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Carotenoid Oxidation in Photosystem II†
Abstract
The oxidation of carotenoid upon illumination at low temperature has been studied in Mn-depleted photosystem II (PSII) using EPR and electronic absorption spectroscopy. Illumination of PSII at 20 K results in carotenoid cation radical (Car+*) formation in essentially all of the centers. When a sample which was preilluminated at 20 K was warmed in darkness to 120 K, Car+* was replaced by a chlorophyll cation radical. This suggests that carotenoid functions as an electron carrier between P680, the photooxidizable chlorophyll in PSII, and ChlZ, the monomeric chlorophyll which acts as a secondary electron donor under some conditions. By correlating with the absorption spectra at different temperatures, specific EPR signals from Car+* and ChlZ+* are distinguished in terms of their g-values and widths. When cytochrome b559 (Cyt b559) is prereduced, illumination at 20 K results in the oxidation of Cyt b559 without the prior formation of a stable Car+*. Although these results can be reconciled with a linear pathway, they are more straightforwardly explained in terms of a branched electron-transfer pathway, where Car is a direct electron donor to P680(+), while Cyt b559 and ChlZ are both capable of donating electrons to Car+*, and where the ChlZ donates electrons when Cyt b559 is oxidized prior to illumination. These results have significant repercussions on the current thinking concerning the protective role of the Cyt b559/ChlZ electron-transfer pathways and on structural models of PSII.
... Reduced cyt b 559 can be oxidized by P þ 680 through a sequential or branched pathway involving a carotenoid and a special chlorophyll denoted Chl Z [41,[46][47][48][49]. This pathway involves slow electron transfer steps and comes into play only when electron transfer from Y Z for some reason is inactivated or slowed down. ...
... In these reactions, Y red D is oxidized by the S 2 and S 3 states while Y Å D is reduced by the S 0 state [25,28]. Cyt b 559 is oxidized by P þ 680 (reactions 4) when the manganese cluster is inactive at low temperatures, during photoactivation or when it is absent [46][47][48][49][50]. This reaction involves a carotenoid and chlorophyll Z . ...
... Thus, once the equilibria between WOC (in the S 2 or S 3 states) and P 680 create an oxidizing equivalent on P 680 , this is likely to oxidize Y Å D in a faster reaction. Cyt b 559 is also in contact [25,36] with these components and is known to reduce P þ 680 via the carotenoidchlorophyll Z pathway [46][47][48][49]64]. It is likely that the slow reduction of Y ox D by cyt b 559 observed at )80°C [52], also involves this pathway. ...
We have investigated the electron transfer from reduced tyrosine Y D (YDred) and cytochrome b559 to the S2 and S3 states of the water oxidizing complex (WOC) in Photosystem II. The EPR signal of oxidized cyt b559, the S 2 state multiline EPR signal and the EPR signal from Y D· were measured to follow the electron transfer to the S2 and S3 states at 245 and 275 K. The majority of the S2 centers was reduced directly from YDred but at 245 K we observed oxidation of cyt b559 in about 20% of the centers. Incubation of the YDredS3 state resulted in biphasic changes of the S2 multiline signal. The signal first increased due to reduction of the S3 state. Thereafter, the signal decreased due to decay of the S2 state. In contrast, the YD· signal increased with a monophasic kinetics at both temperatures. Again, we observed oxidation of cyt b559 in about 20% of the PSII centers at 245 K. This oxidation correlated with the decay of the S2 state. The complex changes can be explained by the conversion of YDredS3 centers (present initially) to YD·S1 centers, via the intermediate YD·S2 state. The early increase of the S2 state multiline signal involves electron transfer from Y Dred to the S3 state resulting in formation of YD·S2. This state is reduced by cyt b559 resulting in a single exponential oxidation of cyt b 559. Taken together, these results indicate that the electron donor to S2 is cyt b559 while cyt b559 is unable to compete with YDred in the reduction of the S3 state in the pre-reduced samples. We also followed the decay of the S 2 and S3 states and the oxidation of cyt b559 in samples where YD was oxidized from the start. In this case cyt b559 was able to reduce both the S2 and the S3 states suggesting that different pathways exist for the electron transfer from cyt b559 to the WOC. The activation energies for the Y DredS2→YD·S1 and YDredS 3→YD·S2 transformations are 0.57 and 0.67 eV, respectively, and the reason for these large activation energies is discussed.
... transport cyclique des e -, CEF, autour du PSII et de ce fait pourrait protéger le PSII contre les effets néfastes de photodommage provoqué par la lumière élevée et la photoinhibition (Allakhverdiev et al., 1997;Stewart and Brudvig, 1998;Hanley et al., 1999;Carpentier, 1999;Tracewell and Brudvig, 2008;Allakhverdiev et al., 2008;Essemine et al., 2012b). ...
... En se basant sur leurs positions, il parait qu'ils peuvent avoir des fonctions différentes. Ils participent pour quencher le taux d'oxygène singulet produit suite au phénomène de recombinaison du charge à l'état triplet de P680* (Telfer et al., 1994), ou pourraient agir comme des transporteurs secondaires des edans le cas où la donation des epar l'eau n'est pas fonctionnelle et ainsi dans le cas de danger de l'accumulation de P680 + , ils jouent un rôle de protection (Hanley et al., 1999;Telfer et al., 2002 andMartinez-Junza et al., 2008;Litvín et al., 2008). (Tracewell et al., 2001, Tracewell and Brudvig, 2003. ...
