Binding of anions to proteorhodopsin affects the Asp97 pK(a).
Proteorhodopsin (PR), a retinal protein of marine proteobacteria, is a light-driven proton pump. Light excitation of PR initiates a photocycle that triggers the translocation of a proton from the cytoplasmic to the extracellular side. Asp97 is located near the retinal-protonated Schiff base and serves as the proton acceptor during the photocycle. The pK(a) of Asp97 is unusually high ( approximately 7.0), especially in comparison with that of its bR equivalent residue Asp85 ( approximately 2.6). We have studied possible anions binding to PR (produced from gene vector eBAC31A08 and expressed in Escherichia coli) and their effect on its absorption maxima, Asp97 pK(a), and the photocycle. We found that chloride, sulfate, trifluoroacetate, trichloroacetate, and tribromoacetate anions bind to PR and regulate Asp97 pK(a). Asp97 has a pK(a) of approximately 7.8 in water, but the value decreases to approximately 7.0 in the presence of sulfate and chloride anions. Halogeno-acetate anions elevated Asp97 pK(a) and compete with chloride anions. The most significant effect was detected with tribromoacetate anions that increase the Asp97 pK(a) to a value of >9.5. The possibility that PR has at least two binding sites for these anions is discussed. In addition, we have demonstrated that these anions bind to PR also at high pH (above Asp97 pK(a)) because they affect the rate of growth and thermal decay of the M intermediate in the photocycle of PR.
Sharaabi
Yamit
Y
Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel.
Brumfeld
Vlad
V
Sheves
Mordechai
M
D000042:Absorption; D000838:Anions / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002712:Chlorides / Q000378:metabolism; D008027:Light; D017494:Proton Pumps / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011522:Protons; D012172:Retinaldehyde / Q000737:chemistry / Q000378:metabolism*; D012243:Rhodopsin / Q000378:metabolism*; D025604:Rhodopsins, Microbial; D012545:Schiff Bases / Q000737:chemistry; D014867:Water / Q000737:chemistry / Q000378:metabolism
D000838:Anions; D002712:Chlorides; D017494:Proton Pumps; D011522:Protons; D025604:Rhodopsins, Microbial; D012545:Schiff Bases; C413548:proteorhodopsin; D014867:Water; D012243:Rhodopsin; D012172:Retinaldehyde