Biophysical Journal 60: 475-490 (1991)
© 1991 the Biophysical Society

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Balashov, S. P. Articles by Ebrey, T. G. Search for Related Content PubMed Articles by Balashov, S. P. Articles by Ebrey, T. G.

Redshift of the purple membrane absorption band and the deprotonation of tyrosine residues at high pH

Origin of the parallel photocycles of trans-bacteriorhodopsin

Department of Physiology and Biophysics, University of Illinois, Urbana, Illinois 61801 USA

ABSTRACT

At high pH (> 8) the 570 nm absorption band of all-trans bacteriorhodopsin (bR) in purple membrane undergoes a small (1.5 nm) shift to longer wavelengths, which causes a maximal increase in absorption at 615 nm. The pK of the shift is 9.0 in the presence of 167 mM KCl, and its intrinsic pK is 8.3. The red shift of the trans-bR absorption spectrum correlates with the appearance of the fast component in the light-induced L to M transition, and absorption increases at 238 and 297 nm which are apparently caused by the deprotonation of a tyrosine residue and red shift of the absorption of tryptophan residues. This suggests that the deprotonation of a tyrosine residue with an exceptionally low pK (pK_a 8.3) is responsible for the absorption shift of the chromophore band and fast M formation. The pH and salt dependent equilibrium between the two forms of bR, "neutral" and "alkaline," bR bR_a, results in two parallel photocycles of trans-bR at high pH, differing in the rate of the L to M transition. In the pH range 10-11.8 deprotonation of two more tyrosine residues is observed with pK's 10.3 and 11.3 (in 167 mM KCL). Two simple models discussing the role of the pH induced tyrosine deprotonation in the photocycle and proton pumping are presented.

It is suggested that the shifts of the absorption bands at high pH are due to the appearance of a negatively charged group inside the protein (tyrosinate) which causes electrochromic shifts of the chromophore and protein absorption bands due to the interaction with the dipole moments in the ground and excited states of bR (Stark effect). This effect gives evidence for a significant change in the dipole moment of the chromophore of bR upon excitation.

Under illumination alkaline bR forms, besides the usual photocycle intermediates, a long-lived species with absorption maximum at 500 nm (P500). P500 slowly converts into bR_a in the dark. Upon illumination P500 is transformed into an intermediate having an absorption maximum at 380 nm (P380). P380 can be reconverted to P500 by blue light illumination or by incubation in the dark.

This article has been cited by other articles:

				M. Ming, M. Lu, S. P. Balashov, T. G. Ebrey, Q. Li, and J. Ding pH Dependence of Light-Driven Proton Pumping by an Archaerhodopsin from Tibet: Comparison with Bacteriorhodopsin Biophys. J., May 1, 2006; 90(9): 3322 - 3332. [Abstract] [Full Text] [PDF]

				L. S. Sanii, A. W. Schill, C. E. Moran, and M. A. El-Sayed The Protonation-Deprotonation Kinetics of the Protonated Schiff Base in Bicelle Bacteriorhodopsin Crystals Biophys. J., July 1, 2005; 89(1): 444 - 451. [Abstract] [Full Text] [PDF]

				C. D. Heyes and M. A. El-Sayed Proton Transfer Reactions in Native and Deionized Bacteriorhodopsin upon Delipidation and Monomerization Biophys. J., July 1, 2003; 85(1): 426 - 434. [Abstract] [Full Text] [PDF]