Effet du stress thermique sur le transport cyclique des électrons autour du PSI dans des mutants d'Arabidopsis thaliana déficients dans certaines voies cycliques (crr2-2 et pgr5) et dans d'autres mutants défectueux en digalactosyle-diacylglycerole, DGDG (dgd1-2 et dgd1-3)
... The molecular organization of Cyt b559 is revealed in the crystal structures of the PS II complexes from cyanobacteria Thermosynechococcus (T) elongatus and T. vulvanus [5][6][7][8][9][10], red alga Cyanidium caldarium [11] and spinach [12]. It is considered that Cyt b559 operates to protect the PS II complex from photoinhibition in unfavorable environmental conditions by providing a pathway of backflow of electrons to the oxidizing equivalents at the donor side of PS II -cycling around PS II [13][14][15][16][17][18][19][20][21][22][23][24][25] (reviewed in Refs. [1][2][3][4]26]). ...
... Cyt b559 is a terminal reductant for oxidized P680 [13][14][15][16]18,21,22,25,60] and gets rapidly rereduced from PQH 2 of the pool [61][62][63][64][65][66]. It is proposed that fast redox equilibrium between Cyt b559 and plastoquinol of the pool is established via, yet unidentified in the structure, quinone binding site located near Cyt b559 called Q D [66]. ...
Transformation of three-component redox pattern of cytochrome (Cyt) b559 in PS II membrane fragments upon various treatments is manifested in decrease of the relative content (R) of the high potential (HP) redox form of Cyt b559 and concomitant increase in the fractions of the two lower potential forms. Redox titration of Cyt b559 in different types of PS II membrane preparations was performed and revealed that (1) alteration of redox titration curve of Cyt b559 upon treatment of a sample is not specific to the type of treatment; (2) each value of RHP defines the individual shape of the redox titration curve; (3) population of Cyt b559 may exist in several stable forms with multicomponent redox pattern: three types of three-component redox pattern and one type of two-component redox pattern as well as in the form with a single Em; (4) transformation of Cyt b559 proceeds as successive conversion between the stable forms with multicomponent redox pattern; (5) upon harsh treatments, Cyt b559 abruptly converts into the state with a single Em which value is intermediate between the Em values of the two lower potential forms. Analysis of the data using the model of Cyt b559-quinone redox interaction revealed that diminution of RHP in a range from 80 to 10% reflects a shift in redox equilibrium between the heme group of Cyt b559 and the interacting quinone, due to a gradual decrease of 90 mV in Em of the heme group at the virtually unchanged Em of the quinone component.
... These compounds absorb in the ultra-violate range (235 nm and 270 nm respectively) where the absorptions are detected using spectrophotometric techniques so as to evaluate the oxidation level [45,46]. This way of conjugated dienes and peroxides determination have been used in a number of studies even though they do have of their own weaknesses [47][48][49][50]. ...
... These compounds absorb in the ultra-violate range (235 nm and 270 nm respectively) where the absorptions are detected using spectrophotometric techniques so as to evaluate the oxidation level [45,46]. This way of conjugated dienes and peroxides determination have been used in a number of studies even though they do have of their own weaknesses [47][48][49][50]. ...
... In our case, the stimulation of H 2 O 2 photoproduction in the PSII preparations induced by the injury of the WOC was mainly due to the increase in the O 2 −• production on the acceptor side of PSII. This conclusion has been made based on the following observations: (1) and TyrZ • in the absence of an Mn cluster can be chlorophylls and carotenoids (their photooxidation has been shown in several works [38][39][40][41][42]), lipids in the lipid belt around D1 and D2 (their presence in the RC has been demonstrated [2,43]), the amino acid residues involved in coordination of the Mn 4 CaO 5 cluster [3], and His located in the vicinity of TyrZ. Apparently, the changes of the acceptor side caused by the modification of the WOC facilitate the photoproduction of O 2 −• . ...
The photoproduction of superoxide anion radical (O2−•) and hydrogen peroxide (H2O2) in photosystem II (PSII) preparations depending on the damage to the water-oxidizing complex (WOC) was investigated. The light-induced formation of O2−• and H2O2 in the PSII preparations rose with the increased destruction of the WOC. The photoproduction of superoxide both in the PSII preparations holding intact WOC and the samples with damage to the WOC was approximately two times higher than H2O2. The rise of O2−• and H2O2 photoproduction in the PSII preparations in the course of the disassembly of the WOC correlated with the increase in the fraction of the low-potential (LP) Cyt b559. The restoration of electron flow in the Mn-depleted PSII preparations by exogenous electron donors (diphenylcarbazide, Mn2+) suppressed the light-induced formation of O2−• and H2O2. The decrease of O2−• and H2O2 photoproduction upon the restoration of electron transport in the Mn-depleted PSII preparations could be due to the re-conversion of the LP Cyt b559 into higher potential forms. It is supposed that the conversion of the high potential Cyt b559 into its LP form upon damage to the WOC leads to the increase of photoproduction of O2−• and H2O2 in PSII.
