Binding of Anions to Proteorhodopsin Affects the Asp97 pKa

摘要

Proteorhodopsin (PR), a retinal protein of marine proteobacteria, is a light-driven proton pump.nLight excitation of PR initiates a photocycle that triggers the translocation of a proton from the cytoplasmicnto the extracellular side. Asp97 is located near the retinal-protonated Schiff base and serves as the protonnacceptor during the photocycle. The pKa ofAsp97 is unusually high (∼7.0), especially in comparison with thatnof its bR equivalent residue Asp85 (∼2.6). We have studied possible anions binding to PR (produced fromngene vector eBAC31A08 and expressed in Escherichia coli) and their effect on its absorption maxima, Asp97npKa, and the photocycle. We found that chloride, sulfate, trifluoroacetate, trichloroacetate, and tribromoa-ncetate anions bind to PR and regulate Asp97 pKa. Asp97 has a pKa of ∼7.8 in water, but the value decreasesnto ∼7.0 in the presence of sulfate and chloride anions. Halogeno-acetate anions elevated Asp97 pKa andncompete with chloride anions. The most significant effect was detected with tribromoacetate anions thatnincrease the Asp97 pKa to a value of >9.5. The possibility that PR has at least two binding sites for thesenanions is discussed. In addition, we have demonstrated that these anions bind to PR also at high pH (abovenAsp97 pKa) because they affect the rate of growth and thermal decay of theMintermediate in the photocyclenof PR.