Akiteru Seki

Interaction of the halobacterial transducer to a halorhodopsin mutant engineered so as to bind the transducer: Cl- circulation within the extracellular channel.

An alkali‐halophilic archaeum, Natronomonas pharaonis, contains two rhodopsins that are halorhodopsin (phR), a light‐driven inward Cl− pump and phoborhodopsin (ppR), the receptor of negative phototaxis functioning by forming a signaling complex with a transducer, pHtrII ( Sudo Y. et al., J. Mol. Biol. 357 [2006] 1274 ). Previously, we reported that the phR double mutant, P240T/F250YphR, can bind with pHtrII. This mutant itself can transport Cl−, while the net transport was stopped upon formation of the complex. The flash‐photolysis data were analyzed by a scheme in which phR→P1→P2→P3→P4→phR. The P3 of the wild‐type and the double mutant contained two components, X‐ and O‐intermediates. After the complex formation, however, the P3 of the double mutant lacked the X‐intermediate. These observations imply that the X‐intermediate (probably the N‐intermediate) is the state having Cl− in the cytoplasmic binding site and that the complex undergoes an extracellular Cl− circulation because of the inhibition of formation of the X‐intermediate.

Role of Arg123 in Light‐driven Anion Pump Mechanisms of pharaonis Halorhodopsin†

Halorhodopsin (HR) acts as a light‐driven chloride pump which transports a chloride ion from the extracellular (EC) to the cytoplasmic space during a photocycle reaction that includes some photointermediates initiated by illumination. To understand the chloride uptake mechanisms, we focused on a basic residue Arg123 of HR from Natronomonas pharaonis (NpHR), which is the only basic residue located in the EC half ion channel. By the measurements of the visible absorption spectra in the dark and the light‐induced inward current through the membrane, it was shown that the chloride binding and transport ability of NpHR completely disappeared by the change of arginine to glutamine. From flashphotolysis analysis, the photocycle of R123Q differed from that of wildtype NpHR completely. The response of the R123H mutant depended on pH. These facts imply that the positive charge at position 123 is essential for chloride binding in the ground state and for the chloride uptake under illumination. On the basis of the molecular structures of HR and the anion‐transportable mutants of bacteriorhodopsin, the effects of the positive charge and the conformational change of the Arg123 side chain as well as the chloride‐pumping mechanism are discussed.