In situ determination of transient pK _a changes of internal amino acids of bacteriorhodopsin by using time-resolved attenuated total reflection Fourier-transform infrared spectroscopy

Abstract

Active proton transfer through membrane proteins is accomplished by shifts in the acidity of internal amino acids, prosthetic groups, and water molecules.

Mechanism of a proton pump analyzed with computer simulations

“…When both the Zundel ion and the negatively charged E194/E204 pair were treated with QM, the proton was attracted by E194/E204 and remained shared by E194/E204 for the entire length of the simulations [41]. This geometry with E194/E204 sharing a proton gives an IR spectrum in reasonable qualitative agreement with experiments [41], bringing support to the initial proposal [87,88] that the E194/E204 dyad, probably in association with water molecules, may act as the extracellular proton release group. E194/E204 may also be important for controlling the mechanical stability of the extracellular region of bacteriorhodopsin [89].…”

Amino acids with an intermolecular proton bond as proton storage site in bacteriorhodopsin

“…Another important consequence of the strong hydrogen bonding interaction is that the COOH band of Glu-194/204 is significantly red-shifted to Ͻ1,700 cm Ϫ1 , falling in the range of amide bands; such strong red-shifts of IR bands are also observed in gas-phase models with a shared proton at both the SCC-DFTB and DFT levels (see SI). This explains why the COOH bands have not been identified in the previous FTIR studies (16,27), which led those authors to pursue alternative models for the PRG.…”

Breaking the Carboxyl Rule

“…Deprotonation of the initially protonated Asp-96 was detected by FTIR during the decay of the M intermediate (16,18,27), which proceeds in a pH-independent manner, but the subsequent reprotonation of the carboxylate during proton uptake from the bulk is pH-dependent (28,29) and is responsible for slowing the N to O transition and overall photocycle turnover at high pH (29 -31). These changes in protonation of the internal carboxyl residue are caused by a transient decrease of its pK a value during the photocycle from above 11 to ϳ 7.5 (28,30,32,33).…”