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Reaction center triplet states in Photosystem I and Photosystem II
Abstract
A photosystem I (PS I) particle has been prepared by lithium dodecyl sulfate digestion which lacks the acceptor X, and iron-sulfur centers B and A. Illumination of these particles at liquid helium temperature results in the appearance of a light-induced spin-polarized triplet signal observed by EPR. This signal is attributed to the triplet state of P-700, the primary donor, formed by recombination of the light induced radical pair P-700+ A1- (where A1 is the intermediate acceptor). Formation of the triplet does not occur if P-700 is oxidized or if A1 is reduced, prior to the illumination. A comparison of the P-700 triplet with that of P-680, the primary donor of Photosystem II, shows several differences. (1) The P-680 triplet is 1.5 mT (15 G) wider than the P-700 triplet. This is reflected by the zero-field splitting parameters, which indicate that P-700 is a slightly larger species than P-680. The zero-field splitting parameters do not indicate that either P-700 or P-680 are dimeric. (2) The P-700 triplet is induced by red and far-red light, while the P-680 triplet is induced only by red light. (3) The temperature dependences of the P-700 triplet and the P-680 triplet are different.
... Formation of the triplet reaction center chlorophyll of PSI ( 3 P700), via recombination of the radical pair P700 + PhQ -, occurs in illuminated PSI complexes and chloroplasts (Frank et al. 1979, Rutherford andMullet 1981). Both 3 P680 and 3 P700 can be detected based on their electron paramagnetic resonance (EPR) signals (e.g. ...
... Both 3 P680 and 3 P700 can be detected based on their electron paramagnetic resonance (EPR) signals (e.g. Rutherford and Mullet 1981). ...
In oxygenic photosynthesis light is captured in series by two protein complexes: photosystem II and photosystem I (PSI and PSII). Electron transfer from PSII to PSI is mediated by the plastoquinone (PQ) pool. Despite being the energy source, light also damages the photosynthetic machinery. Singlet oxygen (1O2), an excited state of O2, may be generated when a charge recombination reaction in PSII re-excites the reaction center chlorophylls (P680), producing a triplet state. In this thesis, massspectroscopy- based detection methods for 1O2 were developed further, to understand the role of this reactive oxygen species in photosynthesis. It was shown that even though both O2 and 1O2 are produced in pumpkin thylakoid membranes, most (if not all) of the 1O2 derives from the ambient dissolved O2, not from the nascent O2 produced due to the water splitting activity of PSII. The result shows that the O2 evolving ability of PSII as such does not render PSII vulnerable to oxidative damage.
Nevertheless, light inactivates PSII, and no consensus about the mechanism(s) of the photoinhibitory damage exists. Therefore, the temperature dependence of the rate constant of PSII photoinhibition was measured under various conditions and compared with temperature dependencies of 1O2 production and recombinations. The results show that in plants and cyanobacteria the rate constant of photoinhibition and production of 1O2 increase similarly with temperature as the miss probability of the oxygen evolving complex. Photoinhibition proceeded under anaerobicity, where no 1O2 is produced, and was unaffected by quenchers of 1O2. We suggest that when a miss occurs, but a recombination does not re-reduce the PSII reaction center, P680+ lives long enough to oxidize a vital component of PSII, causing the photodamage.
Plants have also ways to adjust to different light conditions. An initial fluorescence screen and subsequent high-performance liquid chromatography measurements revealed that 470 nm, 560 nm and 660 nm light favors PSII over PSI and reduces 80–90 % of the PQ pool, whereas 440 nm, 520 nm and 690 nm favors PSI and oxidizes 90–100 % of the pool in Arabidopsis thaliana, when moderate light was used. Light state curvilinearly followed the redox state of the PQ pool; state 2 was reached with 50 % reduction. All tested white lights, including light from the Sun, reduced less than 50 % of the PQ pool. This PSI light character of white light enables plants to respond to the intensity of light via the redox state of the PQ pool.
... 23,[34][35][36][37] Because the recombination to 3 P 700 becomes allowed because of S-T 0 mixing in the radical pair P 700 + A 0 − , the T 0 triplet sublevel of 3 P 700 is populated for all orientations, which leads to a characteristic polarization pattern. 38 At temperatures below ∼100 K, the ZFS parameters indicate that the triplet excitation is localized on one of the two chlorophyll molecules of P 700 23,24,35,39,40 , and recent ENDOR suggests that the triplet state resides on the A-branch chlorophyll. 36 At higher temperatures, the ZFS parameter E becomes smaller, which has been interpreted as resulting from the hopping of the excitation between the two The Journal of Chemical Physics ARTICLE scitation.org/journal/jcp ...
A model is presented describing the effect on spin polarized transient EPR signals caused by incoherent state hopping between two sites. It is shown that the size of the spin state space can be reduced by half to the subspace described by the site-average Hamiltonian and that the dynamics of the system result in a redistribution of population between its eigenstates. Analytical expressions for the rates of population redistribution and the lineshape are derived for the general case in which the back-and-forth rates are unequal. The EPR signals calculated using these expressions are in very good agreement with those obtained by direct numerical solution of the density matrix rate equations. The model is then used to investigate the influence of exciton hopping on triplet state transient EPR spectra. Using the triplet state of the primary donor of Photosystem I as an example, it is shown that the influence of unequal hopping rates becomes more pronounced in the spectrum at longer delay times after the laser flash.
... Higher sensitivity to photooxidative stress in NoM is associated with an increased production of 1 O 2 , rather than with a pleiotropic effect on photosynthetic ET efficiency, since a lack of monomeric LHCs did not cause an over-reduction of photosynthetic ET, especially PQ (Fig. 2D). This suggests that the source of 1 O 2 in NoM is LHCII, most likely because of strongly reduced quenching by PSII RC, rather than charge recombination within PSII RC, which is considered the main source of 1 O 2 in wild type plants [66]. This LHC-type mechanism of photoinhibition is most likely not restricted to NoM mutant plants, since the biosynthesis of PSII RC subunits has been shown to be preferentially reduced in the cold, thus leading to an over-accumulation of antenna proteins [67,68]. ...
Proper assembly of plant photosystem II, in the appressed region of thylakoids, allows for both efficient light harvesting and the dissipation of excitation energy absorbed in excess. The core moiety of wild type supercomplex is associated with monomeric antennae that, in turn, bind peripheral trimeric LHCII complexes. Acclimation to light environment dynamics involves structural plasticity within PSII-LHCs supercomplexes, including depletion in LHCII and CP24. Here, we report on the acclimation of NoM, an Arabidopsis mutant lacking monomeric LHCs but retaining LHCII trimer. Lack of monomeric LHCs impaired the operation of both photosynthetic electron transport and state transitions, despite the fact that NoM underwent a compensatory over-accumulation of the LHCII complement compared to the wild type. Mutant plants displayed stunted growth compared to the wild type when probed over a range of light conditions. When exposed to short-term excess light, NoM showed higher photosensitivity and enhanced singlet oxygen release than the wild type, whereas long-term acclimation under stress conditions was unaffected. Analysis of pigment-binding supercomplexes showed that the absence of monomeric LHCs did affect the macro-organisation of photosystems: large PSI-LHCII megacomplexes were more abundant in NoM, whereas the assembly of PSII-LHCs supercomplexes was impaired. Observation by electron microscopy (EM) and image analysis of thylakoids highlighted impaired granal stacking and membrane organisation, with a heterogeneous distribution of PSII and LHCII compared to the wild type. It is concluded that monomeric LHCs are critical for the structural and functional optimisation of the photosynthetic apparatus.
... For instance, the The two phylloquinones (A 1A and A 1B ) in PSI differ in kinetics and reduction potential and thus in triplet formation through back reactions [68,125,126]. Chlorophyll triplet formation can occur in PSI: under reducing illuminated conditions, in the absence of secondary acceptors, or if the electron acceptor side of PSI is blocked [68,127,128]. Supersaturating light could lead to too few electron donors and acceptors to prevent charge recombination. On the B-side, the energy gap is smaller and triplet formation is favored. ...
The ability to harvest light to drive chemical reactions and gain energy provided microbes access to high energy electron donors which fueled primary productivity, biogeochemical cycles, and microbial evolution. Oxygenic photosynthesis is often cited as the most important microbial innovation—the emergence of oxygen-evolving photosynthesis, aided by geologic events, is credited with tipping the scale from a reducing early Earth to an oxygenated world that eventually lead to complex life. Anoxygenic photosynthesis predates oxygen-evolving photosynthesis and played a key role in developing and fine-tuning the photosystem architecture of modern oxygenic phototrophs. The release of oxygen as a by-product of metabolic activity would have caused oxidative damage to anaerobic microbiota that evolved under the anoxic, reducing conditions of early Earth. Photosynthetic machinery is particularly susceptible to the adverse effects of oxygen and reactive oxygen species and these effects are compounded by light. As a result, phototrophs employ additional detoxification mechanisms to mitigate oxidative stress and have evolved alternative oxygen-dependent enzymes for chlorophyll biosynthesis. Phylogenetic reconstruction studies and biochemical characterization suggest photosynthetic reactions centers, particularly in Cyanobacteria, evolved to both increase efficiency of electron transfer and avoid photodamage caused by chlorophyll radicals that is acute in the presence of oxygen. Here we review the oxygen and reactive oxygen species detoxification mechanisms observed in extant anoxygenic and oxygenic photosynthetic bacteria as well as the emergence of these mechanisms over evolutionary time. We examine the distribution of phototrophs in modern systems and phylogenetic reconstructions to evaluate the emergence of mechanisms to mediate oxidative damage and highlight changes in photosystems and reaction centers, chlorophyll biosynthesis, and niche space in response to oxygen production. This synthesis supports an emergence of H 2 S-driven anoxygenic photosynthesis in Cyanobacteria prior to the evolution of oxygenic photosynthesis and underscores a role for the former metabolism in fueling fine-tuning of the oxygen evolving complex and mechanisms to repair oxidative damage. In contrast, we note the lack of elaborate mechanisms to deal with oxygen in non-cyanobacterial anoxygenic phototrophs suggesting these microbes have occupied similar niche space throughout Earth's history.
