Feikema, Willem Onno
Citation
Feikema, W. O. (2006, September 7). Photosystem II and photoinhibition. Retrieved from
https://hdl.handle.net/1887/4547
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Institutional Repository of the University of Leiden
1
Photosynthesis, an introduction
The discovery of photosynthesisBefore the era of modern science a long held theory about plant growth was that the soil provided the nutrition for higher plants (‘compost theory’). Indeed, nitrogen, phosphorus, sulfur and trace elements required by a higher plant are obtained from the soil [1]. However, in terms of mass, the contribution to growth by these substances is smaller than the contribution by carbon and water and in terms of energy their contribution is none or negative. The medical chemist and physician Jean-Baptiste van Helmont (1577-1644) recognized the requirement of water for growth of higher plants since the growth of a seedling into a tree over a period of 5 years hardly led to a decrease in weight of the pre-weighted dry mass of the soil whereas the mass of the higher plant increased from 2 to 77 kg, thus providing evidence against the compost theory. At the time of Van Helmont no other role for water than directly contributing to the mass of higher plants could be envisioned but besides this limitation van Helmont discovered one of the three components required for photosynthesis: water, light and carbon dioxide [1]. Joseph Priestly (1733-1804) observed for the first time that higher plants produce a gas, ‘dephlogisticated’ air, which is required for animal life [2]. The physician Jan Ingenhousz (1730-1799) recognized that higher plants require light for the production of ‘dephlogisticated’ air by detecting air bubbles of ‘good quality’ on leaves that were placed under water in the light, whereas the air bubbles did hardly emerge and were of ‘bad quality’ if the leaves were placed out of the light [2]. Ingenhousz also showed that the released ‘dephlogisticated’ air was identical to the in 1777 by Lavoisier discovered element oxygen. Jean Senebier (1742-1809) discovered the uptake of CO2 (‘fixed air’) by higher plants and Robert
Development of life
The earth is about 4.5 billion years old and life emerged relatively early in its history during the Archean era, possibly dating back to 4 billion years ago [1]. The earliest known organisms, the bacteria or prokaryotes, are still largely present today. They are unicellular and have a relatively low level of internal organization: all metabolic processes occur in the cytosol and DNA is in direct contact with the cytosol [1]. Besides the prokaryotes, eukaryotes evolved, which according to the oldest fossils known, originated at least 1.5 billion years ago [1]. Eukaryotic cells show a partitioning of metabolic processes in membrane-enclosed organelles. DNA is protected by a double membrane forming the nucleus. The eukaryotic cell structure made that multi-cellular organisms could develop [1].
Anoxygenic photosynthesis
The primary process of photosynthesis in purple bacteria occurs in a protein complex that is referred to as the reaction center (RC). The excitation energy from light harvesting complexes (LHCs) is transferred to a special pair of chlorophyll molecules (P for primary donor) in the RC, which results in a number of events aimed to stabilize the captured energy to generate reductive power. The oxidized primary donor of the RC in purple bacteria (bRC) has a potential of about +0.55 V, which is sufficient to oxidize a variety of substrates [3]. In sulfur bacteria H2S is the ultimate electron donor,
other bacterial photoautotrophs use Fe2+, S
X, formate, oxalate and others [3].
Most electron transport is cyclic [4].
The development of oxygenic photosynthesis and the effects on life
A special event in the history of life was the emergence of cyanobacteria in the Precambrian era, about 3.5 billion years ago. These bacteria were capable of oxidizing water instead of using lower potential reductants like H2S [3]. Cyanobacteria contain a special photosynthetic unit called
photosystem II (PSII) which uses water as the ultimate electron donor. Oxygen is released as a by-product. Prior to the development of these organisms, the atmosphere of the earth may have contained mainly N2, NH3,
H2, H2O, CH4, CO2 and CO [5, 6] and O2 in a vanishingly small
source of electrons had become available [3]. Reductive power is required for CO2 fixation. The oxidation of water by the cyanobacteria resulted in the
release of large quantities of oxygen, which resulted in a series of events that allowed the development of multicellular organisms, a new form of life that was based on the eukaryotic cell plan [9].
