The Protonated Schiff Base of Halorhodopsin from Natronobacterium pharaonis is Hydrolyzed at Elevated Temperatures†

Abstract

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 degrees 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 degrees 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 CI- 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 degrees 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.

Effect of Chloride Binding on the Thermal Trimer−Monomer Conversion of Halorhodopsin in the Solubilized System

“…With respect to NpHR, a weaker predicted Cl -binding site on the extracellular (EC) side than that near the Schiff base has been proposed, with K d values of 5-15 mM (4,19,20) that are close to our result estimated from the structural stability of the trimer (K d app = 22 mM). In addition, Mevorat-Kaplan et al have reported that the hydrolysis reaction of the retinal Schiff base by heating is prevented when Cl -binds to the site on the EC side (18). Similar privation of the bleaching was observed with the addition of not only NaCl but also Na 2 SO 4 in this study (data not shown), supporting the binding of a SO 4 2-with the putative second binding site.…”

“…The first is a minor blue shift of the absorption maximum from 600 nm at 25°C to 580 nm at 40°C, followed by slow bleaching with regard to the absorption maximum shift from 580 to 380 nm. This bleaching is explained by the hydrolysis reaction of the retinal Schiff base (18,32).…”

“…NpHR binds a retinal chromophore to Lys-256 in the seventh helix G through a Schiff base linkage and has a Cl -binding site in the vicinity of the retinal Schiff base (16) with a dissociation constant (K d ) of 1-5 mM (3,9,17). Moreover, another weaker predicted Cl -binding site on the extracellular (EC) side has been proposed (4,(18)(19)(20). However, the relationship between the weaker Cl -binding site and the Cl -channel structure is unknown.…”

Retinal–Protein Interactions in Halorhodopsin from Natronomonas pharaonis: Binding and Retinal Thermal Isomerization Catalysis

“…However, the recovered pigment differed from the native pigment, which suggests that the apomembrane adopts a modified conformation that rebinds the retinal and produces a new conformation of NpHR pigment. 37 In the present report, we studied the binding interaction of retinal isomers with the NpHR opsin. Importantly, we established that the retinal binding to NpHR opsin is regulated by the pH and by the chloride ion concentration.…”

“…In NpHR, the PSB hydrolyzes at elevated temperatures and the rate of hydrolysis is accelerated following light illumination. 37 Upon cooling to 25°C, 80% of the pigment was recovered. However, the recovered pigment differed from the native pigment, which suggests that the apomembrane adopts a modified conformation that rebinds the retinal and produces a new conformation of NpHR pigment.…”