... This narrowing could reflect a change from water to His as the Chl D1 ligand in the D1/T179H mutant. Indeed, the 3 P 700 EPR signal is slightly narrower than the 3 Chl D1 EPR signal [49], and P 700 has His ligands [50]. The T179V mutant seems to have a different proportionality for the Z peaks compared to the Y and X peaks. ...
The monomeric chlorophyll, ChlD1, which is located between the PD1PD2 chlorophyll pair and the pheophytin, PheoD1 is the longest wavelength chlorophyll in the heart of Photosystem II and is thought to be the primary electron donor. Its central Mg²⁺ is liganded to a water molecule that is H-bonded to D1/T179. Here, two site-directed mutants, D1/T179H and D1/T179V, were made in the thermophilic cyanobacterium, Thermosynechococcus elongatus, and characterized by a range of biophysical techniques. The Mn4CaO5 cluster in the water-splitting site is fully active in both mutants. Changes in thermoluminescence indicate that i) radiative recombination occurs via the repopulation of *ChlD1 itself; ii) non-radiative charge recombination reactions appeared to be faster in the T179H-PSII; and iii) the properties of PD1PD2 were unaffected by this mutation, and consequently iv) the immediate precursor state of the radiative excited state is the ChlD1⁺PheoD1⁻ radical pair. Chlorophyll bleaching due to high intensity illumination correlated with the amount of ¹O2 generated. Comparison of the bleaching spectra with the electrochromic shifts attributed to ChlD1 upon QA⁻ formation, indicates that in the T179H-PSII and in the WT*3-PSII, the ChlD1 itself is the chlorophyll that is first damaged by ¹O2, whereas in the T179V-PSII a more red chlorophyll is damaged, the identity of which is discussed. Thus, ChlD1 appears to be one of the primary damage site in recombination-mediated photoinhibition. Finally, changes in the absorption of ChlD1 very likely contribute to the well-known electrochromic shifts observed at ~430 nm during the S-state cycle.
... Recent studies have suggested that 1 O 2 is involved in PSI photoinhibition, although it was assumed that 1 O 2 is not generated in PSI (Hideg and Vass 1995, Cazzaniga et al. 2012, Takagi et al. 2016a). Under the highly reducing conditions of PSI, triplet-state P700 ( 3 P700), which is a source of 1 O 2 production in PSI, is generated by the back reaction of P700 + and A 0 − from P700 + and A 1 − within PSI (Frank et al. 1979, Rutherford and Mullet 1981, Setif et al. 1981, Ikegami et al. 1987, Sétif and Brettel 1990, Polm and Brettel 1998, Rutherford et al. 2012. Currently, the production of 1 O 2 has actually been detected in the PSI-Light harvesting complex I (LHCI) complex. ...
In land plants, photosystem (PS) I photoinhibition limits carbon fixation and causes growth defects. In addition, recovery from PSI photoinhibition takes much longer than PSII photoinhibition when the PSI core-complex is degraded by oxidative damage. Accordingly, PSI photoinhibition should be avoided in land plants, and land plants should have evolved mechanisms to prevent PSI photoinhibition. However, such protection mechanisms have not yet been identified, and it remains unclear whether all land plants suffer from PSI photoinhibition in the same way. In the present study, we focused on the susceptibility of PSI to photoinhibition and investigated whether mechanisms of preventing PSI photoinhibition varied among land plant species. To assess the susceptibility of PSI to photoinhibition, we used repetitive short pulse (rSP) illumination, which specifically induces PSI photoinhibition. Subsequently, we found that land plants possess a wide variety of tolerance mechanisms against PSI photoinhibition. In particular, gymnosperms, ferns, and mosses/liverworts exhibited higher tolerance to rSP illumination-induced PSI photoinhibition than angiosperms, and detailed analyses indicated that the tolerance of these groups could be partly attributed to flavodiiron proteins, which protected PSI from photoinhibition by oxidizing the PSI reaction center chlorophyll (P700) as an electron acceptor. Furthermore, we demonstrate, for the first time, that gymnosperms, ferns, and mosses/liverworts possess a protection mechanism against photoinhibition of PSI that differs from that of angiosperms.
... Dominant population of the T O sublevels is not observed in any case. Note that the zero-field splitting for triplets in P S I and PS II particles are nearly identical to that of monomer Chl [100][101][102]. In light of the data on model compounds, it is a mistake to assert that this proves that the primary electron donor in PS I or PS II is a dimer or is not. ...
In oxygenic photosynthesis sunlight is harvested and funneled as excitation energy into the reaction center (RC) of Photosystem II (PSII), the site of primary charge separation that initiates the photosynthetic electron transfer chain. The chlorophyll ChlD1 pigment of the RC is the primary electron donor, forming a charge-separated radical pair with the vicinal pheophytin PheoD1 (ChlD1⁺PheoD1⁻). To avert charge recombination, the electron is further transferred to plastoquinone QA, whereas the hole relaxes to a central pair of chlorophylls (PD1PD2), subsequently driving water oxidation. Spin-triplet states can form within the RC when forward electron transfer is inhibited or back reactions are favored. This can lead to formation of singlet dioxygen, with potential deleterious effects. Here we investigate the nature and properties of triplet states within the PSII RC using a multiscale quantum-mechanics/molecular-mechanics (QM/MM) approach. The low-energy spectrum of excited singlet and triplet states, of both local and charge-transfer nature, is compared using range-separated time-dependent density functional theory (TD-DFT). We further compute electron paramagnetic resonance properties (zero-field splitting parameters and hyperfine coupling constants) of relaxed triplet states and compare them with available experimental data. Moreover, the electrostatic modulation of excited state energetics and redox properties of RC pigments by the semiquinone QA⁻ is described. The results provide a detailed electronic-level understanding of triplet states within the PSII RC and form a refined basis for discussing primary and secondary electron transfer, charge recombination pathways, and possible photoprotection mechanisms in PSII.
Photosynthetic organisms face a constant dilemma: harvesting as much light as possible while minimizing damage when in excess light conditions. Environmental conditions reproducibly change during the day while sudden light changes superimpose due to shading and require quick responses to ensure maximal growth. Pigment-binding antenna proteins fulfil both roles by ensuring efficient light harvesting, funnelling excitation energy towards the reaction centres while dissipating excess energy in the form of heat when necessary and scavenging high energy species as a further defence. Oxygenic organisms exhibit a highly diversified complement of antenna proteins in striking contrast with the photosynthetic reaction centres, which are highly conserved. This can be attributed to the adaptation to different light regimes found on Earth. Of particular importance is the mechanism called Non-Photochemical Quenching (NPQ) catalyzing energy dissipation by regulating the interactions between chlorophylls and carotenoids, likely by multiple reactions involving excited states. During the evolution different proteins have fulfilled this function including LHCSR, found in most algae, until PSBS evolved in advanced green algae and replaced LHCSR in higher plants.
Over the last few years it has become increasingly clear that considerable homology exists between the acceptor side components of photosystem II (PS II) and those of reaction centers from purple photosynthetic bacteria (Parson and Ke 1982, Crofts and Wraight 1983, Dutton, Chap. 5, Michel and Deisenhofer, Chap. 8.4, both this Vol.). While one might hardly have expected to see familiar faces among the high potential donors of PS II, recent evidence suggests that some of the same old dogs may have learned new tricks.
In photosynthetic reaction centers, reduction of the secondary acceptors leads to triplet charge recombination of the primary radical pair (RP). This process is spin selective and in a magnetic field it populates only the T0 state of the donor triplet state. As a result, the triplet state of the donor has a distinctive spin polarization pattern that can be measured by transient electron paramagnetic resonance (TREPR) spectroscopy. In heliobacterial reaction centers (HbRCs), the primary donor, P800, is composed of two bacteriochlorophyll g′ molecules and its triplet state has not been studied as extensively as those of other reaction centers. Here, we present TREPR and optically detected magnetic resonance (ODMR) data of ³P800 and show that although it can be detected by ODMR it is not observed in the TREPR data. We demonstrate that the absence of the TREPR spectrum is a result of the fact that the zero-field splitting (ZFS) tensor of ³P800 is maximally rhombic, which results in complete cancelation of the absorptive and emissive polarization in randomly oriented samples.