The early chemistry of life used iron in many biochemical processes. The Fe(III)/Fe(II) redox potential is highly variable since it can be fine tuned by different ligands and encompasses almost the entire biologically significant range between -0.5 to +0.6 V for catalysis in most biochemical reactions [9]. Upon the release of oxygen into the atmosphere, oxidation of ferrous iron, Fe(II), to ferric iron, Fe(III), occurred at a large scale. Since Fe(III), in contrast to Fe(II), is not soluble in water, the bio-availability of iron was lost. The accumulation of oxygen into the biosphere induced an even larger change for life: the oxidation of copper from a Cu(I) insoluble compound to the soluble Cu(II) form, whereby copper became available for life. Copper was a good alternative redox mediator in metabolic reactions. Compared to the Fe(III)/Fe(II) redox couple the Cu(II)/Cu(I) redox couple has the advantage of higher redox potentials, in copper enzymes ranging between +0.25 and +0.75 V [9]. So enzymes based on copper are capable of performing oxidation reactions that require this higher potential and thus oxygenic photosynthesis enabled the development of new metabolic pathways. However, Fe(II) was still required and rather than that metabolic pathways based on the Fe(II)/Fe(III) redox couple vanished, microbial life found a way to gather insoluble Fe(III) by developing siderophores (greek for ‘iron carriers’) [10]. Therefore, the accumulation of O2 instead of
changing an iron world into a copper world, changed an iron world into an iron-copper world [9].
carbohydrates (both are produced by photosynthesis) to H2O and CO2,
which ultimately provides about 10 times more energy than anaerobic respiration [12]. Thus, the advent of O2 in the atmosphere paved the way for
multi-cellular organisms to live and to evolve on land. Therefore the evolution of PSII was a landmark in the development of life [9].
Chloroplasts in higher plants and the light and dark reactions
Higher plant photosynthesis occurs in specialized organelles, the chloroplasts (Fig. 1), which are plastids of 5-10 µm diameter that can be observed easily as green grains by light microscopy. They were first observed by van Leeuwenhoek in the 16th century.
thylakoid lumen outer membrane inter membrane space thylakoid membrane stroma granum inner membrane
Figure 1. Schematic representation of the chloroplast. It is enclosed by an outer and
In higher plants and algae, chloroplasts result from endosymbiosis between the cyanobacteria and the eukaryotic predecessors of the higher plants and algae [13-15].
In 1864 the overall reaction for photosynthesis (eq. 1) was given by Boussingault:
6CO2 + 6H2O + light → C6H12O6 + 6O2 (1)
where the energy of the absorbed light is converted into chemical energy [1]. A question that remained unanswered for a long time is the origin of the oxygen molecules. From the overall eq. 1 it is not clear whether the oxygen originates from carbon dioxide or from water. The dominant theory was that CO2 is split into carbon and oxygen and that water is subsequently added to
the carbon. In the 1930s van Niel challenged this hypothesis by his proposal that the oxygen originates from water [1]. This suggestion was based on an analogy to the overall reaction observed in sulfur bacteria, which they perform in an anaerobic environment:
6CO2 + 12H2S + light → C6H12O6 + 6H2O + 12S (2)
where H2S provides electrons that reduce CO2 to carbohydrates and the
element sulphur is produced [1]. In 1937 Hill demonstrated that chloroplasts can generate oxygen in the absence of CO2 if a suitable oxidant (e.g.
ferricyanide) is added. CO2 as the source of oxygen was thereby excluded
and it was proven that oxidation of H2O can be uncoupled from CO2
reduction. The oxidation of water as the source of oxygen was confirmed in the 1940s by use of the heavy oxygen isotope 18O as a tracer [1] by Kamen and Rubin. The O2 released by higher plants was found to contain 18O after
2H2O
4e
-PSII
cyt b
6f
PSI ATP synthaseQB thylakoid lumen 4H+ OEC 2QBH2 2QB 4H+ Cytb559 3H+ stroma ADP ATP 2H+ + 2NADP+ + 4e -QA 4H+ + O 2 4hν 4hν Fd P700 P680 PC 2NADPH
Figure 2. Schematic representation of the thylakoid membrane and the
of water and the generation of high energy ATP according to the following reactions of linear electron transfer:
2H2O + 4hν → O2 + 4H+ + 4e– (3)
4Fdox + 4e– + 4hν → 4Fdred (4)
ADP + Pi → ATP (5)
where (3) and (4) are driven by light-induced electron transport through PSII and photosystem I (PSI). Reaction (5), the phosphorylation of ADP to ATP at the stromal side of the thylakoid membrane, is catalyzed by ATP synthase, which is driven by the energy of a proton gradient across the thylakoid membrane. The production of one molecule ATP from ADP and Pi requires
3 protons to pass through ATP synthase [1]. The reducing power of Fdred can
be used to reduce NADP+ (eq. 6).
2NADP+ + 2H+ + 4Fdred → 2NADPH + 4Fdox (6)
In the dark reactions the reducing power of NADPH is ultimately used to reduce CO2 to carbohydrates, also referred to as carbon fixation. Carbon
fixation occurs in a metabolic process that comprises 13 distinct reactions, which can be subdivided in three phases, the total of which forms a reductive pentose-phosphate cycle. It is commonly referred to as the Calvin cycle [16]. The dark reactions use products of the light reactions. The first phase is the carboxylation phase. The main enzyme is ribulose bis-phosphate carboxylase-oxygenase (rubisco), which catalyzes the reaction of ribulose bisphosphate (which contains 5 carbon atoms) with one CO2 molecule to
Chlorophyll pigments
In the LHCs carotenoid and chlorophyll pigment molecules harvest light energy in the form of excited state energy, which is transferred to the chlorophyll molecules that form the primary donor P of the RC. In higher plants two types of chlorophyll are found, chlorophyll a and chlorophyll b. The structure (Fig. 3) comprises four pyrroles (I-IV) that ligate a central magnesium ion.
