Role of Putative Anion-Binding Sites in Cytoplasmic and Extracellular Channels ofNatronomonas pharaonisHalorhodopsin

Abstract

Natronomonas (Natronobacterium) pharaonis halorhodopsin (NpHR) is an inward light-driven Cl(-) ion pump. For efficient Cl(-) transport, the existence of Cl(-)-binding or -interacting sites in both extracellular (EC) and cytoplasmic (CP) channels is postulated. Candidates include Arg123 and Thr126 in EC channels and Lys215 and Thr218 in CP channels. The roles played by these amino acid residues in anion binding and in the photocycle have been investigated by mutation of the amino acid residues at these positions. Anion binding was assayed by changes in circular dichroism and the shift in the absorption maximum upon addition of Cl(-) to anion-free NpHR. The binding affinity was affected in mutants in which certain EC residues had been replaced; this finding revealed the importance of Arg123. On the other hand, mutants in which certain residues in the CP channel were replaced (CP mutants) did not show changes in their dissociation constants. The photocycles of these mutants were also examined, and in the case of the EC mutants, the transition to the last step was greatly delayed; on the other hand, in the CP mutants, L2-photointermediate decay was significantly prolonged, except in the case of K215Q, which lacked the O-photointermediate. The importance of Thr218 for binding of Cl(-) to the CP channel was indicated by these results. On the basis of these observations, the possible anion transport mechanism of NpHR was discussed.

Structural Mechanism for Light-driven Transport by a New Type of Chloride Ion Pump, Nonlabens marinus Rhodopsin-3

“…Overall architectures viewed parallel to the membrane. Asn-98 in NM-R3) for Cl Ϫ transport (32)(33)(34). In addition, it was proposed that Asp-116 in KR2 (corresponding to Thr-102 in NM-R3) plays an important role in Na ϩ pumping, as estimated from the structure and mutant experiments (Fig.…”

A light-driven sodium ion pump in marine bacteria

“…As KR2 is a light-driven sodium ion pump, NaR probably possesses a sodium-ion binding site. Chloride binds to HR as the RSB counterion, so that the binding can be monitored by colour change (l max change by 420 nm) 30 . In contrast, NaR shows little colour change between the presence and absence of sodium ions ( Fig.…”

A Unique Light-Driven Proton Transportation Signal in Halorhodopsin from Natronomonas pharaonis

“…5 d), due to its less important role in chloride binding. In addition, atomic structures showed that the hydroxyl group (-OH) in Thr126 is not oriented directly toward chloride, and the anion binding preference for these residues was found to occur in the order of Arg123 > Ser130 > Thr126 (36), which explains the detected photocurrent signal in T126A. Therefore, this unique photocurrent signal is tightly associated with chloride.…”