The recent isolation of a complex containing the polypeptides D1, D2 and cytochrome b559, confirmed the proposal that D1 and D2 bind the reaction centre components of PS21,2. The complex was shown to be photochemically active by the photoreduction of phaeophytin and a spin polarised triplet was also detected by epr3. The triplet species originates from a radical pair recombination between the oxidised primary donor chlorophyll P680+ and the reduced phaeophytin acceptor I-4.Redox titrations of the reaction centre preparation using the spin polarised triplet signal confirmed the lack of a quinone electron acceptor but suggested that the non-haem iron still present may act as an electron acceptor when oxidised5.
Recent advances in structure and function of photosystem 1 reaction centre (PS I RC) protein-pigment complex in the inner membranes of green plants and of cyanobacteria are discussed. The complex cooperates with photosystem II reaction centre (PS II RC) complex in the same membranes to convert light energy into electrochemical energy in photosynthetic reaction. The structure of the protein moiety of the PS I RC complex is discussed on the basis of recently identified subunit components and their amino-acid sequences. The sequences show very low homology to those of subunits of RC complexes of PS II or purple bacteria, and suggest that this complex has a structure and an evolutionary origin somewhat different from the latter complexes. The PS I RC produces extremely low redox potential and reduces NADP+. Almost all the prosthetic groups, such as chlorophylls, quinone, and iron-sulphur centres, in this RC have recently been identified. Recent experimental findings on characteristics of the prosthetic groups and their reactions are reviewed. It has become clear that the protein moiety of this RC complex creates special environments for the function of prosthetic groups. Future studies, expected from the modification of structure of this RC now in progress in various laboratories, are discussed.
This chapter discusses the primary reactions of photosystems I and II of algae and higher plants. In photosynthetic organisms, the “primary reactions” fulfil the objective of converting the energy of light into a primary form of chemical energy which lasts for a time compatible with ordinary biochemical processes—that is, milliseconds. In these reactions, a rather large fraction, approximately 40%, of the photon energy is stored as chemical free energy. The primary reactions can be viewed from two major perspectives. Firstly, from a photochemical point of view: pigment molecules are excited to their lowest excited singlet state, which reacts in an electron transfer reaction, the first step of a process of charge separation. Secondly, from a biochemical point of view the reactions take place in highly organized complexes, the reaction centres, which are made up of several classes of molecules that cooperate in fulfilling complementary roles, such as: architectural support, light absorption, energy transfer and electron transfer. All oxygenic organisms, ranging from cyanobacteria to algae and higher plants, contain photosystem I (PS I) and PS II reaction centres, with only minor variations in spite of their large taxonomic and ecological diversity.
Absorption spectra in the near infra-red have been obtained for transient species obtained from chlorophyll a, by flash absorption spectroscopy. The triplet state of Chl a presents an absorption band around 760 nm (ϵ = 7,500 M−1 cm−1) and a very broad band (ϵ = 1,700 M−1 cm−1) around 1100 nm. At low temperature, evidence was obtained for the formation of the triplet state of aggregated Chl a. Its t1/2 of decay is around 0.5 ms instead of 1 ms for the monomeric species. The radical-cation Chl a+ was formed by reaction of 3Chl a with a quinone; it has an absorption band at 840 nm (in cyclohexanol) with a shoulder around 740 nm and no absorption between 1000 and 1650 nm. The cation has also been formed in vivo, as the oxidized state of the photosynthetic primary electron donors, P680 and P700. The spectrum of P+680 resembles that of Chl a+; its absorption maximum is at 820 nm. The spectrum of P+680, maximum at 810 nm, is significantly broader. P+700 has no absorption between 1000 and 1650 nm. These spectra are in favor of recent suggestions on a monomeric nature of P680 and of a dimeric nature for P700.
In this review, the main research developments that have led to the current simplified picture of photosystem I are presented. This is followed by a discussion of some conflicting reports and unresolved questions in the literature. The following points are made: (1) the evidence is contradictory on whether P700, the primary donor, is a monomer or dimer of chlorophyll although at this time the balacnce of the evidence points towards a monomeric structure for P700 when in the triplet state; (2) there is little evidence that the iron sulfur centers FA and FB act in series as tertiary acceptors and it is as likely that they act in parallel under physiological conditions; (3) a role for FX, probably another iron sulfur centrer, as an obligatory electron carrier in forward electron transfer has not been proven. Some evidence indicates that its reduction could represent a pathway different to that involving FA and FB; (4) the decay of the acceptor 'A2-' as defined by optical spectroscopy corresponds with 700+ {Mathematical expression} recombination under some circumstances but under other conditions it probably corresponds with P700+ A1- recombination; (5) P700+ A1- recombination as originally observed by optical spectroscopy is probably due to the decay of the P700 triplet state; (6) the acceptor A1- as defined by EPR may be a special semiquinone molecule; (7) A0 is probably a chlorophyll a molecule which acts as the primary acceptor. Recombination of P700+ A0- gives rise to the P700 triplet state. A working model for electron transfer in photosystem I is presented, its general features are discussed and comparisons with other photosystems are made.
The Photosystem I reaction centre protein CP1, isolated from barley using polyacrylamide gel electrophoresis showed an EPR (Electron Paramgnetic Resonance) spectrum with the polarisation pattern AEEAAE, typical of the primary donor triplet state (3)P700, created via radical pair formation and recombination. (3)P700 could also be detected by Fluorescence Detected Magnetic Resonance (FDMR) at λf > 700 nm even in the presence of a large number of chlorophyll antennae. Its zero field splitting parameters, D=282.5×10(-4) cm(-1) and E=38.5×10(-4) cm(-1), were independent of the detection wavelength, and agreed with ADMR (Absorption Detected Magnetic Resonance) and EPR values. The signs of the (3)P700 D+E and D-E transitions were positive (increase in fluorescence intensity on applying a resonance microwave field). In contrast, in the emission band 685 < λf < 700 nm FDMR spectra with negative D+E and D-E transitions were detected, and the D value was wavelength-dependent. These FDMR results support an excitation energy transfer model for CP1, derived from time-resolved fluorescence studies, in which two chlorophyll antenna forms are distinguished, with fluorescence at 685 < λf < 700 nm (inner core antennae, F690), and λf > 700 nm (low energy antenna sites, F720), in addition to the P700. The FDMR spectrum in F690 emission can be interpreted as that of (3)P700, observed via reverse singlet excitation energy transfer and added to the FDMR spectrum of the antenna triplet states generated via intramolecular intersystem crossing. This would indicate that reversible energy transfer between F690 and P700 occurs even at 4.2 K.
The formation of chlorophyll triplet states during illumination of Photosystem I reaction center samples depends upon the redox state of P-700, X and ferredoxin Centers A and B. When the reaction centers are in the states P-700+A1XFdBFd−A and P-700 A1XFd−BFd−A prior to illumination, we observe electron paramagnetic resonance (EPR) spectra from a triplet species which has zero-field splitting parameters (|D| and |E|) larger than those of either the chlorophyll a or chlorophyll b monomer triplet, and a polarization which results from population of the triplet spin sublevels by an intersystem crossing mechanism. We interpret this triplet as arising from photoexcited chlorophyll antenna species associated with reaction centers in the states P-700+Fd−A and P-700+X−, respectively, which undergo de-excitation via intersystem crossing. When the reaction centers are in the states P-700A1XFd−BFd−A and P-700A1X−Fd−BFd−A prior to illumination, we observe a triplet EPR signal with a polarization which results from population of the triplet spin sublevels by radical pair recombination, and which has a |D| value similar to that of chlorophyll a monomer. We interpret this triplet (the radical pair-polarized triplet) as arising from 3P-700 which has been populated by the process . We observe both the radical pair-polarized triplet and the chlorophyll antenna triplet when the reaction centers are in the state P-700 A1XFd−BFd−A, presumably because the processes P-700+A−1X → P-700+A1X− and have similar rate constants when Centers A and B are reduced, i.e., the forward electron transfer time from A−1 to X is apparently much slower in the redox state P-700 A1XFd−BFd−A than it is in state P-700 A1XFdBFdA. The amplitude of the radical pair-polarized triplet EPR signal does not decrease in the presence of a 13.5-G-wide EPR signal centered at g 2.0 which was recorded in the dark prior to triplet measurements in samples previously frozen under intense illumination. This g 2.0 signal, which has been attributed to phototrapped A−1 (Heathcote, P., Timofeev, K.N. and Evans, M.C.W. (1979) FEBS Lett. 101, 105–109), corresponds to as many as 12 spins per P-700 and can be photogenerated during freezing without causing any apparent attenuation of the radical pair-polarized triplet amplitude. We conclude that species other than A−1 contribute to the g 2.0 signal.