IV III
I II R
Figure 3. Structure of chlorophyll. R = CH3 in chlorophyll a and R = CHO in chlorophyll b. The chlorophyll comprises pyrroles I-IV. The four nitrogen atoms are ligated to a central Mg(II). In pheophytin the central Mg(II) is absent. Reprinted from Ref. [17].
A photon is absorbed from light that is polarized in the plane of the molecule. Capture of a photon with polarization along the pyrrole I-III direction gives rise to the QY transition in chlorophyll a. This transition of
chlorophyll a occurs at about 665 nm wavelength in an organic solvent and at ca. 680 nm if it is bound to the protein matrix [17]. A photon polarized along the pyrrole II-IV direction induces the so-called QX transition, which
occurs at shorter wavelength than the QY transition. The QX transition is
Most bacteriochlorophylls absorb photons of longer wavelengths than chlorophyll a [17], which results in lower excited state energies.
The PSII protein complex of higher plants
Before 1981, research on PSII in chloroplasts, cyanobacteria and algae was hampered by the absence of methods to isolate active PSII. This changed when a method for purification of PSII membranes (granal fragments) from spinach chloroplasts became available in 1981 [18, 19]. These PSII membrane fragments are able to evolve oxygen when a suitable electron acceptor is added. The purification includes thylakoid incubation with a detergent and subsequent centrifugation.
The protein complex of PSII of higher plants comprises at least 23 polypeptides, Fig. 4. The membrane-intrinsic D1 and D2 polypeptides form a hetero-dimer, the heart of the RC where the primary photoreactions occur [20].
Figure 4. Schematic representation of the PSII protein complex, comprising
They are encoded by the chloroplast genes psbA and psbD. At the acceptor side the pheophytin (Pheo) and quinone acceptors (QA and QB) are located.
At the donor side of the D1 polypeptide the oxygen evolving complex (OEC) is located. It contains 4 Mn ions, Ca2+ and 1 Cl– [21] as essential
cofactors. The Mn4 cluster is protected by 3 extrinsic polypeptides. The
24 (PsbP) and 17 (PsbQ) kDa polypeptides provide stability to the Mn4
cluster. Their genes are located on the nuclear genome. These polypeptides are replaced by a cytochrome c (cyt c) in the PSII RC of cyanobacteria. The larger 33 kDa polypeptide, which is encoded by the nuclear gene psbO, also stabilizes the Mn4 cluster, and is required for its assembly.
Cytochrome b559 (cyt b559) consists of two small polypeptides α and β which
are encoded by the psbE and psbF genes and form a hetero-dimer, providing one histidine ligand each to the heme iron [20]. In intact PSII RCs the heme iron is oxidized at low quantum yield by P+
680, presumably via ChlZ (see
Fig. 5), a chlorophyll a monomer, and is rereduced by the plastoquinone pool, maybe via QB. The core proteins CP47 (PsbB) and CP43 (PsbC) serve
as light harvesting antennae and contain 16 and 13 chlorophyll a pigment molecules respectively [22]. CP47 seems to be required for the stable assembly of PSII complexes: neither D1 nor D2 accumulates in thylakoid , although me are encoded by the nucleus. The reason for the two different locations
to unravel their membranes of Synechocystis when the psbB gene is inactivated.
Most PSII polypeptides are encoded by the chloroplast genome so
of the genes are unclear. In Ref. [23] it is proposed that local genes allow a rapid transcription-translation cycle and fast polypeptide transport, thus providing a rapid response mechanism for the dynamics that occur in photosynthesis, whereas nuclear encoding would result in a too slow response cycle [23].
The structure of the PSII RC
A powerful technique to obtain insight in the structure of proteins is X-ray crystallography. Crystallography has been applied to many thousands of proteins and protein complexes that are water-soluble
Figure 5. The structure of the cofactors of the PSII RC in Thermosynechococcus
ulcanus, resolved by X-ray crystallography at 3.7 Å resolution [28]. The protein v
matrix is not shown. QB was absent in the preparation. In PSII of higher plants yt c550 is not present.
