11 research outputs found
Spectroscopic Evidence for a Redox-Controlled Proton Gate at Tyrosine D in Photosystem II
Tyrosine D (TyrD) is one of two well-studied
redox active tyrosines
in Photosystem II. TyrD shows redox kinetics much slower than that
of its homologue, TyrZ, and is normally present as a stable deprotonated
radical (TyrD<sup>•</sup>). We have used time-resolved continuous
wave electron paramagnetic resonance and electron spin echo envelope
modulation spectroscopy to show that deuterium exchangeable protons
can access TyrD on a time scale that is much faster (50–100
times) than that previously observed. The time of H/D exchange is
strongly dependent on the redox state of TyrD. This finding can be
related to a change in position of a water molecule close to TyrD
Stability of the S<sub>3</sub> and S<sub>2</sub> State Intermediates in Photosystem II Directly Probed by EPR Spectroscopy
The stability of the S<sub>3</sub> and S<sub>2</sub> states
of
the oxygen evolving complex in photosystem II (PSII) was directly
probed by EPR spectroscopy in PSII membrane preparations from spinach
in the presence of the exogenous electron acceptor P<i>p</i>BQ at 1, 10, and 20 °C. The decay of the S<sub>3</sub> state
was followed in samples exposed to two flashes by measuring the split
S<sub>3</sub> EPR signal induced by near-infrared illumination at
5 K. The decay of the S<sub>2</sub> state was followed in samples
exposed to one flash by measuring the S<sub>2</sub> state multiline
EPR signal. During the decay of the S<sub>3</sub> state, the S<sub>2</sub> state multiline EPR signal first increased and then decreased
in amplitude. This shows that the decay of the S<sub>3</sub> state
to the S<sub>1</sub> state occurs via the S<sub>2</sub> state. The
decay of the S<sub>3</sub> state was biexponential with a fast kinetic
phase with a few seconds decay half-time. This occurred in 10–20%
of the PSII centers. The slow kinetic phase ranged from a decay half-time
of 700 s (at 1 °C) to ∼100 s (at 20 °C) in the remaining
80–90% of the centers. The decay of the S<sub>2</sub> state
was also biphasic and showed quite similar kinetics to the decay of
the S<sub>3</sub> state. Our experiments show that the auxiliary electron
donor Y<sub>D</sub> was oxidized during the entire experiment. Thus,
the reduced form of Y<sub>D</sub> does not participate to the fast
decay of the S<sub>2</sub> and S<sub>3</sub> states we describe here.
Instead, we suggest that the decay of the S<sub>3</sub> and S<sub>2</sub> states reflects electron transfer from the acceptor side
of PSII to the donor side of PSII starting in the corresponding S
state. It is proposed that this exists in equilibrium with Y<sub>Z</sub> according to S<sub>3</sub>Y<sub>Z</sub> ⇔ S<sub>2</sub>Y<sub>Z</sub><sup>•</sup> in the case of the S<sub>3</sub> state
decay and S<sub>2</sub>Y<sub>Z</sub> ⇔ S<sub>1</sub>Y<sub>Z</sub><sup>•</sup> in the case of the S<sub>2</sub> state decay.
Two kinetic models are discussed, both developed with the assumption
that the slow decay of the S<sub>3</sub> and S<sub>2</sub> states
occurs in PSII centers where Y<sub>Z</sub> is also a fast donor to
P<sub>680</sub><sup>+</sup> working in the nanosecond time regime
and that the fast decay of the S<sub>3</sub> and S<sub>2</sub> states
occurs in centers where Y<sub>Z</sub> reduces P<sub>680</sub><sup>+</sup> with slower microsecond kinetics. Our measurements also demonstrate
that the split S<sub>3</sub> EPR signal can be used as a direct probe
to the S<sub>3</sub> state and that it can provide important information
about the redox properties of the S<sub>3</sub> state
Quantitative LC-MS of PSII core protein phosphorylation of Col-0, Ws-4 and L<i>er</i>-0 accessions.
<p>Data represent means ±SD of four independent preparations.</p>*<p>, Significantly different from Col-0 and L<i>er</i>-0 (Student's t-test P<0.05).</p><p>The plants were grown hydroponically for six weeks at an irradiance of 120 µmol photons m<sup>−2</sup> s<sup>−1</sup> and treated for 3 h at an irradiance of 950 µmol photons m<sup>−2</sup> s<sup>−1</sup>. Thylakoid membranes were isolated in the presence of NaF. The levels of phosphorylated PSII core proteins were analyzed by quantitative mass spectrometry and expressed relative to Col-0.</p
Rapid light response curves of PSII quantum yield and excitation pressure in Col-0, Ws-4 and L<i>er</i>-0 accessions.
<p>Leaves from plants grown hydroponically at an irradiance of 120 µmol photons m<sup>−2</sup> s<sup>−1</sup> were detached and illuminated stepwise using photosynthetically active radiation (PAR) of various intensities. Chlorophyll fluorescence was measured, and the quantum yield of PSII photochemistry <b>Φ</b><sub>PSII</sub> (A) the excitation pressure 1-qP (B) were calculated. The data are plotted <i>versus</i> irradiance and represent means ±SD (n = 7). SD bars are shown when larger than the symbols.</p
Photoinhibition of PSII in Col-0, Ws-4 and L<i>er</i>-0 accessions.
