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
N1 - Funding Information:
ACKNOWLEDGMENTS. We thank Ayumi Imai and Megumi Kobayashi for their technical assistance in preparing the LH1-RC complex from strain DP2, and the Kao Corporation for providing lauryldimethylamine N-oxide. This work was supported by the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT; Grants-in-Aid for Scientific Research B 16H04174, to Z.-Y.W.-O. and C 16K07295, to Y.K.), Takeda Science Foundation, a Sumitomo Foundation Grant for Basic Science Research Projects, the Kurata Memorial Hitachi Science and Technology Foundation (Z.-Y.W.-O.), and a Japan Society for the Promotion of Science Research (JSPS) Fellowship (to T.K.). This work was supported in part by Grant-in-Aid for JSPS KAKENHI Grant 15K00642, MEXT KAKENHI Grant 24107004, and the Strategic Research Base Development Program for Private Universities from MEXT, Japan (to K.I.). K.V.P.N. was supported in part by grants from the Tokyo Ohka Foundation and the Precursory Research for Embryonic Science and Technology program of the Japan Science and Technology Agency.
Publisher Copyright:
© 2017, National Academy of Sciences. All rights reserved.
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
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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 -