to their hydrophobic character, and thus the availability of membrane protein structures is relatively scarce. The crystal structure of the bRC accomplished a major breakthrough when it was resolved for Rhodopseudomonas
viridis [24] and for Rhodobacter (Rb.) sphaeroides [25, 26]. For
cyanobacterial PSII the crystal structure became available in 2001 [27] at a resolution of 3.8 Å. A second structure from a different species at 3.7 Å resolution [28] is shown in Fig. 5. Cyanobacterial structures at 3.2 Å [29] and 3.0 Å [22] have been published recently. The structure of the PSII RC is highly similar to the structure of bRCs, especially at the acceptor side: the intermediary acceptor pheophytin (called Pheo in PSII and I in the bRC) and the primary acceptor QA and secondary acceptor QB with a non-heme iron Fe
located between QA and QB, is highly conserved. The donor side of the PSII
RC is different from bRCs in the presence of the Mn4 cluster which uses
pigments are present, which comprise the primary donor P680. PD1 is
coordinated at the central Mg2+ ion by D1-His198 in the D1 subunit and P D2
is coordinated by D2-His197 in subunit D2 in cyanobacteria [30]. The center to center distances of ChlD1 (BA), PD1, PD2 and ChlD2 are approximately 10 Å
[27, 28].
The primary reactions in PSII
Upon the capture of a photon by a pigment molecule of the light harvesting complex of PSII (LHCII), the excited state is transferred to P680. The excited
state P*680 is short lived owing to the process of charge separation
(multiphasic, 2 - 3 ps and 20 - 25 ps [31]), whereby ChlD1 (also known as
BA) probably functions as the primary donor [30, 32], and results in the
radical pair state P+
680Pheo–. During its lifetime P+680 is probably mainly
localized on PD1 (about 80 %) and partly on PD2 (about 20 %) at ambient
temperatures [30]. The midpoint potential of P+/P is probably 1.3 V [33], much higher than that of chlorophyll a in solution, and is probably required for the ultimate oxidation of water. The electron at Pheo is transferred to quinone QA in about 200 ps and further transfer to the quinone QB occurs in
a few hundred µs [34]. The successive electron transfer reactions must be
rmediate (‘Z’) occurs nearly monophasic with t1/2 ≈ 23
faster than the recombination rate of the back reactions of the previous states [31], which would result in loss of the absorbed energy and the risk of creating the RC triplet state via radical pair mechanism. Re-reduction of P+
680
by a redox active inte
ns for the S0-S1 and S1-S2 transitions of the oxygen evolving complex (see
Fig. 6 for the cycle of S-states) and t1/2 ≈ 50 ns and ≈ 260 ns (biphasic) for
both the S2-S3 and S3-S0 transitions [35, 36]. These slower rates are possibly
due to Coulomb attraction by the Mn4 cluster [35]. ‘Z’ in subunit D1 and its
symmetrical counterpart ‘D’ in subunit D2, were identified as tyrosine radicals by studying PSII in cyanobacteria with electron paramagnetic resonance (EPR) and by selective labeling of tyrosine with deuterium which was fed to the cyanobacteria. Thus these intermediates were named YZ and
YD [37]. Site directed mutagenesis supported this finding, and assigned YZ to
Tyr-161 of the D1 polypeptide and YD to Tyr-161 of D2 [38, 39]. YZ ends up
as a neutral radical (Y••
Z) by proton donation to a nearby histidine, and in turn
oxidizes the Mn4 cluster in 30 - 1000 µs (dependent on the oxidation state of
the Mn4 cluster) [40, 34]. The higher oxidation state of the Mn4 cluster is
The OEC and the Mn4 cluster
TheOEC, which contains the Mn4 cluster (Fig. 5) cycles through 5 oxidation
states in the light, commonly referred to as the Kok cycle of S-states [41], Fig. 6. The procession of S-states is driven by P+
680 via Y••Z. The S4 to S0 state
transition occurs spontaneous without light and is accompanied by the PSII complex. Per S-cycle two molecules of
r [43], and direct evidence was a release of one O2 molecule per
Figure 6. The Kok cycle of S-states in the OEC of PSII. The Mn4 cluster of the OEC cycles through 5 oxidation states during illumination, and is oxidized by P+
680 via Y•Z. Per S-cycle two molecules of H2O are oxidized by the Mn4 cluster, which results in the release of one molecule O2 during the transition of S4 to S0.
H2O are oxidized by the Mn4 cluster. In an ensemble of PSII protein
complexes, the cycle can proceed more or less synchronously by exciting with single turnover light flashes. For RCs in the S1 state, O2 will be released
upon flash number 3. Evolution of oxygen by single turnover flashes can be detected with a Joliot oxygen electrode [42].