... Under these conditions, the side-path donors expected to be oxidized are either Car, Chl Z or Cytb 559 and the dominant reduced electron acceptor formed is Q A − , e.g. [38][39][40]. The light-minus-dark absorption spectra recorded at~77 K from 350 nm to 800 nm is shown in Fig. S2B. ...
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.
... ChlZ D1 and ChlZ D2 are each in direct contact with a beta-carotene molecule, known as Car D1 and Car D2 respectively, seen using crystallography first by Ferreira et al. (2004) and Loll, Kern, Saenger, Zouni, and Biesiadka (2005), but detected and characterized by spectroscopy well before that; see for example (Hanley, Deligiannakis, Pascal, Faller, & Rutherford, 1999;Kwa, Newell, van Grondelle, & Dekker, 1992;Noguchi, Mitsuka, & Inoue, 1994). The position of Car D1 and Car D2 differs in that the former is positioned perpendicular to the membrane plane while the latter is parallel to the membrane plane: ...
Photosystem II is a photochemical reaction center that catalyzes the light‐driven oxidation of water to molecular oxygen. Water oxidation is the distinctive photochemical reaction that permitted the evolution of oxygenic photosynthesis and the eventual rise of eukaryotes. At what point during the history of life an ancestral photosystem evolved the capacity to oxidize water still remains unknown. Here, we study the evolution of the core reaction center proteins of Photosystem II using sequence and structural comparisons in combination with Bayesian relaxed molecular clocks. Our results indicate that a homodimeric photosystem with sufficient oxidizing power to split water had already appeared in the early Archean about a billion years before the most recent common ancestor of all described Cyanobacteria capable of oxygenic photosynthesis, and well before the diversification of some of the known groups of anoxygenic photosynthetic bacteria. Based on a structural and functional rationale, we hypothesize that this early Archean photosystem was capable of water oxidation to oxygen and had already evolved protection mechanisms against the formation of reactive oxygen species. This would place primordial forms of oxygenic photosynthesis at a very early stage in the evolutionary history of life.
... PSII activity, measured as the rates of Q A reduction at 293 (Fig. 2B, inset) and 77 K (Fig. 2B) and as the rate of b-carotene cation radical formation at 15 K (18,19), were comparable with red and far-red excitation ( Fig. 2C and fig. S3). ...
Lower-energy photons do the work, too
Plants and cyanobacteria use chlorophyll-rich photosystem complexes to convert light energy into chemical energy. Some organisms have developed adaptations to take advantage of longer-wavelength photons. Nürnberg et al. studied photosystem complexes from cyanobacteria grown in the presence of far-red light. The authors identified the primary donor chlorophyll as one of a few chlorophyll molecules in the far-red light–adapted enzymes that were chemically altered to shift their absorption spectrum. Kinetic measurements demonstrated that far-red light is capable of directly driving water oxidation, despite having less energy than the red light used by most photosynthetic organisms.
Science , this issue p. 1210
... The kinetics of Tyr D and Tyr Z oxidation were measured by monitoring the EPR signal induction at 3465 G (arrow in Fig. 1) under continuous illumination at three different pH values (Fig. 2). The field position chosen for kinetic measurement is lying outside of the magnetic field range in which signals from Chl and Car cations could contribute (Visser et al. 1977;Hanley et al. 1999). ...
Photosystem II (PS II) contains two redox-active tyrosine residues on the donor side at symmetrical positions to the primary donor, P680. TyrZ, part of the water-oxidizing complex, is a preferential fast electron donor while TyrD is a slow auxiliary donor to P680⁺. We used PS II membranes from spinach which were depleted of the water oxidation complex (Mn-depleted PS II) to study electron donation from both tyrosines by time-resolved EPR spectroscopy under visible and far-red continuous light and laser flash illumination. Our results show that under both illumination regimes, oxidation of TyrD occurs via equilibrium with TyrZ• at pH 4.7 and 6.3. At pH 8.5 direct TyrD oxidation by P680⁺ occurs in the majority of the PS II centers. Under continuous far-red light illumination these reactions were less effective but still possible. Different photochemical steps were considered to explain the far-red light-induced electron donation from tyrosines and localization of the primary electron hole (P680⁺) on the ChlD1 in Mn-depleted PS II after the far-red light-induced charge separation at room temperature is suggested.
Electronic supplementary material
The online version of this article (doi:10.1007/s11120-017-0442-3) contains supplementary material, which is available to authorized users.
... [50] The position of ChlZ-D2 in between cytochrome b 559 and a -carotene (serving as an electron donor under stress condition) close to the P680, makes this chlorophyll a perfect candidate in order to fulfill this function of alternative electron donor. [49,51] The unusual large fluctuation in the SES found in ChlZ-D2 may have implications in its possible photoprotective role, additionally allowing it to serve as an intermediate excitation energy quencher for di↵erent excitation energy pathways. ...
