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1 November 2006 The Protonated Schiff Base of Halorhodopsin from Natronobacterium pharaonis is Hydrolyzed at Elevated Temperatures
Keren Mevorat-Kaplan, Vlad Brumfeld, Martin Engelhard, Mordechai Sheves
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Halorhodopsin from Natronobacterium pharaonis (pHR) is a light-driven chloride pump in which photoisomerzation of a retinal chromophore triggers a photocycle which leads to a chloride anion transport across the plasma membrane. Similarly to other retinal proteins the protonated Schiff base (PSB), which covalently links the retinal to the protein, does not experience hydrolysis reaction at room temperature even though several water molecules are located in the protonated Schiff base (PSB) vicinity. In the present studies we have revealed that in contrast to other studied archaeal rhodopsins, temperature increase to about 70°C hydrolyses the PSB linkage of pHR. The rate of the reaction is affected by Cl concentration and reveals an anion binding site (in addition to the Cl in the SB vicinity) with a binding constant of 100mM (measured at 70°C). We suggest that this binding site is located on the extracellular side and its possible role in the Cl pumping mechanism is discussed. The rate of the hydrolysis reaction is affected by the nature of the anion bound to pHR. Substitution of the Cl anion by Br, I and SCN exhibits similar behavior to that of Cl in the region of 100mM but higher concentrations are needed for N3, HCOO and NO2 to achieve similar behavior. Steady state pigment illumination accelerates the reaction and reduces the energy of activation and the frequency factor. Adjusting the sample temperature to 25°C following the hydrolysis reaction led to about 80% pigment recovery. However, the newly reformed pigment is different from the mother pigment and has different characteristics. It is concluded that the apo-membrane adopts a modified conformation and/or aggregated state which rebinds the retinal to give a new conformation of the pHR pigment.

Keren Mevorat-Kaplan, Vlad Brumfeld, Martin Engelhard, and Mordechai Sheves "The Protonated Schiff Base of Halorhodopsin from Natronobacterium pharaonis is Hydrolyzed at Elevated Temperatures," Photochemistry and Photobiology 82(6), 1414-1421, (1 November 2006).
Received: 15 December 2005; Accepted: 1 April 2006; Published: 1 November 2006

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