Light-driven protonation changes of internal aspartic acids of bacteriorhodopsin: an investigation of static and time-resolved infrared difference spectroscopy using [4-13C]aspartic acid labeled purple membrane

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Abstract
The molecular events during the photocycle of [Halobacterium halobium] bacteriorhodopsin [BR] were studied by the method of time-resolved and static infrared difference spectroscopy. Characteristic spectral changes involving the C.dbd.O stretching vibration of protonated carboxylic groups were detected. To identify the corresponding groups with either Glu or Asp, BR was selectively labeled with [4-13C]Asp. An incorporation of ca. [circa] 70% was obtained. The comparison of the difference spectra in the region of the CO2- stretching vibrations of labeled and unlabeled BR indicates that ionized Asp are influenced during the photocycle, the earliest effect being observed already at the K610 intermediate. Taken together, the results provide evidence that 4 internal Asp undergo protonation changes and that one Glu, remaining protonated, is disturbed. The results are discussed in relation to the various aspects of the proton pumping mechanism, such as retinal isomerization, charge separation, pK changes, and proton pathway.