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 language | English |
|---|---|
| Pages (from-to) | 141-152 |
| Number of pages | 12 |
| Journal | Photosynthesis Research |
| Volume | 120 |
| Issue number | 1-2 |
| DOIs | |
| Publication status | Published - 2014 |
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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 journal › Article
}
TY - JOUR
T1 - Using site-directed mutagenesis to probe the role of the D2 carotenoid in the secondary electron-transfer pathway of photosystem II
AU - Shinopoulos, Katherine E.
AU - Yu, Jianfeng
AU - Nixon, Peter J.
AU - Brudvig, Gary W
PY - 2014
Y1 - 2014
N2 - 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.
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.
KW - Chlorophyll radical
KW - EPR spectroscopy
KW - Near-IR spectroscopy
KW - Photosystem II
KW - Site-directed mutagenesis
KW - β-Carotene radical
UR - http://www.scopus.com/inward/record.url?scp=84898816394&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84898816394&partnerID=8YFLogxK
U2 - 10.1007/s11120-013-9793-6
DO - 10.1007/s11120-013-9793-6
M3 - Article
C2 - 23334888
AN - SCOPUS:84898816394
VL - 120
SP - 141
EP - 152
JO - Photosynthesis Research
JF - Photosynthesis Research
SN - 0166-8595
IS - 1-2
ER -