Using site-directed mutagenesis to probe the role of the D2 carotenoid in the secondary electron-transfer pathway of photosystem II
Author(s)
Shinopoulos, KE
Yu, J
Nixon, PJ
Brudvig, GW
Type
Journal Article
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 b559 (Cyt b559), 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 b559, 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 Synechocystissp. 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 Car•? near-IR spectrum in all three
mutated PSII samples, allowing the assignment of the
spectral signature of CarD2
•?; CarD2
•? 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 Chl•?
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.
(PSII), which occurs when water oxidation is inhibited,
involves redox-active carotenoids (Car), as well as chlorophylls
(Chl), and cytochrome b559 (Cyt b559), 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 b559, 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 Synechocystissp. 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 Car•? near-IR spectrum in all three
mutated PSII samples, allowing the assignment of the
spectral signature of CarD2
•?; CarD2
•? 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 Chl•?
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.
Date Issued
2014-05-01
Date Acceptance
2013-01-02
Citation
Photosynthesis Research, 2014, 120 (1-2), pp.141-152
ISSN
1573-5079
Publisher
Springer Verlag (Germany)
Start Page
141
End Page
152
Journal / Book Title
Photosynthesis Research
Volume
120
Issue
1-2
Copyright Statement
© The Author(s) 2013. This article is published with open access at Springerlink.com
Subjects
Science & Technology
Life Sciences & Biomedicine
Plant Sciences
PLANT SCIENCES
beta-Carotene radical
Chlorophyll radical
Cytochrome b(559)
EPR spectroscopy
Near-IR spectroscopy
Photosystem II
Site-directed mutagenesis
OXYGEN-EVOLVING COMPLEX
BETA-CAROTENE
PRIMARY PHOTOCHEMISTRY
CYTOCHROME B(559)
D1 POLYPEPTIDE
EPR
RESOLUTION
OXIDATION
DONOR
PHOTOINHIBITION
Publication Status
Published