Its role in water oxidation and oxygen release gives manganese a most important function in biology [20]. The observation of an EPR multiline signal after one photonic excitation (S2 state) provided the earliest indication
that the Mn4 cluster plays a direct role in storage of the oxidizing equivalents
that are required for the oxidation of wate
shift in XANES analysis of samples in the S1 state compared to the
istance of 2.7-2.8 Å, at least one but possibly two mono-µ-oxo bridged Mn airs (3.3 Å) and at least one Mn-O-Ca link at approximately 3.4 Å [7]. he origin of the M found in na l early marine MnO2
recipitates of the Archean ocean, where PSII may have evolved in hotosynthetic bacteria that lived on these precipitates [7].
he application of EPR to photosynthesis research
pon charge separation in RCs unpaired electrons are involved in the lectron transfer chain. Selective detection of unpaired electrons is the ecial feature that makes EPR ul technique in ph synthesis search [45]. This technique is principle of the splitting of the nergy levels pulated by unpaired electrons in the presence of an xternally applied magnet ield [46], which is called Zeeman splitting.
oublet states (S = ½) and an introduction to EPR
a one spin system (S = ½), with magnetic quantum number ms = +½
reases the energy vel, whereas the magnetic moment of electrons in the β substate is oriented S2 state [44]. From these observations it has been derived that 2 Mn(III) and
2 Mn(IV) are present in the S1 state, and possibly 1 Mn(III) and 3 Mn(IV) in
the S2 state [7, 34]. The organization of the Mn4 cluster is far from clear. On
the basis of EXAFS studies it was concluded that the Mn4 cluster contains at
least two and probably three di-µ-oxo bridged Mn pairs with a mutual d
p
T n4 cluster may be tura
p p T U e sp a very usef based on the oto re e po e ic f D In
(upspin or α) and ms = -½ (downspin or β), the multiplicity is two according
to the multiplicity rule (M = 2S + 1, where M represents the number of spin states) [46]. The magnetic moment of electrons in the α substate is oriented anti-parallel to the applied magnetic field (B) which inc
le
parallel to B, which lowers the energy level [46]. Without a magnetic field both spin states are degenerate, i.e. have the same energy. In an EPR experiment the applied magnetic field (B) is swept. If the energy difference (∆E) between the two spin states α and β is equal to the photon energy (hν) of the applied radiation, transitions are induced between the spin states according to eq. 7 [46].
Here h is the Planck constant, ν the frequency of the applied microwave radiation, βe the Bohr magneton and g the g-value. A transition from ms = -½
to ms = +½ (or from β to α) corresponds to a stimulated absorption (A) and
from ms = +½ to ms = -½ (or from α to β) to a stimulated emission (E).
The net absorption measured by EPR results from a difference in the population of the different spin states, following Boltzmann distribution. In X-band EPR (at 9-10 GHz), the population ratio Nα/Nβ will
be 998 over 1000 at room temperature (293 K). At 5 K this ratio decreases to 913 over 1000, in principle resulting in a 40-fold enhancement of the signal amplitude.
To reduce noise, phase sensitive detection is often used in EPR, where a odulated magnetic field is superimposed on the applied magnetic field, shape compared to direct
and e EPR signal will diminish, which is called microwave power saturation. attice relaxation time T1
FI) are independent of the strength of the magnetic eld and can provide insight into the structure of the system under study. If the in
e magnetic field, the HFI are isotropic. The interactions can also depend on m
which results in signals that show a first derivative
detected absorption. In EPR the signal amplitude is linear with the square root of the power of the applied microwave radiation under the prerequisite that the Boltzmann distribution of the spin population is maintained. At higher microwave power the Boltzmann distribution can be affected by the transitions that are induced by the applied microwave radiation. The population difference then will decrease (heating of the spin system) th
The onset of the effect is determined by the spin l
and is affected by pathways for energy dissipation in the surroundings of the spin system under investigation. Water and a protein matrix can cause fast T1
relaxation but particularly nearby transition metals can provide enhanced spin relaxation. For a free electron S = ½ system a single absorption at the free electron g value (g = 2.0023) would occur [46]. However, usually the unpaired electron is confined to atomic or molecular orbitals, and the electron spin interacts with nearby nuclear spins. This hyperfine interaction sensed by an electron causes a shift in the magnetic field at which absorption occurs.
For example, nuclear spin states with mi = -½ and mi = +½ give rise to a
splitting of the EPR lines. Consequently two lines can be observed. Such hyperfine interactions (H
fi
the orientation of the system relative to the applied magnetic field, which results in an anisotropic HFI tensor [46].
Another feature that influences the spectrum is g anisotropy. The orbital movement of the electron causes a local magnetic field that couples to the magnetic moment caused by the electron spin. This interaction also results in an orientation-dependent tensor [46]. Contrary to HFI, EPR line shapes that arise from g anisotropy depend on the strength of the applied magnetic field. At magnetic fields for X-band spectroscopy, the g anisotropy is often poorly resolved [47]. When a spin system is confined to small molecules dissolved in a low viscosity medium like water or organic solvents, HFI and g anisotropy will be averaged out by the fast tumbling rate of the molecule if the rotations are close to or faster than the frequency differences between the various possible EPR transitions (in the order of 109 s-1). This results in
narrow spectra from which isotropic HFI coupling constants and isotropic g values can readily be obtained. Molecules in viscous solution e.g. glycerol, or large protein complexes like PSII in buffer with a relatively low viscosity, have lower tumbling rates (on the order of 107 s-1 or lower). Here anisotropy
is not averaged out, resulting in broad overall spectra that are characteristic of a powder, a static ensemble of randomly oriented molecules. Such spectra are therefore called powder spectra [46].
