Role of Putative Anion-Binding Sites in Cytoplasmic and Extracellular Channels of Natronomonas pharaonis Halorhodopsin

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• 作者：Maki Sato ; Megumi Kubo ; Tomoyasu Aizawa ; Naoki Kamo ; Takashi Kikukawa ; Katsutoshi Nitta ; and Makoto Demura
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：March 29, 2005
• 年：2005
• 卷：44
• 期：12
• 页码：4775 - 4784
• 全文大小：327K
• 年卷期：v.44,no.12(March 29, 2005)
• ISSN：1520-4995

文摘

Natronomonas (Natronobacterium) pharaonis halorhodopsin (NpHR) is an inward light-drivenCl^- ion pump. For efficient Cl^- transport, the existence of Cl^--binding or -interacting sites in bothextracellular (EC) and cytoplasmic (CP) channels is postulated. Candidates include Arg123 and Thr126in EC channels and Lys215 and Thr218 in CP channels. The roles played by these amino acid residuesin anion binding and in the photocycle have been investigated by mutation of the amino acid residues atthese positions. Anion binding was assayed by changes in circular dichroism and the shift in the absorptionmaximum upon addition of Cl^- to anion-free NpHR. The binding affinity was affected in mutants inwhich certain EC residues had been replaced; this finding revealed the importance of Arg123. On theother hand, mutants in which certain residues in the CP channel were replaced (CP mutants) did not showchanges in their dissociation constants. The photocycles of these mutants were also examined, and in thecase of the EC mutants, the transition to the last step was greatly delayed; on the other hand, in the CPmutants, L2-photointermediate decay was significantly prolonged, except in the case of K215Q, whichlacked the O-photointermediate. The importance of Thr218 for binding of Cl^- to the CP channel wasindicated by these results. On the basis of these observations, the possible anion transport mechanism ofNpHR was discussed.