Light harvesting from the Sun by antenna complexes surrounding the reaction center of Photosystem II represents the first step of the natural oxygenic photosynthesis performed by plants, algae and cyanobacteria. The excitation energy derived from the sunlight is absorbed by the chlorophylls of the antenna and subsequently conveyed to the reaction center of Photosystem II through resonant energy transfer mechanisms. In the special pair of chlorophylls of the reaction center the charge separation occurs, eventually leading to the oxidation of water molecules into protons, electrons and molecular oxygen. The adsorption properties of the antenna chlorophylls are ad hoc modulated by the protein environment to guarantee fast energy transfer and minimize side and back reactions. At the same time these properties are influenced by the molecular fluctuations occurring at physiological temperature. In the present work, combining classical molecular dynamics simulations with the Charge Density Coupling method, we estimated the impact of the thermal fluctuations on the site energy shift of the chlorophylls embedded in the Photosystem II complex. Our results show how the effect of the molecular fluctuations is not homogeneous throughout the complex, although the symmetry of the homodimer is maintained. Many peripheral chromophores undergo fluctuations larger then 10 kJ/mol around the average values. Possible physiological roles of such fluctuations are discussed.
... Both peripheral chlorophylls are required for photoautotrophic growth as mutations that impair their binding cannot assemble functional PSII (Lince & Vermaas, 1998;Ruffle et al., 2001). ChlZ D1 and ChlZ D2 are each in direct contact with a beta-carotene molecule, known as Car D1 and Car D2 respectively, seen using crystallography first by Ferreira et al. (2004) and Loll et al. (2005), but detected and characterized by spectroscopy well before that, see for example (Kwa et al., 1992;Noguchi et al., 1994;Hanley et al., 1999). The position of Car D1 and Car D2 differs in that the former is positioned perpendicular to the membrane plane while the latter is parallel to the membrane plane: however, one of the beta-rings of each carotenoid links to ChlZ D1 and ChlZ D2 via strictly conserved tryptophan residues (D1-W105 and D2-W104, respectively), located in the unique protein fold between the 1 st and 2 nd transmembrane helices described above, and therefore absent in L and M. ...
The evolution of Photosystem II changed the history of life by oxygenating the Earth's atmosphere. However, there is currently no consensus on when and how oxygenic photosynthesis originated. Here we present a new perspective on the evolution of oxygenic photosynthesis by studying the evolution of D1 and D2, the core subunits of Photosystem II, as a function of time. A Bayesian relaxed molecular clock approach was applied to the phylogeny of Type II reaction center proteins using geochemical constraints and calibrations derived from the fossil record of Cyanobacteria and photosynthetic eukaryotes. The resulting age estimates were interpreted in the context of the structure and function of photochemical reaction centers. Firstly, we point out it is likely that the ancestral protein to D1 and D2 gave rise to a photosystem that was capable of water oxidation. Secondly, our results indicate that the gene duplication event that led to the divergence of D1 and D2 is likely to have occurred more than a billion years before the emergence of the last common ancestor of extant Cyanobacteria. Thirdly, we show that it is unlikely that Cyanobacteria obtained photosynthesis via horizontal gene transfer. Furthermore, the data strongly suggest that the origin of photosynthesis in the early Archaean was necessarily followed by a rapid diversification of reaction center proteins, which included the divergence of D1 and D2. It is therefore concluded that primordial forms of water oxidation could have originated relatively soon after the emergence of photosynthesis.
... This would be possible if the 1-electron redox potential of the added b-Car is a more negative than that of the Edge et al. 2000;Ishikita and Knapp 2005) have been reported to transfer electrons only to P680 ? , the strongest oxidant of PS II (E m 0 & 1.1 V; Hanley et al. 1999;Vrettos et al. 1999) and not, of course, to plastoquinones. A direct reduction, therefore, of Q A and of intersystem plastoquinones by exogenously added b-Car seems highly unlikely. ...
Singlet-excited oxygen (1O
2*) has been recognized as the most destructive member of the reactive oxygen species (ROS) which are formed during oxygenic photosynthesis by plants, algae, and cyanobacteria. ROS and 1O
2* are known to damage protein and phospholipid structures and to impair photosynthetic electron transport and de novo protein synthesis. Partial protection is afforded to photosynthetic organism by the β-carotene (β-Car) molecules which accompany chlorophyll (Chl) a in the pigment-protein complexes of Photosystem II (PS II). In this paper, we studied the effects of exogenously added β-Car on the initial kinetic rise of Chl a fluorescence (10–1000 μs, the OJ segment) from the unicellular cyanobacterium Synechococcus sp. PCC7942. We show that the added β-Car enhances Chl a fluorescence when it is excited at an intensity of 3000 μmol photons m−2 s−1 but not when excited at 1000 μmol photons m−2 s−1. Since β-Car is an efficient scavenger of 1O
2*, as well as a quencher of 3Chl a
* (precursor of 1O
2*), both of which are more abundant at higher excitations, we assume that the higher Chl a fluorescence in its presence signifies a protective effect against photo-oxidative damages of Chl proteins. The protective effect of added β-Car is not observed in O2-depleted cell suspensions. Lastly, in contrast to β-Car, a water-insoluble molecule, a water-soluble scavenger of 1O
2*, histidine, provides no protection to Chl proteins during the same time period (10–1000 μs).