Singlet states (S = 0)
Two electrons that occupy the same orbital have opposite spin states according to the Pauli principle (ms = +½ and ms = -½ ) and thus form a
two-spin system with total spin S = 0, which is a singlet according to the multiplicity rule. Zeeman splitting does not occur if a magnetic field is applied. The magnetic moments associated with the opposite spin states cancel one another, which results in a diamagnetic system. Therefore S = 0 is EPR silent.
Triplet states (S = 1) and radical pair mechanism
In Fig. 7 the sublevel energies of a triplet state (S = 1) without an externally applied magnetic field are shown. The triplet eigenstates are TX, TY and TZ,
Figure 7. Scheme of the zero field sublevel energies of the triplet state, resulting
from the dipolar interaction between two electrons and characterized by ZFS parameters D and E. X, Y and Z correspond to the triplet axes within the molecule. The dotted line represents the energy of the triplet state in absence of electron dipolar interactions (i.e. when D and E are zero).
between the two electrons and are characterized by the zero field splitting (ZFS) parameters D and E. These are the so-called zero-field energy levels. When a magnetic field is applied, the energy levels will be split further. In the high-field approximation the new triplet eigenstates are designed as T– , T0 and T+ and are a linear combination of TX, TY and TZ. This linear
combination is dependent on the orientation of the magnetic field with respect to the triplet axes. When the magnetic field is along one of these
xes, the energy levels can be approxim
Y + 1/3D X Z D 2E - 2/3D
a ated by some relatively simple
Table 1. In 3Chl the two different orientations relative
ef. [47], becoming realistic only when a moderate or high magnetic eld (e.g. > 400 Gauss) is applied. T (cm-1), energy; B (Gauss), magnetic field;
FS parameters, see Fig. 7. equations as shown in
to the magnetic field (X and Y) correlate, by coincidence, more or less to the QX and QY absorption axes of ground state chlorophyll if D and E are
assumed to be positive. The Z orientation is perpendicular to the porphyrin plane.
Table 1. High field approximations of the T+, T0 and T- triplet sublevel energies for all three canonical orientations of the triplet axes X, Y and Z in the magnetic field according to R
fi
g, triplet g-value; βe, Bohr magneton; D and E (cm-1), Z
B//X B//Y B//Z T+ gβeB - 1/6D + 1/2E gβeB - 1/6D - 1/2E gβeB + 1/3D
T0 1/3D - E 1/3D + E -2/3D
Formation of the donor triplet state 3P is depicted in Fig. 8. Upon
photo-duced charge se +I– (or
+Pheo– in PSII) can form. Normally the electron in I– is rapidly transferred
downstream acceptors, but this can be blocked, e.g. if the primary cceptor quinone QA is reduced or removed from its b et. Because
f optical selection rules the two-spin system at P is created with a total spin = 0 (i.e. a singlet), but undergoes a decrease in orbital overlap by transfer
n dephasing or spin evolution.
- +
in paration in photosynthetic RCs a radical pair P P
to
a inding pock
o
S
of one electron to the intermediary acceptor I. Consequently the exchange interaction between the two electrons decreases and if the lifetime of the P+I–
radical pair is long enough the spin state of the system can change under the influence of locally different magnetic interactions that are sensed by the separated electrons. This process is called spi
Subsequent back-transfer from I to P can lead to the formation of a triplet state. This entire process is called radical pair mechanism (RPM) and the resulting triplet state can be detected by EPR. After photoinduced charge separation the radical pair dephases with frequency ω according to eq. 8 after [48]. ] [ 2 ) ( j j j i i i eB Am A m g h B =
π
∆β
+ Σ − Σω
(8)This equation describes spin evolution at low magnetic dipole-dipole and exchange interactions within the radical pair. B represents the externally applied magnetic field, h is the Planck constant and βe the Bohr magneton.
