Using site-directed mutagenesis to probe the role of the D2 carotenoid in the secondary electron-transfer pathway of photosystem II

Katherine E. Shinopoulos, Jianfeng Yu, Peter J. Nixon, Gary W Brudvig

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Secondary electron transfer in photosystem II (PSII), which occurs when water oxidation is inhibited, involves redox-active carotenoids (Car), as well as chlorophylls (Chl), and cytochrome b 559 (Cyt b 559), and is believed to play a role in photoprotection. CarD2 may be the initial point of secondary electron transfer because it is the closest cofactor to both P680, the initial oxidant, and to Cyt b 559, the terminal secondary electron donor within PSII. In order to characterize the role of CarD2 and to determine the effects of perturbing CarD2 on both the electron-transfer events and on the identity of the redox-active cofactors, it is necessary to vary the properties of CarD2 selectively without affecting the ten other Car per PSII. To this end, site-directed mutations around the binding pocket of CarD2 (D2-G47W, D2-G47F, and D2-T50F) have been generated in Synechocystis sp. PCC 6803. Characterization by near-IR and EPR spectroscopy provides the first experimental evidence that CarD2 is one of the redox-active carotenoids in PSII. There is a specific perturbation of the Caṙ+ near-IR spectrum in all three mutated PSII samples, allowing the assignment of the spectral signature of Car D2 ̇+; Car D2 ̇+ exhibits a near-IR peak at 980 nm and is the predominant secondary donor oxidized in a charge separation at low temperature in ferricyanide-treated wild-type PSII. The yield of secondary donor radicals is substantially decreased in PSII complexes isolated from each mutant. In addition, the kinetics of radical formation are altered in the mutated PSII samples. These results are consistent with oxidation of CarD2 being the initial step in secondary electron transfer. Furthermore, normal light levels during mutant cell growth perturb the shape of the Chl̇+ near-IR absorption peak and generate a dark-stable radical observable in the EPR spectra, indicating a higher susceptibility to photodamage further linking the secondary electron-transfer pathway to photoprotection.

Original languageEnglish
Pages (from-to)141-152
Number of pages12
JournalPhotosynthesis Research
Volume120
Issue number1-2
DOIs
Publication statusPublished - 2014

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Mutagenesis
Photosystem II Protein Complex
site-directed mutagenesis
Carotenoids
Site-Directed Mutagenesis
photosystem II
electron transfer
carotenoids
Electrons
Oxidation-Reduction
photostability
cytochrome b
Paramagnetic resonance
oxidation
Synechocystis sp. PCC 6803
Synechocystis
chlorophyll
ferricyanides
Oxidation
mutants

Keywords

  • Chlorophyll radical
  • EPR spectroscopy
  • Near-IR spectroscopy
  • Photosystem II
  • Site-directed mutagenesis
  • β-Carotene radical

ASJC Scopus subject areas

  • Plant Science
  • Cell Biology
  • Biochemistry
  • Medicine(all)

Cite this

Using site-directed mutagenesis to probe the role of the D2 carotenoid in the secondary electron-transfer pathway of photosystem II. / Shinopoulos, Katherine E.; Yu, Jianfeng; Nixon, Peter J.; Brudvig, Gary W.

In: Photosynthesis Research, Vol. 120, No. 1-2, 2014, p. 141-152.

Research output: Contribution to journalArticle

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AU - Brudvig, Gary W

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AB - Secondary electron transfer in photosystem II (PSII), which occurs when water oxidation is inhibited, involves redox-active carotenoids (Car), as well as chlorophylls (Chl), and cytochrome b 559 (Cyt b 559), and is believed to play a role in photoprotection. CarD2 may be the initial point of secondary electron transfer because it is the closest cofactor to both P680, the initial oxidant, and to Cyt b 559, the terminal secondary electron donor within PSII. In order to characterize the role of CarD2 and to determine the effects of perturbing CarD2 on both the electron-transfer events and on the identity of the redox-active cofactors, it is necessary to vary the properties of CarD2 selectively without affecting the ten other Car per PSII. To this end, site-directed mutations around the binding pocket of CarD2 (D2-G47W, D2-G47F, and D2-T50F) have been generated in Synechocystis sp. PCC 6803. Characterization by near-IR and EPR spectroscopy provides the first experimental evidence that CarD2 is one of the redox-active carotenoids in PSII. There is a specific perturbation of the Caṙ+ near-IR spectrum in all three mutated PSII samples, allowing the assignment of the spectral signature of Car D2 ̇+; Car D2 ̇+ exhibits a near-IR peak at 980 nm and is the predominant secondary donor oxidized in a charge separation at low temperature in ferricyanide-treated wild-type PSII. The yield of secondary donor radicals is substantially decreased in PSII complexes isolated from each mutant. In addition, the kinetics of radical formation are altered in the mutated PSII samples. These results are consistent with oxidation of CarD2 being the initial step in secondary electron transfer. Furthermore, normal light levels during mutant cell growth perturb the shape of the Chl̇+ near-IR absorption peak and generate a dark-stable radical observable in the EPR spectra, indicating a higher susceptibility to photodamage further linking the secondary electron-transfer pathway to photoprotection.

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KW - β-Carotene radical

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