... . This mechanism occurs only when there is overlap of the wave functions of the D and the A. Dexter's mechanism is dominant in the triplettriplet ET and hence plays an important function in photosynthesis (the photoprotective role of Cars is realized through 3 Chl → 3 Cars ET). Contrary to the Förster's mechanism, the ET rate constant is independent from the oscillator strength of both transitions (this is evident from the normalization conditions) (32). ...
Photosynthesis is the primary process by which energy is fed into the biological world. In its course, complex machinery performs highly effective transformation of light energy. Quite generally, the process consists of two parts: light-dependent, including the reactions necessary for the conversion of light into chemical energy (ATP) and reducing power (NADPH), and carbon-fixation, where the latter compounds are used to incorporate CO2 into simple sugars. The major part of the photosynthetic organisms utilizes water as a main electron source in a process called oxygenic Photosynthesis.
The significance and complexity of photosynthesis have been a matter of systematic research from the pure molecular mechanisms up to the physiological and even ecological aspects. The results from these studies find extensive application in fields like agronomy and environmental protection. Moreover, in the light of the global warming and the energy crisis faced by humanity, the detailed understanding of photosynthesis becomes crucial not only for the preservation of the vulnerable ecosystems, but also for the prevention of the world economy collapse. In this respect, a large field related to photosynthesis research deals with the design and development of eco-friendly light energy conversion systems mimicking the photosynthetic apparatus.
In order to precisely understand Nature’s engineering approaches the working mechanism of each part of the photosynthetic apparatus has to be studied in detail. In this regard, the subject of the current work is one of the main participants in the light-dependent phase of oxygenic photosynthesis, Photosystem I (PS I). This complex carries an immense number of cofactors: chlorophylls (Chl), carotenoids, quinones, etc, which together with the protein entity exhibit several exceptional properties. First, PS I has an ultrafast light energy trapping kinetics with a nearly 100% quantum efficiency. Secondly, both of the electron transfer branches in the reaction center are suggested to be active. Thirdly, there are some so called 'red' Chls in the antenna system of PS I, absorbing light with longer wavelengths than the reaction center. These 'red' Chls significantly modify the trapping kinetics of PS I.
The purpose of this thesis is to obtain better understanding of the above-mentioned, specific features of PS I. This will not merely cast more light on the mechanisms of energy and electron transfer in the complex, but also will contribute to the future developments of optimized artificial light-harvesting systems. In the current work, a number of PS I complexes isolated from different organisms (Thermosynechococcus elongatus, Chlamydomonas reinhardtii, Arabidopsis thaliana) and possessing distinctive features (different macroorganisation – monomers, trimers, monomers with a semibelt of peripheral antenna attached; presence of 'red' Chls) is investigated. The studies are primarily focused on the electron transfer kinetics in each of the cofactor branches in the PS I reaction center, as well as on the effect of the antenna size and the presence of 'red' Chls on the trapping kinetics of PS I. These aspects are explored with the help of several ultrafast optical spectroscopy methods: i) time-resolved fluorescence – single photon counting and synchroscan streak camera; and ii) ultrafast transient absorption. Physically meaningful information about the molecular mechanisms of the energy trapping in PS I is gained with the help of kinetic modeling.
Chapter 1 is a broad background introduction to the major issues in the light energy trapping kinetics (in particular of PS I) that still remain to be elucidated.
Chapter 2 summarizes the main experimental techniques and data analysis strategies used in the current work.
Chapter 3 represents a broad description of one of the methods used here – synchroscan streak camera – for time-resolved detection of fluorescence signals. The chapter covers the main tests that were performed during the installation of the set-up and improvements that were made during this work in order to obtain high quality data with.
Chapter 4 is a thorough investigation of the light energy trapping kinetics in higher plant core and intact PS I particles, and stroma membranes from A thaliana. The kinetic analysis of the experimental data confirms the previously proposed 'charge recombination' model for the trapping kinetics in PS I. No bottleneck in the energy flow from the bulk antenna compartments to the reaction center has been found. For both particles, a trap-limited kinetics is realized, with an apparent charge separation lifetime of about 6 ps. No 'red' Chls are found in the PS I-core complex from A. thaliana. Rather, the observed 'red'-shifted fluorescence (700-710 nm range) originates from the reaction center. In contrast, two 'red' Chls compartments, located in the peripheral light-harvesting complexes, are resolved in the intact PS I particles (decay lifetimes 33 and 95 ps, respectively). These two 'red' states have been attributed to the two 'red' states found in Lhca 3 and Lhca 4 respectively. The influence of the 'red' Chls on the slowing of the overall trapping kinetics in the intact PS I complex is estimated to be approximately four times larger than the effect of the bulk antenna enlargement.
