The protonated Schiff base of halorhodopsin from Natronobacterium pharaonis is hydrolyzed at elevated temperatures.

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.

Spin labeling of Natronomonas pharaonis halorhodopsin: probing the cysteine residues environment.

Halorhodopsin from Natronomonas pharaonis (pHR) is a light-driven chloride pump that transports a chloride anion across the plasma membrane following light absorption by a retinal chromophore which initiates a photocycle. Analysis of the amino acid sequence of pHR reveals three cysteine residues (Cys160, Cys184, and Cys186) in helices D and E. Here we have labeled the cysteine residues with nitroxide spin labels and studied using electron paramagnetic resonance (EPR) spectroscopy their mobility, accessibility to various reagents, and the distance between the labels. It was revealed by following the d(1)/d parameter that the distance between the spin labels is ca. 13-15 Angstrom. The EPR spectrum suggests that one label has a restricted mobility while the other two are more mobile. Only one label is accessible to hydrophilic paramagnetic broadening reagents leading to the conclusion that this label is exposed to the water phase. All three labels are reduced by ascorbic acid and reoxidized by molecular oxygen. The rate of the oxidation is accelerated following retinal irradiation indicating that the protein experiences conformation alterations in the vicinity of the labels during the pigment photocycle. It is suggested that Cys186 is exposed to the bulk medium while Cys184, located close to the retinal ionone ring, exhibits an immobilized EPR signal and is characterized by a hydrophobic environment.

Effect of anions on the photocycle of halorhodopsin. Substitution of chloride with formate anion.

Halorhodopsin from Natronomonas pharaonis is a light-driven chloride pump which transports a chloride anion across the plasma membrane following light absorption by a retinal chromophore which initiates a photocycle. It was shown that the chloride anion bound in the vicinity of retinal PSB can be replaced by several inorganic anions, including azide which converts the chloride pump into a proton pump and induces formation of an M-like intermediate detected in the bR photocycle but not in native halorhodopsin. Here we have studied the possibility of replacing the chloride anion with organic anions and have followed the photocycle under several conditions. It is revealed that the chloride can be replaced with a formate anion but not with larger organic anions such as acetate. Flash photolysis experiments detected in the formate pigment an M-like intermediate characterized by a lifetime much longer than that of the O intermediate. The lifetime of the M-like intermediate depends on the pH, and its decay is significantly accelerated at low pH. The decay rate exhibited a titration-like curve, suggesting that the protonation of a protein residue controls the rate of M decay. Similar behavior was detected in N. pharaonis pigments in which the chloride anion was replaced with NO(2)(-) and OCN(-) anions. It is suggested that the formation of the M-like intermediate indicates branching pathways from the L intermediate or basic heterogeneity in the original pigment.