The suggestion that the electron acceptor A1 in plant photosystem I (PSI) is a quinone molecule is tested by comparisons with the bacterial photosystem. The electron spin polarized (ESP) EPR signal due to the oxidized donor and reduced quinone acceptor (P
870+Q-) in iron-depleted bacterial reaction centers has similar spectral characteristics as the ESP EPR signal in PSI which is believed to be due to P
700+A
1-, the oxidized PSI donor and reduced A1. This is also true for better resolved spectra obtained at K-band (∼24 GHz). These same spectral characteristics can be simulated using a powder spectrum based on the known g-anisotropy of reduced quinones and with the same parameter set for Q- and A1-. The best resolution of the ESP EPR signal has been obtained for deuterated PSI particles at K-band. Simulation of the A1- contribution based on g-anisotropy yields the same parameters as for bacterial Q- (except for an overall shift in the anisotropic g-factors, which have previously been determined for Q-). These results provide evidence that A1 is a quinone molecule. The electron spin polarized signal of P700+ is part of the better resolved spectrum from the deuterated PSI particles. The nature of the P700+ ESP is not clear; however, it appears that it does not exhibit the polarization pattern required by mechanisms which have been used so far to explain the ESP in PSI.
In order to determine the origin of the photoexcited pheophytin triplet state (3Phe) in plant Photosystem II preparations ( complexes) an absorption and fluorescence detected magnetic resonance (ADMR, FDMR) study was conducted in zero magnetic field at low temperatures. The ADMR signal intensity dependence on excitation light flux was linear showing that 3Phe is formed by a 1-photon process. Upon successive exposure of the sample to strong white light it was shown that 3Phe is not directly correlated with the primary donor triplet state 3P680. This is consistent with the absence of a double resonance signal connecting 3Phe with 3P680 down to an instrumental sensitivity of ) below 10−6. An intermediate state of photoinhibition between intact and fully degraded reaction centres is responsible for the formation of 3Phe. It may be a conformationally changed state of the reaction centre protein, most likely having a larger distance between P680 and the pheophytin whose triplet state is observed. Factor analysis of the absorption spectra of a series of gradually degraded samples yielded two spectral components which were interpreted as the spectra of fully intact and completely degraded reaction centres.
Triplet formation was observed upon excitation with a laser flash of membrane fragments of Heliobacterium chlorum by means of kinetic analysis of the absorbance difference spectra. At low redox potential charge recombination between the photooxidized primary donor P-798+ and the reduced primary acceptor A−0 yielded the triplet of P-798, both at room temperature and at 5 K. The exponential time constant for this recombination was found to be 17 ns at 300 K and 55 ns at 5 K. The yield of triplet formation from the state P-798+A−0 was 20–25% and 25–35%, respectively. At low temperature the quantum yield for the formation of the primary radical pair was a factor of 2 lower in reaction centers in which the (first) secondary electron acceptor X was in the reduced state, than in reaction centers where X was in the oxidized state before illumination. Absorbance difference spectra were measured for the formation of the triplet of P-798, for P-798+A−0 and for P-798+X− formation. Electrochromic bandshifts of bacteriochlorophyll (BChl) g were only observed in the spectra of P-798+X−. In addition to the reaction center triplet an antenna triplet was observed at low temperature, located on the long wavelength absorbing BChl g-808. The quantum yield of formation of this triplet was approximately 1–2%, independent of the redox state of the reaction center.
Optically detected magnetic resonance (ODMR) of P700 triplet state has been performed in large photosystem I (PSI) particles. The `intactness' of the P700 environment in the PSI particles has been proved by the comparison of the spectra with those obtained in thylakoids and leaves. Effect of degradation of the samples on the spectra has also been tested. When detected in `native' PSI centers the microwaves induced triplet-minus-singlet absorption spectra (T−S) show very sharp features which allow to estimate the minimum number of bands involved in the change of the electronic state of the primary donor (singlet to triplet), and can be interpreted in terms of interactions among pigments in the reaction center. The analogy with the reaction centers of purple bacteria is discussed. The previously published spectra (H.J. den Blanken, A.J. Hoff, Biochim. Biophys. Acta 724 (1983) 52–61; J. Vrieze, P. Gast, A.J. Hoff, J. Phys. Chem. 100 (1996) 9960–9967) obtained in particles having smaller antenna size, resemble the ones obtained in our degraded samples: it is suggested that the isolation procedure to small particles may produce an increase in the heterogeneity of the environment of the primary donor and/or a change of the mutual orientations and interactions among pigments. The fluorescence detected magnetic resonance (FDMR) in large PSI particles supports an excitation energy transfer model derived from time resolved optical studies (R. Van Grondelle, J.P. Dekker, T. Gillbro, V. Sundstrom, Biochim. Biophys. Acta 1187 (1994) 1–65) and adapted for helium temperature, characterized by the presence of antenna pools emitting at 690, 720 and 735nm, respectively. The FDMR signal of P700 changes sign (from positive to negative) when detected at emission wavelength shorter than 700nm. Comparison of FDMR of P700 triplet state in large PSI-200 particles, in thylakoids and, for the first time, in leaves has been done.
Time-resolved EPR studies were done on broken spinach chloroplasts under reducing conditions at low temperature (10 K). A dramatic dependence of signal dynamics and lineshape in the g 2.00 region on the reduction state of Photosystem I is demonstrated. Computer simulations of the spin-polarized lineshapes obtained in this work suggest that the primary electron acceptor in Photosystem I, a species known as A1, could be a chlorophyll a dimer.
The yield of the triplet state of the primary electron donor of Photosystem I of photosynthesis (PT-700) and the characteristic parameters (g value, line shape, saturation behavior) of the ESR signal of the photoaccumulated intermediary acceptor A have been measured for two types of Photosystem I subchloroplast particles: Triton particles (TSF 1, about 100 chlorophyll molecules per P-700) that contain the iron-sulfur acceptors FX, FB and FA, and lithium dodecyl sulfate (LDS) particles (about 40 chlorophyll molecules per P-700) that lack these iron-sulfur acceptors. The results are: (i) In Triton particles the yield of PT-700 upon illumination is independent of the redox state of A and of FX,B,A and is maximally about 5% of the active reaction centers at 5 K. The molecular sublevel decay rates are kx = 1100 s−1 ± 10%, ky = 1300 s−1 ± 10% and kz = 83 s−1 ± 20%. In LDS particles the triplet yield decreases linearly with concentration of reduced intermediary acceptors, the maximal yield being about 4% at 5 K assuming full P-700 activity. (ii) In Triton particles the acceptor complex A consists of two acceptors A0 and A1, with A0 preceding A1. In LDS particles at temperatures below −30°C only A0 is photoactive. (iii) The spin-polarized ESR signal found in the time-resolved ESR experiments with Triton particles is attributed to a polarized P-700-A−1 spectrum. The decay kinetics are complex and are influenced by transient nutation effects, even at low microwave power. It is concluded that the lifetime at 5 K of P-700A0A−1 must exceed 5 ms. We conclude that PT-700 originates from charge recombination of P-700A−0, and that in Triton particles A0 and A1 are both photoaccumulated upon cooling at low redox potential in the light. Since the state P-700AF−X does not give rise to triplet formation the 5% triplet yield in Triton particles is probably due to centers with damaged electron transport.
ESR studies at 4.2 K have been conducted in Photosystem I particles prepared with SDS (CP 1 particles) which were not poised at a low redox potential. Continuous illumination induces the appearance of a triplet state, spin-polarized according to a charge-recombination process. The decay kinetics of the triplet zero-field sublevels were derived from a study of the flash-induced ESR signals at the different triplet peaks: kx = 1150±350 s−1, ky = 1050±350 s−1, kz ⩽ 130 s−1. From comparison with the absorption data and from an ESR kinetic study in the g 2.0 region, it was concluded that this triplet is the P-700 triplet whose decay accounts for the whole of the optical signal observed at low temperature with k ≈ 900 s−1 () and which is formed through a back-reaction between P-700+ and the reduced primary acceptor A−1. This interpretation of the observed optical decay was recently proposed (Setif, P., Hervo, G. and Mathis, P. (1981) Biochim. Biophys. Acta 638, 257–267), contrasting with a previous assignment to the P-700+-A−1 decay (Mathis P., Sauer, K. and Remy, R. (1978) FEBS Lett. 88, 275–278). A prolonged illumination induces an irreversible charge separation in about half the reaction centers. This was interpreted as indicating the presence in these centers of an electron acceptor following A1, which is reduced with a very weak quantum efficiency, whereas the back-reaction between P-700+ and A−1 is the major decay process. The chemical nature of this acceptor is unknown.
10% of the chlorophyll associated with a ‘native’ Photosystem (PS) I complex (110 chlorophylls/P-700) is chlorophyll (Chl) b. The Chl b is associated with a specific PS I antenna complex which we designate as LHC-I (i.e., a light-harvesting complex serving PS I). When the native PS I complex is degraded to the core complex by LHC-I extraction, there is a parallel loss of Chl b, fluorescence at 735 nm, together with 647 and 686 nm circular dichroism spectral properties, as well as a group of polypeptides of 24-19 kDa. In this paper we present a method by which the LHC-I complex can be dissociated from the native PS I. The isolated LHC-I contains significant amounts of Chl b (Chl ). The long-wavelength fluorescence at 730 nm and circular dichroism signal at 686 nm observed in native PS I are maintained in this isolated complex. This isolated fraction also contains the low molecular weight polypeptides lost in the preparation of PS I core complex. We conclude that we have isolated the PS I antenna in an intact state and discuss its in vivo function.