Σ indicates summation over all the nuclear spin states mi and mj. Ai and Aj are
the hyperfine coupling constants respectively for the electron at the molecule with nuclei i and j respectively. If the process of dephasing can be completed
l pair state vs. the singlet radical pair state. By application of an external
m n ulated ixes with the singlet energy
l l, T+ and T- are not populated as they hifted out of
r n let sta tion o nd T0
triplet states predicts a theoretical spin population ratio of 1:1.
within the radical pair lifetime, a theoretical distribution can be predicted for the resulting percentage of triplet vs. singlet radical pair states. At zero-magnetic field the three triplet sublevels are nearly degenerate and close in energy with the singlet energy level. The four radical pair states mix accordingly and populate into a ratio of 3:1 respectively for the triplet radica
ag etic field, T0 is pop since this level m
eve whereas are s
Figure 8. Electron transfer steps in RCs where electron transfer to downstream
acceptors is blocked. Spin evolution occurs with frequency ω in the radical pair P+I–. Recombination to the ground state can occur directly from the radical pair singlet st P 1(P•+ I•¯) 3(P•+ I•¯) ω (B) 3P 1P*I hν 3 ps kS kISC kT 3O 2 1O 2
ate via kS. The radical pair triplet state can recombine to the triplet ate of the primary donor via kT. The triplet state of the prim
e ground state with intersystem crossing rate constant kISC a
triplet oxygen, thereby generating harmful singlet oxygen. Note that ω depends on
elated to the
st ary donor can decay to
th nd via energy transfer
to
the magnetic field according to eq. 8 and note that for PS II 3P should be interpreted as 3B
A (ChlD1 in Fig. 5).
The triplet yield is determined, apart from the spin evolution rate, by two decay channels: 1) decay from the singlet radical pair state by recombination to the ground state via kS either directly or with P* repopulation and 2) decay
of the radical pair state to the triplet state of the primary donor (3P in bRCs or 3B
A in PS II) via kT, in Fig. 8. In bRCs, at 5 K, kT is usually about ten
times larger than kS, which explains the high triplet yield in RCs after
0.04 -0.02 0.00 0.02 -0.04 0. 0.02 0.04 00 0 150 300 -0.02 0.00 0.02 0.04 -0.04 -0.02 Z X Y en er gy ( cm -1) B//X rgy ( cm -1 ) B//Y Z X Y
A
Z X Y en er gy ( cm -1 ) B//Z enemagnetic field, G
Figure 9. PSII triplet state 3BX-band EPR spectrum of the RC triplet state of PSII with D = 288x10-4 cm-1
and E = 44x10-4 cm-1 (Fig. 9C), similar to Ref. [52].
In RPM the un
external magnetic field has various co nt.
1) It leads to the characteristic polarization pattern of absorption (A) and
emission (E) of 3P, AEEAAE [45, 51, 54], see also Fig. 9B and C. This
polarization pattern is unique for RPM and can be readily identified by EPR.
2) Microwave induced transitions result in enhanced signal amplitude
compared to systems where the spin population follows the Boltzman distribution. Transitions between T0 and T+ result in enhanced absorption
and transitions between T0 and T- in enhanced emission. At 5 K, a Boltzman
distribution of spin populations at X-band energy (0.308 cm-1) results in a ratio of about 913 over 1000, whereas a RPM generated spin polarized triplet state in a magnetic field yields a theoretical population ratio of 0 over 1000, leading to a more than tenfold increase in signal amplitude. Compared to Boltzmann systems measured at room temperature, triplet systems generated by RPM show an even higher signal enhancement (theoretically up to 500 times). 3) Contrary to spin systems that follow a Boltzmann distribution, the EPR signal amplitude of single T0 polarization in RPM generated triplet
states is independent of temperature, were it not that in photosynthetic RCs kS can increase with increasing temperature, resulting in lower triplet yield
and consequently lower signal amplitude. 4) Spin lattice relaxation will destroy the effect of spin polarization, resulting in reduction of signal amplitude. Contrary to spin systems where spin population follows the Boltzmann distribution, fast spin-lattice relaxation can be counterproductive in measurements of spin polarized triplet states. 5) Microwave induced depolarization, the consequence of inducing transitions by the application of microwave radiation, can accelerate the loss of signal amplitude.
In itself, the triplet state is not harmful to RCs, and returns to the singlet ground state either via intersystem crossing (ISC) equilibrium constant kISC,
or via a channel where the triplet energy is transferred to oxygen (Fig. 8). Oxygen has a triplet ground state, whereas the singlet state is higher in energy. Triplet chlorophyll can transfer its energy to 3O
2 if oxygen is nearby,
which forms 1O2. The singlet excited state of O2 is highly reactive and leads
to destruction of the RC [55].
