Abstract
Proton translocation across membranes is vital to all kingdoms of life. Mechanistically, it relies on characteristic proton flows and modifications of hydrogen bonding patterns, termed protonation dynamics, which can be directly observed by fast magic angle spinning (MAS) NMR. Here, we demonstrate that reversible proton displacement in the active site of bacteriorhodopsin already takes place in its equilibrated dark-state, providing new information on the underlying hydrogen exchange processes. In particular, MAS NMR reveals proton exchange at D85 and the retinal Schiff base, suggesting a tautomeric equilibrium and thus partial ionization of D85. We provide evidence for a proton cage and detect a preformed proton path between D85 and the proton shuttle R82. The protons at D96 and D85 exchange with water, in line with ab initio molecular dynamics simulations. We propose that retinal isomerization makes the observed proton exchange processes irreversible and delivers a proton towards the extracellular release site.
Highlights
Proton translocation across membranes is vital to all kingdoms of life
The proton cage extends to R82 which is involved in a hydrogen bonding network with water molecules and the carboxyl group of D85 (Fig. 1b)[4,5]
The observed proton exchange between the RSB, water molecules, D85 and R82 indicate the existence of a pathway across the proton cage at the BR active site (Figs. 1a, b, 2, 3a)
Summary
Proton translocation across membranes is vital to all kingdoms of life. Mechanistically, it relies on characteristic proton flows and modifications of hydrogen bonding patterns, termed protonation dynamics, which can be directly observed by fast magic angle spinning (MAS) NMR. Direct proton detection was employed in a temperature range from 100 K to 290 K to monitor protons in the BR dark-adapted state (mixture of 13-cis,15-syn and 13-trans,15-anti retinal configurations) at key sites of the proton transport pathway, after introducing a subset of exchanging protons into the interior of the perdeuterated samples upon illumination It is well-established that the structural rearrangements during the photocycle are required for complete proton transport through the BR pore from the uptake to the release site, which is exploited here for reprotonation of sites involved in the pathway[9,23,24,25]. Ab initio molecular dynamics simulations allowed for an estimation of proton distribution between a H2O molecule and two carboxyl groups, a simplified model for proton displacement in the D85 environment
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