Chapter 5 is a study of the light energy trapping kinetics in cyanobacterial PS I complexes – monomers and trimers isolated from T. elongatus, addressing the same questions as in the previous chapter. It demonstrates the adequacy of the 'charge recombination' model for describing the trapping kinetics. Based on this model the reaction center excited state can be resolved. The overall trapping kinetics in the studied complex is shown to be trap-limited even though the presence of the 'red' Chls induces a substantial slowing down (~60%). Two kinetically different 'red' Chl pools were resolved. Both of these 'red' pools originate from the same groups of pigments in either of the two aggregation states. This indicates that careful separation of the trimers into monomers does not disturb substantially the 'red' Chls and we can thus exclude their location at the monomer-monomer interface. Acceleration of the secondary electron transfer step in the studied complexes as compared to PS I from mesophilic organisms is observed.
Chapter 6 represents a sub-ps time-resolved fluorescence study performed on His-tagged intact PS I core complexes isolated from C. reinhardtii. The higher time-resolution of the experimental set-up used (<1 ps) allows indebt investigation of the intra-antenna excitation energy transfer. In order to account for these processes a new, branched model with two sequentially linked antenna compartments in each branch was used. The model successfully describes the experimental data and delivers valuable information about the spectral properties of the different PS I antenna pools. In addition, the data analysis further confirms the previously proposed ′charge recombination′ model for the description of the trapping kinetics in PS I.
Chapter 7 deals with the branching of the electron transfer reactions in the RC of PS I. The RC is composed of two cofactor branches related by a pseudo-C2 symmetry axis. The ultimate electron donor P700 (a pair of chlorophylls) and the tertiary acceptor FX (FeS cluster), are both located on this axis, while each of the two branches is made up of a pair of chlorophylls (ec2 and ec3) and a phylloquinone. Based on the observed biphasic reduction of FX it has been suggested that both branches in PS I are competent in electron transfer but the nature and rates of the initial electron transfer steps has not been characterized. This part of the current work reports an ultrafast transient absorption study of C. reinhardtii mutants in which specific amino acids forming H-bonds with either ec3A (PsaA-Y696F) or ec3B (PsaB-Y676F) are exchanged. The analysis of the experimental data shows that the rate of primary CS is lowered independently in each of the mutant PS I complexes, providing direct evidence that the primary ET is initiated separately and independently in each branch. Furthermore, the data prove that the initial CS events occur within the ec2/ec3 pairs, generating ec2+-ec3– radical pairs, followed by rapid reduction by P700. The results on this study are of great practical importance since they reveal the solution used by Nature to optimize the light trapping kinetics from large antenna systems.
... The difference spectra in Fig. S3 show the creation of a peak near 980 nm upon illumination clearly due to car + , which is known[11] to be created via low temperature illumination. The absorbance of this peak allows us, from the ε max of car + of 2.2x10 6 M -1 cm -1 to estimate[12] that there is ~0.003 car + per PS II created in this process. Other absorbance changes near 1500 nm are likely to be of vibrational overtone absorptions of the sample, with only a weak feature near 1550 nm appearing in the difference spectrum that is easily associated with cyt b 559 . ...
... It is well accepted that Cyt b 559 is not involved in the linear electron transport chain from water to plastoquinone; however, it participates in the cyclic electron transport around PSII as a protective mechanism during the process of photoinhibition (Shinopoulos and Brudvig 2012). It is generally believed that the HP form of Cyt b 559 avoids oxidation of the PSII electron donor side by donation of an electron to highly oxidizing P680 + from Car D2 and ChlZ D2 (Hanley et al. 1999, Tracewell et al. 2011, whereas the LP form of Cyt b 559 prevents overeduction of the PSII electron acceptor side by acceptance of an electron from pheophytin (Barber and De Las Rivas 1993) or plastoquinol (Buser et al. 1992). In addition to the involvement of Cyt b 559 in the cyclic electron transport around PSII, several lines of evidence have been provided on the enzymatic function of Cyt b 559 (for a review, see Pospíšil 2011). ...
Metal ions play a crucial role in enzymatic reactions in all photosynthetic organisms such as cyanobacteria, algae and plants. It well known that metal ions maintain the binding of substrate in the active site of the metalloenzymes and control the redox activity of enzyme in the enzymatic reaction. A large pigment-protein complex Photosystem II (PSII), known to serve as water-plastoquinone oxidoreductase, contains three metal centers comprising non-heme iron, heme iron of cytochrome b559 and water splitting manganese complex. Metal ions bound to PSII proteins maintain the electron transport from water to plastoquinone and regulate the pro-oxidant and antioxidant activity in PSII. In this review, the attention is focused on the role of PSII metal centers in 1) the formation of superoxide anion and hydroxyl radicals by sequential one-electron reduction of molecular oxygen and the formation of hydrogen peroxide by incomplete two-electron oxidation of water, and 2) the elimination of superoxide anion radical by one-electron oxidation and reduction (superoxide dismutase activity) and hydrogen peroxide by two-electron oxidation and reduction of water (catalase activity). The balance between the formation and elimination of reactive oxygen species by PSII metal centers is discussed as an important aspect in the prevention of photo-oxidative damage of PSII proteins and lipids.
... Under physiological conditions it has been proposed that the population of the photo-induced excited state of some S-states which are part of the OEC turnover, might represent the principal source of photo-oxidative damage both by visible as well as ultraviolet radiation [318,337]. At the same time, under conditions that promote a relatively long lived P 680 + , alternative electron transfer chains, involving one of thecarotene, one of the distal Chls bound to D1-D2 (Chl Z , Fig. 1B) and possibly Cyt b 559 , have been observed within the PSII reaction centre [327,[338][339][340][341][342]. Those might be seen as "safety valves" which can sustain a low yield of electron transfer in PSII when the donor side is impaired and, by reducing P 680 + , lowering the yield of oxidative damage. ...