ique population of the triplet T0 sublevel in the presence of an
Photoinhibition, two possible mechanisms
Photoinhibition is the process whereby the yield of photosynthesis drops irreversibly by light-induced damage to the photosynthetic apparatus. Photoinhibition is caused mainly by high light intensities. In higher plants photoinhibition results in bleaching of leaves and can result in death [56, 57]. Photoinhibition in higher plants occurs mainly by damage to the PSII protein complex [57]. At least two mechanisms for damage in PSII may exist [55]. First, after charge separationelectron donation to P+
680 may become
impaired or retarded by malfunctions within the OEC, which may be caused by direct light-induced damage to the Mn4 cluster [58]. Consequently, the
lifetime of P+680, usually on the order of hundreds of nanoseconds,
will be longer. The high redox potential of this state can lead to damaging reactions with the protein matrix, which is called donor-side photoinhibition [55]. In addition, when the downstream linear electron transfer to PSI can not keep up with the number of charge separations in PSII, electron transfer in PSII is blocked by reduction of the plastoquinone pool. Subsequent charge separations can lead to a doubly reduced state of QA [59]. Via the light induced formation of the
triplet state in the photosynthetic RC by RPM [52, 60], singlet oxygen, 1O 2,
can be generated, which catalyses degradation and is called acceptor-side photoinhibition [55, 59, 61]. The D1 subunit is the main target of both types of photoinhibition, possibly since the RC triplet state and the high potential state P+
680 is located in or close to the D1 subunit.
Solution 1. Reduction of 1O
2 damage by RC triplet state quenching
s of Rb. sphaeroides and is well
must be sensitive
69] it was suggested that zeaxanthin, formed by light- This process is found in photosynthetic RC
understood. A carotenoid molecule (spheroidene) is positioned at about 10 Å from the primary donor P. This is too distant for close van der Waals contact which was shown to be required for high quantum yield triplet energy transfer between porphyrin donor and carotenoid (Car) acceptor pairs. However, in Ref. [63] it was shown that spheroidene quenches the primary donor triplet state with very high quantum yield above 35 K. The crystal structure of Rb. sphaeroides [25] revealed that between spheroidene and the primary donor an accessory bacteriochlorophyll BB is located at 4 Å from
spheroidene. It was shown that BB is an intermediate in triplet transfer from
the primary donor to spheroidene [64]. Quenching of the high energy triplet state 3P by formation of 3Car with high efficiency was demonstrated by EPR
[64]. The energy level of 3Car is too low to induce excitation of 3O
2 to 1O2
and it decays in 5 ± 2 µs [65] to the ground state thereby generating only heat [66]. For PSII in higher plants and cyanobacteria the situation is quite different. Two carotenoid molecules (β-carotene) are present in the D1D2Cyt b559 preparation, as was shown by HPLC [67], but no 3Car is
formed if 3P
680 is present [68].
Solution 2. Non-photochemical quenching by the xanthophyll cycle
Long term acclimation to changes in light intensity can be achieved by adapting the size of the pigment antenna pool per RC by changes in gene expression or proteolysis [62]. However, antenna systems
Figure 10. Schematic representation of the xantophyll cycle showing the
de-epoxidation of violaxanthin to zeaxanthin in excess light and the reverse reaction in limiting light. Antheraxanthin is the mono-epoxidized intermediate for both reactions. Red awn after [69].
O HO HO OH antheraxanthin zeaxanthin
limiting
light
O HO OH OH violaxanthinexcess
light
O r f excessclic electron transport route by PSI and in anoxygenic photosynthetic induced de-epoxidation of violaxanthin is an effective quencher o
excitation energy. Two principles for the quenching activity of zeaxanthin have been proposed. First, direct quenching by downhill energy transfer from excited chlorophyll a to zeaxanthin may occur, possibly facilitated by a structural change in the pigment protein complex that is activated by a change in (lumenal) pH. The second possible mechanism is indirect quenching. Xanthophylls (such as zeaxanthin) bind to the LHC protein complexes or to enzymes which regulate their morphology and may thereby induce or control the organisation of the antenna pigments, which results in the observed fluorescence quenching [70]. However, at present it is not clear which of the two models predominates.
Solution 3. Cyclic electron transfer within the RC
Another way of preventing either RC triplet states or an extended lifetime of P+
680 is by a flow of electrons from the PSII RC acceptor side to the donor
bacteria, which occurs via intermediates outside the RC, and whereby the ns via cofactors present within the RC and the captured energy is not reserved. CET in PSII of higher plants indeed exists, possibly via cyt b559,
molecule and th nt. However, electron transfer to fast enough to compete with electron donation Z, Mn4
[71].
his wo
Chapter 2 the activation o se to light in the iatom
. tricornutum is analyzed and a possible role for tyrosine ET is udied. In Chapter 3 the influence of high potential quinone on the spin state f cyt b559 is investigated in PSII membrane fragments and compared with a
odel em. In Chapter 4 the triplet state in PSII RCs with singly duced primary acceptor quinone QA is identified by time-resolved
tural MnO2 precipitates in the early ocean,
] K.M. Towe, Environmental oxygen conditions during the origin and early evolution of
captured energy is used to generate ATP [4]. Photoprotective CET in PSII ru p a β-carotene e ChlZ pigme P+ 680 is not nd H via Y a 2O T rk
In f CET in respon marine d
Y in C P D st o m syst re direct-detection EPR. References
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