Probing structure–function relationships in early events in photosynthesis using a chimeric photocomplex

Kenji V.P. Nagashima, Mai Sasaki, Kanako Hashimoto, Shinichi Takaichi, Sakiko Nagashima, Long Jiang Yu, Yuto Abe, Kenta Gotou, Tomoaki Kawakami, Mizuki Takenouchi, Yuuta Shibuya, Akira Yamaguchi, Takashi Ohno, Jian Ren Shen, Kazuhito Inoue, Michael T. Madigan, Yukihiro Kimura, Zheng Yu Wang-Otomo

Research output: Contribution to journalArticle

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Abstract

The native core light-harvesting complex (LH1) from the thermophilic purple phototrophic bacterium Thermochromatium tepidum requires Ca2+ for its thermal stability and characteristic absorption maximum at 915 nm. To explore the role of specific amino acid residues of the LH1 polypeptides in Ca-binding behavior, we constructed a genetic system for heterologously expressing the Tch. tepidum LH1 complex in an engineered Rhodobacter sphaeroides mutant strain. This system contained a chimeric pufBALM gene cluster (pufBA from Tch. tepidum and pufLM from Rba. sphaeroides) and was subsequently deployed for introducing site-directed mutations on the LH1 polypeptides. All mutant strains were capable of phototrophic (anoxic/ light) growth. The heterologously expressed Tch. tepidum wild-type LH1 complex was isolated in a reaction center (RC)-associated form and displayed the characteristic absorption properties of this thermophilic phototroph. Spheroidene (the major carotenoid in Rba. sphaeroides) was incorporated into the Tch. tepidum LH1 complex in place of its native spirilloxanthins with one carotenoid molecule present per αβ-subunit. The hybrid LH1-RC complexes expressed in Rba. sphaeroides were characterized using absorption, fluorescence excitation, and resonance Raman spectroscopy. Site-specific mutagenesis combined with spectroscopic measurements revealed that α-D49, β-L46, and a deletion at position 43 of the α-polypeptide play critical roles in Ca binding in the Tch. tepidum LH1 complex; in contrast, α-N50 does not participate in Ca2+ coordination. These findings build on recent structural data obtained from a high-resolution crystallographic structure of the membrane integrated Tch. tepidum LH1-RC complex and have unambiguously identified the location of Ca2+ within this key antenna complex.

Original languageEnglish
Pages (from-to)10906-10911
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume114
Issue number41
DOIs
Publication statusPublished - Oct 10 2017

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Photosynthesis
Carotenoids
Peptides
Rhodobacter sphaeroides
Light
Proteobacteria
Raman Spectrum Analysis
Multigene Family
Site-Directed Mutagenesis
Hot Temperature
Fluorescence
Amino Acids
Mutation
Membranes
Growth

Keywords

  • Ca binding
  • Light harvesting
  • Photosynthesis
  • Q transition
  • Thermochromatium tepidum

ASJC Scopus subject areas

  • General

Cite this

Probing structure–function relationships in early events in photosynthesis using a chimeric photocomplex. / Nagashima, Kenji V.P.; Sasaki, Mai; Hashimoto, Kanako; Takaichi, Shinichi; Nagashima, Sakiko; Yu, Long Jiang; Abe, Yuto; Gotou, Kenta; Kawakami, Tomoaki; Takenouchi, Mizuki; Shibuya, Yuuta; Yamaguchi, Akira; Ohno, Takashi; Shen, Jian Ren; Inoue, Kazuhito; Madigan, Michael T.; Kimura, Yukihiro; Wang-Otomo, Zheng Yu.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 114, No. 41, 10.10.2017, p. 10906-10911.

Research output: Contribution to journalArticle

Nagashima, KVP, Sasaki, M, Hashimoto, K, Takaichi, S, Nagashima, S, Yu, LJ, Abe, Y, Gotou, K, Kawakami, T, Takenouchi, M, Shibuya, Y, Yamaguchi, A, Ohno, T, Shen, JR, Inoue, K, Madigan, MT, Kimura, Y & Wang-Otomo, ZY 2017, 'Probing structure–function relationships in early events in photosynthesis using a chimeric photocomplex', Proceedings of the National Academy of Sciences of the United States of America, vol. 114, no. 41, pp. 10906-10911. https://doi.org/10.1073/pnas.1703584114
Nagashima, Kenji V.P. ; Sasaki, Mai ; Hashimoto, Kanako ; Takaichi, Shinichi ; Nagashima, Sakiko ; Yu, Long Jiang ; Abe, Yuto ; Gotou, Kenta ; Kawakami, Tomoaki ; Takenouchi, Mizuki ; Shibuya, Yuuta ; Yamaguchi, Akira ; Ohno, Takashi ; Shen, Jian Ren ; Inoue, Kazuhito ; Madigan, Michael T. ; Kimura, Yukihiro ; Wang-Otomo, Zheng Yu. / Probing structure–function relationships in early events in photosynthesis using a chimeric photocomplex. In: Proceedings of the National Academy of Sciences of the United States of America. 2017 ; Vol. 114, No. 41. pp. 10906-10911.
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AU - Nagashima, Sakiko

AU - Yu, Long Jiang

AU - Abe, Yuto

AU - Gotou, Kenta

AU - Kawakami, Tomoaki

AU - Takenouchi, Mizuki

AU - Shibuya, Yuuta

AU - Yamaguchi, Akira

AU - Ohno, Takashi

AU - Shen, Jian Ren

AU - Inoue, Kazuhito

AU - Madigan, Michael T.

AU - Kimura, Yukihiro

AU - Wang-Otomo, Zheng Yu

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N2 - The native core light-harvesting complex (LH1) from the thermophilic purple phototrophic bacterium Thermochromatium tepidum requires Ca2+ for its thermal stability and characteristic absorption maximum at 915 nm. To explore the role of specific amino acid residues of the LH1 polypeptides in Ca-binding behavior, we constructed a genetic system for heterologously expressing the Tch. tepidum LH1 complex in an engineered Rhodobacter sphaeroides mutant strain. This system contained a chimeric pufBALM gene cluster (pufBA from Tch. tepidum and pufLM from Rba. sphaeroides) and was subsequently deployed for introducing site-directed mutations on the LH1 polypeptides. All mutant strains were capable of phototrophic (anoxic/ light) growth. The heterologously expressed Tch. tepidum wild-type LH1 complex was isolated in a reaction center (RC)-associated form and displayed the characteristic absorption properties of this thermophilic phototroph. Spheroidene (the major carotenoid in Rba. sphaeroides) was incorporated into the Tch. tepidum LH1 complex in place of its native spirilloxanthins with one carotenoid molecule present per αβ-subunit. The hybrid LH1-RC complexes expressed in Rba. sphaeroides were characterized using absorption, fluorescence excitation, and resonance Raman spectroscopy. Site-specific mutagenesis combined with spectroscopic measurements revealed that α-D49, β-L46, and a deletion at position 43 of the α-polypeptide play critical roles in Ca binding in the Tch. tepidum LH1 complex; in contrast, α-N50 does not participate in Ca2+ coordination. These findings build on recent structural data obtained from a high-resolution crystallographic structure of the membrane integrated Tch. tepidum LH1-RC complex and have unambiguously identified the location of Ca2+ within this key antenna complex.

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