Oxygenic photosynthesis is indispensable both for the development and maintenance of life on earth by converting light energy into chemical energy and by producing molecular oxygen and consuming carbon dioxide. This latter process has been responsible for reducing the CO2 from its very high levels in the primitive atmosphere to the present low levels and thus reducing global temperatures to levels conducive to the development of life. Photosystem I and photosystem II are the two multi-protein complexes that contain the pigments necessary to harvest photons and use light energy to catalyse the primary photosynthetic endergonic reactions producing high energy compounds. Both photosystems are highly organised membrane supercomplexes composed of a core complex, containing the reaction centre where electron transport is initiated, and of a peripheral antenna system, which is important for light harvesting and photosynthetic activity regulation. If on the one hand both the chemical reactions catalysed by the two photosystems and their detailed structure are different, on the other hand they share many similarities. In this review we discuss and compare various aspects of the organisation, functioning and regulation of plant photosystems by comparing them for similarities and differences as obtained by structural, biochemical and spectroscopic investigations.
Existence of the alternative charge separation pathway in Photosystem II under the far-red light was induced photochemistry in Photosystem II was proposed by us on the basis of induced electron transfer reactions at cryogenic temperature 5 K. Here we extend these studies to the higher temperature range of 77-295 K with help of electron paramagnetic resonance spectroscopy. Induction of the S2 state multiline signal, oxidation of cytochrome b559 and chlorophyllZ was studied in Photosystem II membrane preparations from spinach after application of a laser flashes in visible (532 nm) or far-red (730-750 nm) spectral regions. Temperature dependence of the S2 state multiline signal induction after single flash at 730-750 nm (Tinhibition ~ 240 K) was found to be different than that at 532 nm (Tinhibition ~ 157 K). No contaminant oxidation of the secondary electron donors cytochrome b559 or chlorophyllZ was observed. Photoaccumulation experiments with extensive flashing at 77 K showed similar results, with no or very little induction of the secondary electron donors. Thus, the partition ratio defined as (yield of YZ/CaMn4O5-cluster oxidation):(yield of Cytb559/ChlZ/CarD2 oxidation) was found to be 0.4 at under visible light and 1.7 at under far-red light at 77 K. Our data show that different products of charge separation after far-red light exists in the wide temperature range which further support the model of the different primary photochemistry in PSII with localization of hole on the ChlD1 molecule.
Resonance Raman spectroscopy is one of the most powerful techniques in analytical science due to its molecular selectivity, high sensitivity, and the fact that, in contrast to IR absorption spectroscopy, the presence of water does not hamper or mask the results. Originating in physics and chemistry, the use of Raman spectroscopy has spread and now includes a variety of applications in different disciplines, including biology. In this chapter, we introduce the basic principles of Raman and resonance Raman scattering, and show resonance Raman can be applied to study carotenoid molecules, in complex biological or chemical matrices. We describe the type of information that can be extracted from resonance Raman spectra, illustrating the power of this method by a series of example applications.
Laser excitation of a single precursor, namely 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (HHEMP), has been used for generating the radical cations and radical anions of various carotenoids in methanol. In the presence of oxygen, laser excitation of HHEMP undergoes an efficient α-cleavage reaction (Norrish type I) to form acyl radicals, which react with O2, in a nearly diffusion-controlled reaction, to form their corresponding strong oxidizing acylperoxyl radicals (RO2•) (E = ~1.1 V (v SHE)), which are capable of oxidizing almost all carotenoids. Under argon-saturated conditions and in the presence of strong base (0.01 M NaOH or tetrabutylammonium hydroxide (TBAOH)), the initially formed 2-hydroxy-2-propyl radical (ACH•), generated after LFP of HHEMP, is deprotonated to form the strong reducing acetone ketyl radical (AC•-) (E {acetone/ AC•-} = -2.1 V (v SHE)), which is capable of reducing all carbonyl-containing carotenoids. To validate this new proposed approach, retinal and β-apo-8'-carotenal (APO), with known spectroscopic data, were investigated in methanol, acetonitrile and tetrahydrofuran (THF). In addition, the radical ions of newly investigated carotenoids, namely 4-oxo-β-apo-15'-carotenoic acid (4-oxo-15'), crocetindial, 4-oxo-β-apo-10'-carotenoic acid ethyl ester (4-oxo-10') and 4-oxo-β-apo-8'-carotenoic acid ethyl ester (4-oxo-8') have been reported. Moreover, the scope of this approach has been extended to investigate the radical ions of chlorophyll b.
