Blue Halorhodopsin from Natronobacterium pharaonis: Wavelength Regulation by Anions

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Abstract
Halorhodopsin, the chloride pump from Natronobacterium pharaonis (pharaonis hR), was isolated under conditions of low ionic strength. The quotient between the optical densities of pharaonis hR in 4 M NaCl at 280 and 577 nm amounts to 1.1, indicating a high purity of the protein and integrity of the chromophore. Gel filtration chromatography of the purified pharaonos hR at neutral pH and in the absence of inorganic salts leads to a shift to the absorption maximum to 600 nm (pharaonis hRblue). The purple color can be regained by the addition of anions such as chloride, iodide, azide, nitrate, and also fluoride. The absorption maxima are dependent on the nature of the anion and the pH. At pH 7, sulfate does not influence the 600-nm absorption, while at pH 4.5 the absorption maximum is shifted to 581 nm. The blue form of halorhodopsin (pharaonis hRblue) was titrated with different sodium salts, such as chloride, azide, and nitrate. The half-maximal binding is in the millimolar range, with Br < Cl < NO3 < N3 < BrO3. Deprotonation of the Schiff base can be accomplished by treatment of pharaonis hR or pharaonis hRblue with base. The apparent pK of the Schiff base in pharaonis hRblue was determined to be 8.5. The pK shifted to 8.0 in the presence of 150 mM Na2SO4, whereas the pK of pharaonis hR in 150 mM NaCl is 9.6. In the photocycle of the chloride- and nitrate-containing pharaonis hR, a species similar to hR520 could be detected. Pharaonis hR reconstituted with azide shows photochemical reactions similar to the photocycle of bacteriorhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)

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