The protein composition of the photosynthetic membrane from the cyanobacterium, Anacystis nidulans R2, was analyzed by acrylamide gel electrophoresis following solubilization with lithium dodecyl sulfate. Autoradiograms of 35S-labelled membranes revealed over 90 bands by this procedure. The effect of solubilization conditions on protein resolution was analyzed by modifying temperature and sulfhydryl concentrations. Labelling cells with 59Fe yielded nine iron-containing bands on these gels. Three of these bands, at 33, 19, and 14 kDa, were also heme proteins as determined by tetramethylbenzidine staining, and represent cytochromes f, b6 and c-552, respectively. The remaining iron proteins are highly sensitive to solubilization conditions, especially the presence of 2-mercaptoethanol, and we suggest that these bands may be Fe-S proteins. Lactoperoxidase-catalyzed iodination of the membranes indicated that at least 41 proteins have surface-exposed domains. Some of the known proteins with external surfaces include cytochrome c-552 and the chlorophyll-binding proteins of Photosystems I and II. Neither cytochrome f nor b6 appear to be accessible to external labelling. When this structural information was combined with the isolation of functional submembrane complexes, we constructed a topological model of the membrane. Using this model we have discussed the protein architecture of the cyanobacterial membrane.
The X-band (9.5 GHz) electron spin polarized (ESP) electron paramagnetic resonance (EPR) signal observed in iron-depleted bacterial reaction centers is due to P+-870Q− (P+-870 is the oxidized primary donor, a special pair of bacteriochlorophyll molecules, and Q− is the reduced primary quinone acceptor). This signal contains information about the dynamic interactions and spatial relationships between the photosynthetic primary reactants as they undergo electron transfer. Interpretations of the X-band ESP EPR spectrum have not been unique because of insufficient spectral resolution and the simulations require a large number of adjustable parameters. Therefore, we have acquired ESP EPR spectra at X-band and Q-band (35 GHz) from iron-depleted bacterial reaction centers in which P-870, I (intermediary bacteriopheophytin acceptor), and Q were selectively deuterated (protonated). The effects of selective isotopic substitution and microwave frequency on the ESP EPR spectra of P+-870Q− (i) could not be explained by the polarization transfer mechanism alone, and (ii) allowed the determination of the polarization contributions of 2(P+-870) and 2(Q−) to the composite ESP EPR spectrum. The resolution of these components suggests that the spin-correlated radical pair mechanism makes a significant contribution to the ESP spectrum from 2(P-870I)2Q. The results presented here should provide a stringent test for computer simulations of the correct mechanism of ESP production.
The triplet state of the primary donor of photosystem II particles prepared from a mutant of Chlamydomonas reinhardtii has been studied at 1.2 K with absorbance-detected ESR in zero-magnetic field (ADMR). Two sets of resonances with slightly different zero-field splitting parameters |D| and |E| were observed, |D| = 285.5, |E| = 38.8 and |D| = 288.8 × 10−4 cm−1, |E| = 42.2 × 10−4 cm−1, respectively. Both sets of |D| and |E| values are close to those found for PT-700, as are the sublevel decay rates kx = 930 ± 40, ky = 1088 ± 50 and kz = 110 ± 5 s−1. The AMDR-detected triplet-minus-singlet absorbance difference spectrum of PT-680 is very similar to that of PT-700 and closely resembles that of covalently connected Chl a dimers in vitro. We conclude that P-680 is a Chl a dimer whose general structure is similar to that of P-700.
Illumination at 230 K of dithionite-reduced particles results in the appearance of an EPR detectable radical 13 G wide with g = 2.0033. This radical is formed in a ratio of 2.28 (±0.5)/P700. Investigation of the time course of formation shows two components are present. One (A1) has g = 2.0051 and the other (Aog= 2.0024. Reduction of A1 results in an increase in reaction centre triplet formation, subsequent reduction of Ao results in a decrease of triplet formation to the base level. We propose that these components function sequentially in the transfer of electrons from P700 to the iron—sulphur acceptors.
Two large membrane protein complexes, Photosystem I and II (PS I and PS II), perform the first step in the conversion of the light energy from the sun into chemical energy: the light-induced transmembrane charge separation. They function in series; PS II provides the strong positive redox potential for water oxidation, while PS I generates a strong negative redox potential, which makes it able to reduce ferredoxin and deliver the electrons for the reduction of hydrogen in the form of NADPH. The structural comparison of PS I and PS II sheds light on the evolution of oxygenic photosynthesis. Both Photosystems show similarities in their core structure, indicating that they have been derived from a common ancestor. Striking differences in the arrangement and coordination of cofactors and in their protein environment, however, may contain the secret to the functional differences between the Photosystems. In this article, we address how the oxygen-evolving complex may have developed, and the main similarities and differences in the electron carriers and the organization of the antenna systems of these two complexes.
The photosystem I reaction center is reviewed from the standpoint of electron acceptors, polypeptide composition, and structural organization. Experimental difficulties in the current model are highlighted, and current results on the polypeptide location of each acceptor are reviewed. The amount of iron and labile sulfide, the types of iron-sulfur clusters, and the number of ∼ 64-kDa polypeptides are considered in light of a model for the reaction center. In particular, the presence of [2Fe-2S] clusters in photosystem I has significance in the structure and organization of Fx on the reaction center. Since four cysteinyl ligands are assumed to hold an iron-sulfur cluster, a photosystem I subunit may consist of two ∼ 64-kDa polypeptides bridged by a [2Fe-2S] cluster. The reaction center would consist of a symmetrical pair of these subunits positioned so that two [2Fe-2S] clusters are in magnetic interaction, thereby constituting Fx. The reaction center core would therefore incorporate four ∼ 64-kDa polypeptides and have a molecular weight in excess of 250 kDa
Zusammenfassung In der vorliegenden Übersicht wird der Versuch unternommen, den derzeitigen Erkenntnisstand auf dem Gebiet der photosynthetischen Primärreaktionen, d. h. Ladungstrennung in den Pigmentsystemen, Elektronentransport und ATP-Synthese, zu umreißen. Für ein besseres Verständnis der funktionellen Vorgänge im Verlaufe der Lichtreaktion der Photosynthese wird auf die molekulare Organisation der Thylakoidmembranen der Chloroplasten eingegangen. Dabei werden Zusammenhänge aufgezeigt, die Einsichten in die Struktur-Funktionsbeziehungen gestatten.
The contents of plastoquinone and PS II electron carriers (QB and Z) of four PS II preparations from the thermophilic cyanobacterium Synechococcus sp. were determined. (1) The oxygen-evolving preparations and the PS II reaction center complexes contained about three and two plastoquinone molecules per PS II, respectively. CP2-b, which represents the functional core of the reaction center complex (Yamagishi, A. and Katoh, S. (1985) Biochim. Biophys. Acta 807, 74–80), had a reduced amount of plastoquinone which is just comparable with the QA content, whereas no plastoquinone was detected in CP2-c, the antenna chlorophyll-protein containing only the 40 kDa subunit. (2) The four preparations were essentially free from vitamin K1. (3) The occurrence of QB and Z in the PS II preparations were determined by measuring oxidation kinetics of Q−A, as well as effects of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, ferricyanide and benzidine on the Q−A oxidation with repetitive flash technique. EPR measurements were also carried out to determine Signal IIs and IIf. QB and Z were present in the oxygen-evolving preparations, while the PS II reaction center complexes had Z, but no or only a residual amount of QB. CP2-b lacked both QB and Z. (4) Q−A was oxidized partly by a back electron transport to Z+ and partly by ferricyanide added in the reaction center complexes. No back reaction between Q−A and P-680+ was detected in CP2-b, which instead showed absorption changes indicative of the triplet formation. It is concluded that three plastoquinone molecules (but not vitamin K1) function as QA, QB and Z and, besides them, there is no extra bound plastoquinone molecule which serves as an additional electron donor or acceptor in the vicinity of the PS II reaction center. The results also suggest that QA, pheophytin and P-680 are located in the interior of the reaction center complexes, but are exposed on removal of the 40 kDa subunit.
The kinetics of the quenching of the triplet state of P680 and the formation of singlet oxygen (IO2) have been measured under various conditions using isolated photosystem II reaction centres (PSII RCs). The results support the hypothesis that light-induced generation of 1O2 by PSII RCs is due to the formation of the P680 triplet state by the recombination reaction between oxidized P680 and reduced pheophytin. This suggests that singlet oxygen is likely to bring about protein damage, particularly to the D1 polypeptide, and that this detrimental reaction thus underlies the vulnerability of PSII to photoinhibitory damage.
