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 language | English |
|---|---|
| Pages (from-to) | 10906-10911 |
| Number of pages | 6 |
| Journal | Proceedings of the National Academy of Sciences of the United States of America |
| Volume | 114 |
| Issue number | 41 |
| DOIs | |
| Publication status | Published - Oct 10 2017 |
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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 journal › Article
}
TY - JOUR
T1 - Probing structure–function relationships in early events in photosynthesis using a chimeric photocomplex
AU - Nagashima, Kenji V.P.
AU - Sasaki, Mai
AU - Hashimoto, Kanako
AU - Takaichi, Shinichi
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
PY - 2017/10/10
Y1 - 2017/10/10
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.
AB - 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.
KW - Ca binding
KW - Light harvesting
KW - Photosynthesis
KW - Q transition
KW - Thermochromatium tepidum
UR - http://www.scopus.com/inward/record.url?scp=85030755377&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85030755377&partnerID=8YFLogxK
U2 - 10.1073/pnas.1703584114
DO - 10.1073/pnas.1703584114
M3 - Article
C2 - 28935692
AN - SCOPUS:85030755377
VL - 114
SP - 10906
EP - 10911
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
SN - 0027-8424
IS - 41
ER -