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

Active proton transfer through membrane proteins is accomplished by shifts in the acidity of internal amino acids, prosthetic groups, and water molecu les. The recently introduced step-scan attenuated total reflection Fourier- transform infrared (ATR/FT-IR) spectroscopy was employed to determine trans ient pK(a) changes of single amino acid side chains of the proton pump bact eriorhodopsin, The high pK(a) of D96 (>12 in the ground state) drops to 7.1 +/- 0.2 (in 1 M KCl) during the lifetime of the N intermediate, quantitati ng the role of D96 as the internal proton donor of the retinal Schiff base, We conclude from experiments on the pH dependence of the proton release re action and on point mutants where each of the glutamates on the extracellul ar surface has been exchanged that besides D85 no other carboxylic group ch anges its protonation state during proton release. However, E194 and E204 i nteract with D85, the primary proton acceptor of the Schiff base proton. Th e C=O stretching vibration of D85 undergoes a characteristic pH-dependent s hift in frequency during the M state of wild-type bacteriorhodopsin with a pK(a) of 5.2 (+/- 0.3) which is abolished in the single-site mutants E194Q and E204Q and the quadruple mutant E9Q/E74Q/E194Q/E204Q. The double mutatio n E9Q/E74Q does not affect the lifetime of the intermediates, ruling out an y participation of these residues in the proton transfer chain of bacterior hodopsin, This study demonstrates that transient changes in acidity of sing le amino acid residues can be quantified in situ with infrared spectroscopy .