The importance of algae, both as a contribution to the understanding of living things and in practical terms, hardly needs stressing today. Despite the previous emphasis on photosynthesis research in land plants there is now a large corpus of work on algae. This chapter intends to bring much of the dispersed literature together, so as to achieve an integrated framework from which conclusions can be drawn to further stimulate research. Organisms from the borderline of groups loosely called prokaryotes, plants, and animals have been discussed along with how the majority of algae are influenced by the light climate properties. The structure and function of the photosynthetic membrane have been described. Various kinds and levels of light harvesting available to algae are reviewed briefly. A more detailed analysis of some biochemical and biophysical aspects of light harvesting are also given. Light is essential to all photosynthetic autotrophs. But it is only to the extent that light is limiting to growth that light-harvesting strategies become important. It is therefore necessary to consider under what conditions light does become limiting for algal growth. Strategies of light harvesting are discussed in terms of general ecological, taxonomic, morphological, and cytological aspects. The chapter looks into photosynthetic pigments, reaction centre complexes, and pigment protein (light-harvesting) complexes with details of the principles of light harvesting in light of quantum chemistry and transfer of excitation energy, structure and function, distribution of excitation energy between the photosystems, and interaction of the light-harvesting apparatus with other photosynthetic processes.
The reaction center is the key component for the primary events in the photochemical conversion of light into chemical energy. After excitation by light, a charge separation that spans the cell membrane is formed in the reaction center in a few hundred picoseconds with a quantum yield of essentially one. A conserved pattern in the cofactors and core proteins of reaction centers from different organisms can be defined based on comparisons of the three dimensional structure of two types of reaction centers. Different functional aspects of the reaction center are discussed, including the properties of the bacteriochlorophyll or chlorophyll dimer that constitutes the primary electron donor, the pathway of electron transfer, and the different functional roles of the electron acceptors. The implication of these results on the evolution of the reaction center is presented.
D1/D2-cyt b559 complexes from Pisum sativum were investigated by absorption detected magnetic resonance (ADMR) in zero field. Two different triplet states were detected and further characterized by temperature dependent and time resolved ADMR, microwave holeburning studies, and the magnetic field dependence of the ADMR signals. Their microwave induced absorption (MIA) spectra showed maxima at 683 and 680 nm. They were identified as the triplet states of the primary electron donor P-3(680) and of a pheophytin a molecule possibly connected with partially degraded D1/D2-complexes. From comparison with bacterial reaction centers (RC) it can be concluded that the triplet state of the primary donor in plant photosystem II (P-3(680)) is located on a monomeric chlorophyll a molecule.
Chlorophyll d (Chl d) is the major pigment in the antenna proteins of both photosystems (PSI and II) of the oxyphotobacterium Acaryochloris marina. This fact suggests that photosynthesis based upon Chl d rather than Chl a may be an interesting alternative in oxygenic photosynthesis. While a great deal of spectroscopic information relative to Chl a is available, both in vivo and in vitro, the literature on Chl d is scarce. In particular, the triplet state of Chl d has not been studied in vitro to date. Although triplet states do not represent the main excitation path in the photosynthetic process, they are involved in light stress events both in the antenna complexes and in the reaction centers and may also be used as endogenous paramagnetic probes of the molecular environment. In this paper we make use of both time-resolved EPR and ODMR to characterize, for the first time, the Chl d triplet state in the polar solvent methyl-tetrahydrofuran. Comparison with the spectra of Chl a obtained under the same experimental conditions is also discussed.
Photosystem I particles from spinach were studied with linear-dichroic absorbance-detected magnetic resonance in zero-magnetic field. The microwave-induced triplet-minus-singlet (T−S) spectra and the linear-dichroic (LD) T−S spectra were recorded for the |D| + |E| and the |D| − |E| microwave transitions of the triplet state of the primary donor, 3P700. From these data the directions of the optical transition moments contributing to the T−S spectra in the 600−750 nm region, with respect to the triplet x- and y-axes of 3P700 were obtained. The orientation of the optical QY-transition moment of P700 relative to the triplet x- and y-axis is found to be 35 ± 2° and 56 ± 1°, respectively. A comparison is made with data obtained for monomeric chlorophyll (Chl) a in two glasses. The orientation of the QY-transition moment with respect to the in-plane triplet x- and y-axes of 3P700 differs from monomeric Chl a in the two glasses. This difference is ascribed to the different environments of P700 and Chl a, rather than to the dimeric structure of P700. In addition to the QY-absorption band of P700, the T−S and LD(T−S) spectra contain features that are ascribed to transitions involving accessory Chls. The contribution of the QY transition of the primary acceptor to a band at 687 nm in the T−S spectrum is discussed, and a comparison is made with the signal of the primary acceptor in the T−S spectrum of Heliobacterium chlorum.
Electron paramagnetic resonance (EPR) spectroscopy, employing light modulation (LM) in the submillisecond range together with laser excitation-diode detection (DD) in the submicrosecond range has been carried out on the core complex of photosystem I (CC I) purified from Lemna gibba. The photophysical and photochemical processes that occur in CC I have been investigated as a function of the acceptor's redox state and of temperature, with use of different modes of EPR detection. Photoexcitation of the system in which all the acceptors are in their normal redox state P700A0A1A2A3A4 gives rise to triplet spectra mainly typical of ChlaT(triplet state chlorophyll a) in the internal antenna. When some of the acceptors are reduced (P700A0A1[A2A3A 4]•-), the observed triplet species, by employing the LM method, are those of the reaction center P700 and β-carotene. The latter is sensitized by ChlaT of the internal antenna. With the DD method, the triplet state of Chla is also observed and is accompanied by a decrease of the triplet yield of the other species. These differences in triplet yield are discussed in terms of triplet-triplet annihilation (ChlaT + β-caroteneT) and the cycling times of the partially reduced and nonreduced systems with respect to the two EPR methods employed.
PHOTOSYSTEM I (PSI) is the photochemical reaction complex involved in the generation of a low potential reductant in oxygen-evolving photosynthetic organisms. The primary photochemical reaction in PSI has been identified as the photo-oxidation of a reaction centre chlorophyll complex P700 (ref. 1). This photo-oxidation occurs both at room temperature and in the frozen state at temperatures as low as 4.2 K. At cryogenic temperatures this photo-oxidation has generally been found to be irreversible2. Malkin and Bearden3 showed that the electron acceptor for this photo-oxidation reaction is a bound ferredoxin. This ferredoxin has two iron–sulphur centres with very low redox potentials (Em10 = −550 and −590 mV)4–6. It has been proposed that these centres are the primary electron acceptor of PSI3,4. More recent evidence suggests it may not be7–11, and we have shown11 that when the bound ferredoxin is chemically reduced, illumination at low temperature results in the photo-oxidation of P700 without any change in the bound ferredoxin, this oxidation is reversible. In photosynthetic bacteria a component with an EPR signal at g = 1.82 has been identified as the primary electron acceptor of the photochemical system12,13 and it is possible that a similar component might be involved in chloroplast photochemical reactions. McIntosh et al.
14 obtained kinetic evidence for the presence in PSI particles of a component with an EPR signal at g = 2.06 and g = 1.76 which showed a reversible redox change on illumination of the particles at low temperature parallel to that of P700. The changes observed were small and it was not possible to obtain a spectrum of the component. Using a more highly purified PSI preparation we have now obtained strong evidence that this component is the primary electron acceptor of PSI.
The technique of flash kinetic spectrophotometry was used to demonstrate a broad absorption band around 430 nm, which was kinetically different from P700, in several photosystem-I particles from spinach and blue-green algae. The component represented by this absorption band, designated as "P430", was bleached as fast as P700. Its recovery in the dark was accelerated by ferredoxin and by various artificial electron acceptors with redox potentials as low as -521 mV. The recovery kinetics have been demonstrated to be identical to those of a concomitant reduction of several of the artificial electron acceptors used. Tentatively, "P430" has been proposed as a possible primary electron acceptor of photosystem I.
In vitro and in vivo triplet state electron paramagnetic resonance (epr) spectra of bacteriochlorophylls (Bchls) show important differences in (a) electron spin polarization (esp), and (b) zero field splitting (ZFS) parameters. The unusual esp and ZFS properties of the observed in vivo triplet state are best interpreted as arising from a short-lived radical pair precursor (not directly observable by epr) formed in or with the special pair of bacteriochlorophyll molecules involved in the primary photo-act.
Data are presented which suggest the existence of a light-harvesting pigment-protein complex which is functionally and structurally associated with photosystem I (PSI) reaction centers. These observations are based on techniques which allow isolation of PSI using minimal concentrations of Triton X-100. Properties of density and self aggregation allowed purification of a "native" PSI complex.The isolated PSI particles appear as 106 A spherical subunits when viewed by freeze fracture microscopy. When incorporated into phosphatidyl choline vesicles, the particles lose self-aggregation properties and disperse uniformly within the lipid membrane.The isolated PSI preparation contains 100 +/- 10 chlorophylls/P(700) (Chl a/b ratio greater than 18); this represents a recovery of 27% of the original chloroplast membrane Chl. These particles were enriched in Chl a forms absorbing at 701 to 710 nm. Chl fluorescence at room temperature exhibited a maximum at 690 nm with a pronounced shoulder at 710 nm. At 77 K, peak fluorescence emission was at 736 nm; in the presence of dithionite an additional fluorescence maximum at 695 nm was obtained at 77 K. This dual fluorescence emission peak for the PSI particles is evidence for at least two Chl populations within the PSI membrane subunit. The fluorescence emission observed at 695 nm was identified as arising from the core of PSI which contains 40 Chl/P(700) (PSI-40). This core complex, derived from native PSI particles, was enriched in Chl a absorbing at 680 and 690 nm and fluorescing with maximal emission at 694 nm at 77 K. PSI particles consisting of the PSI core complex plus 20 to 25 Chl antennae (65 Chl/P(700)) could also be derived from native PSI complexes. These preparations were enriched in Chl a forms absorbing at 697 nm and exhibited a 77 K fluorescence emission maximum at 722 nm.A comparison of native PSI particles which contain 110 Chl/P(700) (PSI-110) and PSI particles containing 65 Chl/P(700) (PSI-65) provides evidence for the existence of a peripheral Chl-protein complex tightly associated in the native PSI complex. The native PSI subunits contain polypeptides of 22,500 to 24,500 daltons which are not found in the PSI-65 or PSI-40 subfractions. It is suggested that these polypeptides function to bind 40 to 45 Chl per structural complex, including the Chl which emits fluorescence at 736 nm.A model for the organization of Chl forms is presented in which the native PSI membrane subunit consists of a reaction center core complex plus two regions of associated light-harvesting antennae. The presence of energy "sinks" within the antennae is discussed.
