Control of bacteriorhodopsin color by chloride at low pH. Significance for the proton pump mechanism.
The chromophore of bacteriorhodopsin undergoes a transition from purple (570 nm absorbance maximum) to blue (605 nm absorbance maximum) at low pH or when the membrane is deionized. The blue form was stable down to pH 0 in sulfuric acid, while 1 M NaCl at pH 0 completely converted the pigment to a purple form absorbing maximally at 565 Other acids were not as effective as sulfuric in maintaining the blue form, and chloride was the best anion for converting blue membrane to purple membrane at low pH. The apparent dissociation constant for Cl- was 35 mM at pH 0, 0.7 M at pH 1 and 1.5 M at pH 2. The pH dependence of apparent Cl- binding could be modeled by assuming two different types of chromophore-linked Cl- binding site, one pH-dependent. Chemical modification of bacteriorhodopsin carboxyl groups (probably Asp-96, -102 and/or -104) by 1-ethyl-3-dimethlyaminopropyl carbodiimide, Lys-41 by dansyl chloride, or surface arginines by cyclohexanedione had no effect on the conversion of blue to purple membrane at pH 1. Fourier transform infrared difference spectroscopy of chloride purple membrane minus acid blue membrane showed the protonation of a carboxyl group (trough at 1392 cm -1 and peak at 1731 cm -1). The latter peak shifted to 1723 cm -1 in D2O. Ultraviolet difference spectroscopy of chloride purple membrane minus acid blue membrane showed ionization of a phenolic group (peak at 243 nm and evidence for a 295 nm peak superimposed on a tryptophan perturbation trough). This suggests the possibility of chloride-induced proton transfer from a tyrosine phenolic group to a carboxylate side-chain. We propose a mechanism for the purple to acid blue to chloride purple transition based on these results and the proton pump model of Braiman et al. (Biochemistry 27 (1988) 8516-8520).
Renthal
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R
Division of Earth and Physical Science, University of Texas, San Antonio 78285.
Shuler
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K
Regalado
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R
D000838:Anions; D001436:Bacteriorhodopsins; D001665:Binding Sites; D055598:Chemical Phenomena; D002621:Chemistry; D002712:Chlorides / Q000378:metabolism / Q000494:pharmacology*; D003116:Color; D004591:Electrophoresis, Polyacrylamide Gel; D005583:Fourier Analysis; D006217:Halobacterium / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D011522:Protons; D013053:Spectrophotometry; D013055:Spectrophotometry, Infrared; D013056:Spectrophotometry, Ultraviolet; D013464:Sulfuric Acids
D000838:Anions; D002712:Chlorides; D011522:Protons; D013464:Sulfuric Acids; D001436:Bacteriorhodopsins; C033158:sulfuric acid