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/F250Y^phR, 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→P ₄→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.