This chapter discusses the detection and isolation of P700. The pigment designated P700 has been detected in all normal photosynthetic algae and green plants studied. It occurs in small concentrations (1/200–1/1000) relative to the bulk chlorophyll(s) and is characterized by absorption bands around 430 and 700 nm. The location of these bands at slightly longer wavelengths than those of the light-harvesting chlorophyll a, and the similar solubility properties of the two pigments suggest that P700 is a form of chlorophyll a segregated as a result of environmental influences. To measure P700 by means of light-dark spectroscopy, a monochromatic (700 or 430 nm) detecting light (Idet) is passed through a sample and monitored by a photocell. The change of transmission of Idet is observed upon illumination of the sample by a second actinic light (Iact), which is blocked from the photocell. Chemically induced P700 spectra are most conveniently measured in preparations enriched in P700 but can also be obtained using sonicated chloroplast or cell preparations.
The ordering of the zero field triplet spin sublevels in several monomeric chlrophylls has been determined by magneto- photoselection techniques. For all the chlorophylls which we have examined D > 0. The sign of E changes for the chlorophylls with a chlorin ring structure (i.e. chlorophyll a and related molecules) compared to those with a tetrahydroporphin ring structure (i.e., bacteriochlorophyll a and related molecules).
Photosystem II particles which retained high rates of herbicide-sensitive activity were used to examine the site(s) of action of various herbicides. A polypeptide of 32–34 kdaltons was identified as the triazine-herbicide binding site based upon: (a) parallel loss of atrazine activity and the polypeptide during either trypsin treatment or selective detergent depletion of protein in the Photosystem II complex, and (b) covalent labeling of the polypeptide by a 14C-labeled photoaffinity triazine.In Photosystem II particles depleted of the 32–34-kdalton polypeptide, electron transport was still active and was slightly sensitive to DCMU and largely sensitive to dinoseb (urea and nitrophenol herbicides, respectively). On the basis of this result it is proposed that the general herbicide binding site common to atrazine, DCMU and dinoseb is formed by a minimum of two polypeptides which determine affinity and/or mediate herbicide-induced inhibition of electron transport on the acceptor side of Photosystem II.
The low temperature EPR signal of the excited triplet state of bacteriochlorophyll has been quantitatively studied in reaction centers from Rhodopseudomonas spheroides (carotenoid free R 26 mutant). Using laser flash excitation the light saturation curve of the triplet signal has been compared with that of the free-radical formation due to photooxidation of P870 under identical optical conditions. This comparison shows that the quantum yield of triplet formation is nearly the same as that of the photochemical bleaching of bacteriochlorophyll.
Photooxidation of P700 at cryogenic temperatures is coupled to the photo-reduction of two membrane-bound iron-sulphur centres. This process is irreversible over short time periods at 20K. We have now shown that if the iron-sulphur centres are chemically reduced before freezing, P700 photo-oxidation is no longer coupled to reduction of the iron-sulphur centres. This photooxidation is completely reversible. We therefore conclude that the iron-sulphur centres are secondary electron acceptors and that an unknown primary electron acceptor transfers electrons from P700 to the iron-sulphur centres.
The Photosystem I primary reaction, as measured by electron paramagnetic resonance changes of P-700 and a bound iron-sulfur center, has been studied at 15 degrees K in P-700-chlorophyll alpha-protein complexes isolated from a blue-green alga. One complex, prepared with sodium dodecyl sulfate shows P-700 photooxidation only at 300 degrees K, whereas a second complex, prepared with Triton X-100, is photochemically active at 15 degrees K as well as at 300 degrees K. Analysis of these two preparations shows that the absence of low-temperature photoactivity in the sodium dodecyl sulfate complex reflects a lack of bound iron-sulfur centers in this preparation and supports the assignment of an iron-sulfur center as the primary electron acceptor of Photosystem I.
We report light-induced electron paramagnetic resonance triplet spectra from samples of chloroplasts or digitonin photosystem I particles that depend upon the dark redox state of the bound acceptors of photosystem I. If the reaction centers are prepared in the redox state P-700 A X- FdB-FdA-, then upon illumination at 11K we observe a polarized chlorophyll triplet species which we interpret as arising from radical pair recombination between P-700+ and A-. This chlorophyll triplet is apparently the analog of the PR state of photosynthetic bacteria [Parson, W.W. & Cogdell, R.J. (1975) Biochim. Biophys. Acta 416, 105-149]. If the reaction centers are prepared in the dark redox state P-700 A X FdB-FdA-, then upon illumination at 11K we observe a different triplet species of uncertain origin, possibly pheophytin or carotenoid. This species is closely associated with the photosystem I reaction center and it traps excitation when P-700 is oxidized.
Triton-solubilized Photosystem I particles from spinach chloroplasts exhibit largely reversible P-700 absorption changes over the temperature range from 4.2 K to room temperature. For anaerobic samples treated with dithionite and neutral red at pH 10 and illuminated during cooling, a brief (1 microseconds) saturating flash produces absorption changes in the long wavelength region that decay in 0.95 +/- 0.2 ms from 4.2 to 50 K. Above 80 K a faster (100 +/- 30 microseconds) component dominates in the decay process, but this disappears again above about 180 K. The major decay at temperatures above 200 K occurs in about 1 ms. The difference spectrum of these absorption changes between 500 and 900 nm closely resembles that of P-700. Using ascorbate and 2.6-dichlorophenolindophenol as the reducing system with a sample of Photosystem I particles cooled in darkness to 4.2 K, a fully reversible signal is seen upon both the first and subsequent flashes. The decay time in this case is 0.9 +/- 0.3 ms.
Flash-induced absorption changes of Triton-solubilized Photosystem I particles from spinach were studied under reducing and/or illumination conditions that serve to alter the state of bound electron acceptors. By monitoring the decay of P-700 following each of a train of flashes, we found that P-430 or components resembling it can hold 2 equivalents of electrons transferred upon successive illuminations. This requires the presence of a good electron donor, reduced phenazine methosulfate or neutral red, otherwise the back reaction of P-700+ with P-430 occurs in about 30 ms. If the two P-430 sites, designated Centers A and B, are first reduced by preilluminating flashes or chemically by dithionite under anaerobic conditions, then subsequent laser flashes generate a 250 microseconds back reaction of P-700+, which we associate with a more primary electron acceptor A2. In turn, when A2 is reduced by background (continuous) illumination in presence of neutral red and under strongly reducing conditions, laser flashes then produce a much faster (3 microseconds) back reaction at wavelengths characteristic of P-700. We associate this with another more primary electron acceptor, A1, which functions very close to P-700. The organization of these components probably corresponds to the sequence P-700-A1-A2-P-430[AB]. The relation of the optical components to acceptor species detected by EPR, by electron-spin polarization or in terms of peptide components of Photosystem I is discussed. Preliminary experiments with broken chloroplasts suggest that an analogous situation occurs there, as well.
Abstract— …After a short-term solubilization with sodium dodecyl sulphate, chloroplast membranes of tobacco were separated by polyacrylamide gel electrophoresis into three chlorophyll-protein complexes. In addition to the two major complexes termed I and IIc corresponding respectively to P700 chlorophyll a-protein and light-harvesting chlorophyll a/b-protein described by Thornber (1975), a relatively stable complex termed IIa has been observed. This new complex has an apparent molecular weight of 70,000 daltons and possesses Chl a and b.
Complexes I, IIa and IIC have been isolated and precise spectroscopic analyses have been performed. Fourth derivative analyses of low temperature absorption spectra suggest that complex IIa seems more representative than IIC of chlorophyll a forms present in intact thylakoid membranes.
Moreover, the electrophoretic study reveals that CPI and CPII are composed of only one polypeptidic subunit with respective molecular weights of 68,000 and 24,000 daltons.
