Abstract:
Bacteriorhodopsin from Haloquadratum walsbyi (HwBR) exhibits remarkable acid tolerance, maintaining an unshifted absorption maximum down to pH 2.0 and retaining proton‑pumping activity at pH 4.0 (Hsu et al., 2015). This property is abolished by the W94F mutation, which prolongs the post‑M intermediate by approximately tenfold and eliminates the detectable O‑intermediate state (Yu et al., 2022). These findings suggest a mechanistic link between acid tolerance and photocycle recovery; however, the structural basis of this relationship has remained elusive.
Here, we present a complete time‑resolved structural analysis of HwBR during photocycle recovery under acidic conditions (pH 4.0). Using our newly developed Pre‑Aligned Incremental Rotation (PAIR) pump–probe crystallography protocol, we reconstructed a structural “movie” spanning the entire photocycle of wild‑type HwBR with 2.1–2.4 Å spatial resolution and 2 ms temporal resolution.
The analyses revealed that the unique R82–T201 hydrogen‑bond shield, specific to acid‑tolerant bacteriorhodopsin, breaks within the first 2 ms and reforms shortly thereafter. Retinal re‑isomerization and O‑intermediate formation were directly observed at 14–16 ms after photoactivation, followed by restoration of the pentameric hydrogen‑bond network surrounding the chromophore at 70–72 ms. Final recovery to the ground state was marked by re‑establishment of the proton‑transfer pathway and its associated hydrogen‑bond network. Importantly, the amphipathic Trp94 residue was found to delineate the boundary between the retinal‑binding pocket and the proton‑releasing group region, while directly accelerating the re‑isomerization of 13‑cis retinal back to the all‑trans form.
To further elucidate the role of Trp94, we determined the ground‑state structure of W94F‑HwBR at 1.83 Å resolution. Comparative analysis revealed disruption of the extracellular hydrogen‑bond network in the mutant, supporting Trp94 as a critical stabilizer of the proton‑releasing group region. This disruption explains the impaired acid tolerance and prolonged photocycle recovery observed in W94F.
Together, these results establish a complete 2‑ms resolution cinegraphic record of structural changes in HwBR, providing a framework that links hydrogen‑bond network organization, photocycle recovery kinetics, and acid tolerance in microbial rhodopsins.
Keywords – Time-resolved X-ray crystallography, Light-driven proton pump, Bacteriorhodopsin