Crystal Structure of the 11-cis Isomer of Pharaonis Halorhodopsin: Structural Constraints on Interconversions among Different Isomeric States

Siu-Kit Chan, Haruki Kawaguchi, Hiroki Kubo, Midori Murakami, Kunio Ihara, Kosuke Maki, Tsutomu Kouyama

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Like other microbial rhodopsins, the light driven chloride pump halorhodopsin from Natronomonas pharaonis (pHR) contains a mixture of all-trans/15-anti and 13-cis/15-syn isomers in the dark adapted state. A recent crystallographic study of the reaction states of pHR has shown that reaction states with 13-cis/15-syn retinal occur in the anion pumping cycle that is initiated by excitation of the all-trans isomer. In this study, we investigated interconversions among different isomeric states of pHR in the absence of chloride ions. The illumination of chloride free pHR with red light caused a large blue shift in the absorption maximum of the retinal visible band. During this "red adaptation", the content of the 11-cis isomer increased significantly, while the molar ratio of the 13-cis isomer to the all-trans isomer remained unchanged. The results suggest that the thermally activated interconversion between the 13-cis and the all-trans isomers is very rapid. Diffraction data from red adapted crystals showed that accommodation of the retinal chromophore with the 11-cis/15-syn configuration was achieved without a large change in the retinal binding pocket. The measurement of absorption kinetics under illumination showed that the 11-cis isomer, with a λmax at 565 nm, was generated upon excitation of a red-shifted species (λmax = 625 nm) that was present as a minor component in the dark adapted state. It is possible that this red-shifted species mimics an O-like reaction state with 13-cis/15-syn retinal, which was hypothesized to occur at a late stage of the anion pumping cycle.

Original languageEnglish
Pages (from-to)4092-4104
Number of pages13
JournalBiochemistry
Volume55
Issue number29
DOIs
Publication statusPublished - Jul 26 2016
Externally publishedYes

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Halorhodopsins
Isomers
Chlorides
Crystal structure
Lighting
Anions
Microbial Rhodopsins
Light
Ions
Chromophores
Diffraction
Pumps
Crystals
Kinetics

ASJC Scopus subject areas

  • Biochemistry

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Crystal Structure of the 11-cis Isomer of Pharaonis Halorhodopsin : Structural Constraints on Interconversions among Different Isomeric States. / Chan, Siu-Kit; Kawaguchi, Haruki; Kubo, Hiroki; Murakami, Midori; Ihara, Kunio; Maki, Kosuke; Kouyama, Tsutomu.

In: Biochemistry, Vol. 55, No. 29, 26.07.2016, p. 4092-4104.

Research output: Contribution to journalArticle

Chan, Siu-Kit ; Kawaguchi, Haruki ; Kubo, Hiroki ; Murakami, Midori ; Ihara, Kunio ; Maki, Kosuke ; Kouyama, Tsutomu. / Crystal Structure of the 11-cis Isomer of Pharaonis Halorhodopsin : Structural Constraints on Interconversions among Different Isomeric States. In: Biochemistry. 2016 ; Vol. 55, No. 29. pp. 4092-4104.
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AU - Kubo, Hiroki

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AU - Maki, Kosuke

AU - Kouyama, Tsutomu

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AB - Like other microbial rhodopsins, the light driven chloride pump halorhodopsin from Natronomonas pharaonis (pHR) contains a mixture of all-trans/15-anti and 13-cis/15-syn isomers in the dark adapted state. A recent crystallographic study of the reaction states of pHR has shown that reaction states with 13-cis/15-syn retinal occur in the anion pumping cycle that is initiated by excitation of the all-trans isomer. In this study, we investigated interconversions among different isomeric states of pHR in the absence of chloride ions. The illumination of chloride free pHR with red light caused a large blue shift in the absorption maximum of the retinal visible band. During this "red adaptation", the content of the 11-cis isomer increased significantly, while the molar ratio of the 13-cis isomer to the all-trans isomer remained unchanged. The results suggest that the thermally activated interconversion between the 13-cis and the all-trans isomers is very rapid. Diffraction data from red adapted crystals showed that accommodation of the retinal chromophore with the 11-cis/15-syn configuration was achieved without a large change in the retinal binding pocket. The measurement of absorption kinetics under illumination showed that the 11-cis isomer, with a λmax at 565 nm, was generated upon excitation of a red-shifted species (λmax = 625 nm) that was present as a minor component in the dark adapted state. It is possible that this red-shifted species mimics an O-like reaction state with 13-cis/15-syn retinal, which was hypothesized to occur at a late stage of the anion pumping cycle.

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