The photochemical reduction of pheophytin and bacteriopheophytin has been shown in vitro [l-3] . In reaction centers of photosynthetic bacteria bacteriopheophytin a [4-91 and bacteriopheophytin b [ 10, 1 l] act as an intermediary electron carrier between bacteriochlorophyll dimer and the ‘primary’ electron acceptor, a complex of ubiquinone and Fe. When the ubiquinone is in the reduced form the photoaccumulation of reduced bacteriopheophytin can be observed [5-111. In various species of green plants 1.5-2.3 molecules of pheophytin have been found per 100 molecules of chlorophyll [ 121 . In photosystem II of green plants the photoreduction of the primary electron acceptor, Q (plastoquinone), is accompanied by a blue shift of absorption bands at 54.5 nm and 685 nm [13,14] which can belong to a bound or aggregated form of pheophytin in reaction centers of photosystem II [ 141. The photoreduction of pheophytin may be observed in photosystem II preparations from pea chloroplasts at 20°C [ 151. In this work a reversible reduction of pheophytin in the primary light reaction of photosystem II in pea subchloroplast particles at redox potentials (Eh) from -50 mV to -550 mV (when Q is in the reduced form) is demonstrated. This photoreaction is observed at -170°C as well as at 20°C and is accompanied by a 2-3 fold decrease in the chlorophyll fluorescence yield.
In preparations of photochemical reaction centers from Rhodopseudomonas spheroides R-26, lowering the recox potential so as to reduce the primary electron acceptor prevents the photochemical transfer of an electron from bacteriochlorophyll to the acceptor. Measuring absorbance changes under these conditions, we found that a 20-ns actinic flash converts the reaction center to a new state, P-F, which then decays with a half-time that is between 1 and 10 ns at 295 degrees K. At 25 degrees K, the decay half-time is approx. 20 ns. The quantum yield of state P-F appears to be near 1.0, both at 295 and at 15 degrees K. State P-F could be an intermediate in the photochemical electron-transfer reaction which occurs when the acceptor is in the oxidized form. Following the decay of state P-F, we detected another state, P-R, with a decay half-time of 6 mus at 295 degrees K and 120 mus at 15 degrees K. The quantum yield of state P-R is approx. 0.1 at 295 degrees K, but rises to a value nearer 1.0 at 15 degrees K. The kinetics and quantum yields are consistent with the view that state P-R forms from P-F. State P-R seems likely to be a side-product, rather than an intermediate in the electron-transfer process. The decay kinetics indicate that state P-F cannot be identical with the lowest excited singlet state of the reaction center. One of the two states, P-F or P-R, probably is the lowest excited triplet state of the reaction center, but it remains unclear which one.
An electron paramagnetic resonance signal was observed at 25 degrees K in whole spinach chloroplasts after illumination at 77 degrees K. The light-induced epr spectrum had g-values (g(x) = 1.86, g(y) = 1.94, g(z) = 2.05) and a temperature dependence that were characteristic of the reduced state of a plant-type ferredoxin. The light-induced epr spectrum was also observed in broken spinach chloroplasts from which soluble ferredoxin was removed. Chemical analyses showed that both whole and broken spinach chloroplasts contained amounts of nonheme iron and "acid-labile sulfide" consistent with the presence of a bound iron-sulfur protein, at a level of about one molecule per 75 chlorophyll molecules. These results support the conclusion that chloroplasts contain a bound ferredoxin that may serve as a primary low-potential electron acceptor in photosynthesis.
ESR studies at liquid helium temperatures have been conducted on chromatophore and subchromatophore preparations from Chromatium D. If the primary electron acceptor of reaction center bacteriochlorophyll is chemically in a reduced state before illumination, the light activated exited state bacteriochlorophyll is prevented from undergoing oxidation. This is evidenced under these conditions by the absence of the familiar g ≅ 2 signal. Instead, a new ESR spectrum is generated in the light. This is comprised of both absorption and emission bands. The oxidation-reduction potential dependence and kinetics of the ESR changes, activated by laser pulses, suggest the signals represent bacteriochlorophyll in the triplet state. This state could be a primary intermediate in the early light activated transitions of photosynthesis.
The presence of a bound electron transport component in spinach chloroplasts with an EPR spectrum characteristic of a ferredoxin has been confirmed. The ferredoxin is photoreduced at 77 °K or at room temperature, it is not reduced in the dark by Na2S2O4. The distribution of the ferredoxin in subchloroplast particles has been investigated. The ferredoxin is enriched in Photosystem I particles and it is proposed that it functions as primary electron acceptor for Photosystem I.The EPR spectra indicate the presence of two components which are photoreduced sequentially. It is proposed that they may represent two active centres of a single protein.
Triplet states of reaction center (bacterio-) chlorophylls have been observed by low temperature EPR in a wide variety of photosynthetic preparations ranging from whole cells to purified reaction centers. The reaction center triplet state is seen under conditions where electron transfer from the reaction center chlorophyll is prevented by reduction of the primary electron acceptor. The redox potential dependence shows one-electron titrations with midpoint potentials of −50 mV in Rhodopseudomonas spheroides and approx. −120±20 mV in R. rubrum and Chromatium D. Kinetic measurements with bacterial reaction centers show an approx. 6-μs triplet decay time. Observed zero-field splitting parameters D and E are appreciably lower in the reaction center triplets than in isolated chlorophyll, suggesting that the reaction center photo-active (bacterio-) chlorophyll is not monomeric. EPR lineshapes of the triplet signals (equal mixtures of microwave absorption and emission) show almost complete spin polarization demonstrating that inter-system crossing is a highly selective process.
A comparison has been made between Signal I, the photo-electron spin resonance signal associated with the primary light conversion act in photosynthesis, and free-radical signals generated in various chlorophyll species in vitro. The esr signals obtained from chlorophyll.monomer, (Chl.L)(+.), chlorophyll dimer, (Chl(2))(+.), and chlorophyll oligomer, (Chl(2))(n) (+.), are broader than Signal I, whereas the chlorophyll-water adduct, (Chl.H(2)O)(n) (+.), gives a signal very much narrower than Signal I. The unusually narrow signal from (Chl.H(2)O)(n) (+.) has been ascribed to spin migration, or to unpaired spin delocalization over a large number of chlorophyll molecules. The linewidth of Signal I can be accounted for by a similar delocalization process. A theoretical relationship between the esr linewidth and the number of chlorophyll molecules, N, over which an unpaired spin is delocalized, takes the form DeltaH(N) = 1/ radicalN.DeltaH(M), where DeltaH(M) is the linewidth of monomer (Chl.L)(+.). This relationship for N = 2 accounts well for the linewidths of Signal I in green algae, blue-green algae, and photosynthetic bacteria in both the (1)H- and (2)H-forms. The linewidth of Signal I (as well as the optical properties of reaction-center chlorophyll) are consistent with unpaired spin delocalization over an entity containing two chlorophyll molecules, (Chl.H(2)O.Chl)(+.).
A light-induced spin-polarized triplet state has been detected in a purified Photosystem II preparation by electron paramagnetic resonance spectroscopy at liquid helium temperature. The electron spin polarization pattern is interpreted to indicate that the triplet originates from radical pair recombination between the oxidized primary donor chlorophyll, P-680+, and the reduced intermediate pheophytin, I-, as has been previously demonstrated in bacterial reaction centers. The dependence of the triplet signal on the redox state of I and the primary acceptor, Q, are consistent with the origin of the triplet signal from the triplet state of P-680. Redox-poising experiments indicate the presence of an endogenous donor (or donors) which operates at 3-5 K and 200 K. The zero field-splitting parameters of the triplet are very similar to those of monomeric chlorophyll a however, this alone does not allow a distinction to be made between monomeric and dimeric structures for P-680.
Magnesium tetraphenylchlorin, a synthetic model for chlorophyll, exhibits significant variations in the unpaired spin densities of its cation radicals with concomitant changes in oxidation potentials as a function of solvent and axial ligand. Similar effects are observed for chlorophyll (Chl) a and its cation radicals. Oxidation potentials for Chl --> Chl(+.) as high as +0.9 V (against a normal hydrogen electrode) are observed in nonaqueous solvents, with linewidths of the electron spin resonance signals of monomeric Chl(+.) ranging between 9.2 and 7.8 G in solution. These changes in electronic configuration and ease of oxidation are attributed to mixing of two nearly degenerate ground states of the radicals theoretically predicted by molecular orbital calculations. Comparison of the properties of chlorophyll in vitro with the optical, redox, and magnetic characteristics attributed to P-680, the primary donor of photosystem II which mediates oxygen evolution in plant photosynthesis, leads us to suggest that P-680 may be a ligated chlorophyll monomer whose function as a phototrap is determined by interactions with its (protein?) environment.
Triton-fractionated photosystem-I particles poised at -625 mV, where the two bound iron-sulfur proteins are reduced, have been studied by optical and electron paramagnetic resonance spectroscopies from 293 to 5 K. At 5-9 K, these particles exhibit two decay components with lifetimes of 1.3 and 130 msec in the laser pulse-induced absorption and electron paramagnetic resonance signal changes. Spectral properties of the 130-msec decay component reflect the charge separation between P-700 and some iron-sulfur center having a broad optical absorbance in the 400- to 550-nm region and a previously reported electron paramagnetic resonance signal with g = 1.78, 1.88, and 2.08. Spectral properties of the 1-msec decay component indicate photoinduced charge separation between P-700 and a chlorophyll a dimer having absorption bands at 420, 450, and 700 nm. It is assumed that these two acceptors participate in the electron transfer from P-700(*) to the bound iron-sulfur proteins.