<p>Leaves detached from plants grown hydroponically at an irradiance of 120 µmol photons m<sup>−2</sup> s<sup>−1</sup> (GL) were exposed for 3 h to GL or high light (HL = 950 µmol photons m<sup>−2</sup> s<sup>−1</sup>) treatments in the presence or absence of lincomycin (LN). (A) The <i>F</i><sub>v</sub><i>/F</i><sub>m</sub> parameter was determined in leaves after 5 min dark adaptation. The levels of <i>F</i><sub>v</sub><i>/F</i><sub>m</sub> were calculated relative to a value of 0.82, obtained in 16 h dark-adapted Col-0 leaves, and plotted as means ±SD (n = 3). (B) Thylakoid membranes were isolated from the treated leaves and subjected to western blotting with anti-D1 and with anti-Lhcb2 (as loading control) (0.25 µg Chl/lane). (C) Transmission electron micrographs of chloroplast ultrastructure from plants exposed to 3 h HL. Scale bar: 1 µm.</p
Time course of PSII protein phosphorylation and dephosphorylation in Col-0, Ws-4 and L<i>er</i>-0 accessions.
<p>(A) <i>In vivo</i> steady-state PSII protein phosphorylation. Intact plants grown hydroponically were illuminated for 0, 1.5 and 3 h with high light (HL = 950 µmol photons m<sup>−2</sup> s<sup>−1</sup>), followed by isolation of thylakoids in the presence of NaF, and western blot analysis with Zymed antibody. A western blot of the same samples was performed with anti-D1 antibody, and used as a loading control. (B) Plot of data shown in panel A, where quantification levels are expressed relative to phosphorylation in Col-0. (C) <i>In vitro</i> PSII protein dephosphorylation. Thylakoid membranes were isolated from 3 h HL treated plants in the presence of NaF, and incubated in darkness at 22° or 44°C for the indicated periods of time. Samples were blotted using a Zymed antibody. The western blot with anti-D1 antibody of samples from 3 h HL plants and incubated in darkness at 22° or 44°C for 60 min indicates equal loading of the gels. (D) Plot of C, representing % remaining D1 phosphorylation, 100% = initial phosphorylation level in each accession. The plotted data in (B) and (D) are means ±SD (n = 3). SD bars are shown when larger than the symbols.</p
Fundamental parameters of the O-J-I-P fluorescence induction curves recorded on attached leaves of Col-0, Ws-4 and L<i>er</i>-0 accessions.
<p>The significant differences are indicated by asterisk (Student's t test P<0.05).</p><p>The plants were grown on soil for 15–17 days at an irradiance of 120 µmol photons m<sup>−2</sup> s<sup>−1</sup>. The leaves were adapted to darkness for 15 min before the measurement (n = 20–25).</p
Leaf biomass, chlorophyll content and PSI to PSII ratio of Col-0, Ws-4, and L<i>er</i>-0 accessions.
a<p>Leaf chlorophyll content was measured spectrophotometrically after extraction in ethanol, and expressed on leaf area and on fresh weight basis.</p>b<p>PSI/PSII ratio was measured by EPR spectroscopy in isolated thylakoids.</p><p>The data were expressed as means ±SD (n = number of replicates). The parameters were also expressed relative to Col-0. *, Significantly different from Col-0 (Student's t-test P<0.05).</p><p>The parameters were measured on plants grown hydroponically for six weeks at an irradiance of 120 µmol photons m<sup>−2</sup> s<sup>−1</sup>.</p
Analysis by Blue-native gel electrophoresis of thylakoid protein complexes from Col-0, Ws-4 and L<i>er</i>-0 accessions.
<p>Leaves detached from plants grown hydroponically at an irradiance of 120 µmol photons m<sup>−2</sup> s<sup>−1</sup> (GL) were exposed to high light (HL = 950 µmol photons m<sup>−2</sup> s<sup>−1</sup>) in the absence or presence of lincomycin (LN) for 3 h. As control, 16 h dark-adapted plants were used. Thylakoid membranes were isolated and solubilized mildly with n-dodecyl-ß-D-maltoside, and the various types of Chl protein complexes were separated by Blue-native gel electrophoresis. (A) Representative unstained Blue-native gel (8 µg Chl per lane). (B) (C). The PSII complexes together with various combinations of LHCs were identified based on western bloting with anti-Lhcb2, D1 and CP43 antibodies, and based on previous reports <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046206#pone.0046206-Aro1" target="_blank">[2]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046206#pone.0046206-Fristedt3" target="_blank">[33]</a>. Representative western blot with anti-D1 antibody of gel as in (A). (D) Representative western blot with anti-Lchb2 of gel as as in (A).</p
Thylakoid lipid composition in Col-0, Ws-4 and L<i>er</i>-0 accessions.
<p>Membrane lipids were extracted from plants grown on soil for six weeks and analyzed by LC-MS. (A) The content of each lipid class was expressed as a molar ratio to chlorophyll. (B) Lipid species comprising more than 2% of the lipid class are shown as mol % of the total lipid class. Numbers indicate total number of carbons and double bonds in lipid fatty acids. The plotted data represent means ±SD (n = 3). An asterisk indicates significant difference from Col-0 (Student's t-test P<0.05).</p