to Schiff base distance) was suggested to facilitate transfer of the Schiff base proton to the aspartate. The transfer is driven, presumably, by the new environment of the protonated Schiff base that is unfavorable for a charged group. In model b), from data to 1.62 Å resolution7
and 60% occupancy for L, the hydrogen-bond of the Schiff base to wat402, lost in K, is re-established. This is accomplished by twists in two of the retinal double-bonds and an increase in the bond angle at C13
, so as to maintain, roughly, the contour of the all-trans retinal in spite of the C13
bond rotation. Protonation of Asp85 in this model is therefore via wat402, and driven by the relaxation of the strain in the retinal permitted by loss of interaction between the Schiff base and the aspartate, as follows from a previous suggestion31
, once they lose their electric charges. In model c), from data to 2.4 Å resolution12
(but in a larger unit cell than the others) and 20% occupancy for L, the Schiff base is turned to the cytoplasmic side together
with wat402, maintaining the hydrogen-bond of the Schiff base. Movement of wat402 to the other side of the retinal is made possible by rotation of the Leu93 side-chain to make room for it. The proton transfer in this model is driven, presumably, by the unfavorable free energy of the uncompensated charge of the anionic Asp85 left behind, but the path of the proton will be tortuous and may have to pass via the OH group of Thr89.
Contrary to expectation, as discussed below in more detail, the various kinds of non-crystallographic information about the L state have not decided unambiguously in favor of any one these models. The FTIR O-H stretch bands in L in the wild-type protein and several mutants are consistent only with model c), because they suggest50–52
that the Schiff base becomes hydrogen-bonded to a water molecule specifically in the cytoplasmic region. The chemical shift of 15
N labeled Schiff base in L by solid-state NMR30
is consistent only with models b) and c) because it indicates the existence of strong Schiff base counter-ion interaction. The NMR results suggest30
twists around the C13
=NZ double bonds in L, consistent with model b). Theoretical calculations40
, on the other hand, preclude such twists, and are in conflict with the NMR and crystallographic data. The calculated barrier to proton transfer from the Schiff base to Asp85 agrees with the measured rates, but the predicted structure for L is not consistent with any of the three crystallographic models.
In view of this controversy, we made a new attempt to determine the structural changes in the L state. The data we report here produces an improved model, because i) crystals with higher diffraction quality were used, ii) the structure of L was determined in the partial occupancy refinement with a BR model from the same crystal, and iii) the statistical significance of the changes could be assessed from six independent determinations. The new model is consistent with model b), but with some differences.