Bacteriorhodopsin (BR) is a model protein for light-driven proton pumps, where the vectorial active proton transport results in light-energy conversion. To clarify the microscopic mechanism of primary proton transfer from retinal Schiff base (SB) to Asp85 in BR, herein we performed quantum-mechanical metadynamics simulations of the whole BR system (~3800 atoms). The simulations showed a novel proton transfer mechanism, viz. hydroxide ion mechanism, in which the deprotonation of specific internal water (Wat452) yields the protonation of Asp85 via Thr89, after which the resulting hydroxide ion accepts the remaining proton from retinal SB. Furthermore, systematic investigations adopting four sequential snapshots obtained by the time-resolved serial femtosecond crystallography revealed that proton transfer took 2-5.25 μs on the photocycle. The presence of Wat401, which is the main difference between snapshots at 2 and 5.25 μs, is found to be essential in assisting the primary proton transfer. SIGNIFICANCE: Bacteriorhodopsin (BR), the benchmark of light-driven proton pumps, has attracted much attention from diverse areas in terms of energy conversion. Despite the significant experimental and theoretical efforts, the microscopic mechanism of the proton transfers in BR is not completely unveiled. In this study, quantum-mechanical molecular dynamics simulations of whole BR system were performed to elucidate the primary proton transfer in the L intermediate state with the latest snapshots obtained from X-ray free electron laser. As a result, it is found that the hydroxide ion originating from the specific internal water, which appears at the active site only in the L state, acts as a carrier for the primary proton transfer, demonstrating the importance of hydroxide ions in proton pumps.

Bacteriorhodopsin (BR) is a model protein for light-driven proton pumps, where the vectorial active proton transport results in light-energy conversion. To clarify the microscopic mechanism of primary proton transfer from retinal Schiff base (SB) to Asp85 in BR, herein we performed quantum-mechanical metadynamics simulations of the whole BR system (~3800 atoms). The simulations showed a novel proton transfer mechanism, viz. hydroxide ion mechanism, in which the deprotonation of specific internal water (Wat452) yields the protonation of Asp85 via Thr89, after which the resulting hydroxide ion accepts the remaining proton from retinal SB. Furthermore, systematic investigations adopting four sequential snapshots obtained by the time-resolved serial femtosecond crystallography revealed that proton transfer took 2-5.25 μs on the photocycle. The presence of Wat401, which is the main difference between snapshots at 2 and 5.25 μs, is found to be essential in assisting the primary proton transfer. SIGNIFICANCE: Bacteriorhodopsin (BR), the benchmark of light-driven proton pumps, has attracted much attention from diverse areas in terms of energy conversion. Despite the significant experimental and theoretical efforts, the microscopic mechanism of the proton transfers in BR is not completely unveiled. In this study, quantum-mechanical molecular dynamics simulations of whole BR system were performed to elucidate the primary proton transfer in the L intermediate state with the latest snapshots obtained from X-ray free electron laser. As a result, it is found that the hydroxide ion originating from the specific internal water, which appears at the active site only in the L state, acts as a carrier for the primary proton transfer, demonstrating the importance of hydroxide ions in proton pumps.