World demand for energy is rapidly increasing and finding sufficient supplies of clean energy for the future is one of the major scientific challenges of today. This book presents the latest knowledge and chemical prospects in developing hydrogen as a solar fuel. Using oxygenic photosynthesis and hydrogenase enzymes for bio-inspiration, it explores strategies for developing photocatalysts to produce a molecular solar fuel. The book begins with perspective of solar energy utilization and the role that synthetic photocatalysts can play in producing solar fuels. It then summarizes current knowledge with respect to light capture, photochemical conversion, and energy storage in chemical bonds. Following chapters on the natural systems, the book then summarizes the latest developments in synthetic chemistry of photo- and reductive catalysts. Finally, important future research goals for the practical utilization of solar energy are discussed. The book is written by experts from various fields working on the biological and synthetic chemical side of molecular solar fuels to facilitate advancement in this area of research.
Photosystem II is the pigment–protein complex that oxidises water and reduces plastoquinone in oxygenic photosynthesis. Each Photosystem II complex contains four core proteins surrounded by 13 low-molecular-weight proteins. The 5-kDa PsbT protein is one of the subunits found at the interface of functional Photosystem II dimers. We previously observed that deletion of PsbT in the cyanobacterium Synechocystis sp. PCC 6803 slowed electron transfer and increased the susceptibility of Photosystem II to photodamage and that the addition of bicarbonate (or H C O 3 − ) could prevent this photodamage. Bicarbonate is an essential Photosystem II cofactor that binds to a non-heme iron found between the QA and QB plastoquinone electron acceptors. Formate can bind to the non-heme iron thereby displacing bicarbonate and this results in blocked electron transfer and impaired oxygen evolution. We show Synechocystis sp. PCC 6803 cells lacking PsbT exhibit an enhanced sensitivity to formate addition, while formate inhibition of oxygen evolution in these cells is reversed by the addition of bicarbonate. Our results suggest that H C O 3 − binding is weakened in ΔPsbT cells and that PsbT plays a key role in maintaining the HCO3 –-iron-quinone electron-acceptor complex of Photosystem II and consequently moderates the susceptibility of Photosystem II to high-light-induced photodamage.
The sections in this article are
Introduction
Structure, Function and Manipulation
Biosynthesis and Regulation
Conclusions and Future Directions
Acknowledgements
Carotenoid pigments provide fruits and flowers with distinctive red, orange and yellow colours as well as a number of aromas, which make them commercially important in agriculture, food, health and the cosmetic industries. Carotenoids comprise a large family of C40 polyenes that are critical for the survival of plants and animals alike. β-carotene and its derivatives contain unmodified β-ionone groups, which serve as precursors for vitamin A and are therefore essential dietary components for mammals. Significant progress has been made towards producing staple food crops with elevated provitamin A carotenoids, an important first step in alleviating worldwide vitamin A deficiency. Recent insights into the regulatory processes that control carotenoid composition and content may further advance biofortification projects.
Earlier the catalase-insensitive formation of organic hydroperoxides (via the interaction of organic radicals produced due to redox activity of P680(+?) (or TyrZ(?)) with molecular oxygen) has been found in Mn-depleted PS2 preparations (apo-WOC-PS2) by Khorobrykh et al. (Biochemistry 50:10658-10665, 2011). The present work describes a second pathway of the photoproduction of organic peroxides on the donor side of PS2. It was shown that illumination of CaCl2-treated PS2 membranes (deprived of the PS2 extrinsic proteins without removal of the Mn-containing water-oxidizing complex) (CaCl2-PS2) led to the photoproduction of highly lipophilic organic hydroperoxides (LP-OOH) (in amount corresponding to 1.5 LP-OOH per one reaction center of PS2) which significantly increased upon the addition of exogenous electron acceptor potassium ferricyanide (to 4.2 LP-OOH per one reaction center). Addition of catalase (200?U/ml) before illumination inhibited ferricyanide-induced photoproduction of hydroperoxides while no effect was obtained by adding catalase after illumination or by adding inactivated catalase before illumination. The hydroperoxide photoproduction was inhibited by the addition of exogenous electron donor for PS2, diphenylcarbazide or diuron (inhibitor of the electron transfer in PS2). The addition of exogenous hydrogen peroxide to the CaCl2-PS2 led to the production of highly lipophilic organic hydroperoxides in the dark (3.2 LP-OOH per one reaction center). We suggest that the photoproduction of highly lipophilic organic hydroperoxides in CaCl2-PS2 preparations occurs via redox activity of H2O2 produced on the donor side of PS2.
Upon anthracene-sensitizing, triplet excitation dynamics of β-carotene (β-Car) were studied in n-hexane, in methanol, and in acetonitrile, respectively, by ns flash photolysis spectroscopy. In n-hexane, only the bleaching of the ground state absorption (GSB) and the excitation triplet (3Car*) absorption were observed, and there were no cationic species detected. In both methanol and acetonitrile, similar excitation dynamics were observed, i.e., 3Car* having a similar lifetime to that in n-hexane, and the immediate generation of the cation dehydrodimer (#[Car]2+) upon excitation following transformation into the radical cation Car•+, since Car•+ has much longer lifetime in acetonitrile than in methanol. The results prove that both solvent and carotenoid structure determine the triplet excitation mechanism.
At low temperature (5 K) light-induced charge separation in photosystem II (PS II) is followed by charge stabilization due to reduction of P680+ by secondary donors such as carotenoid (Car) or chlorophyll (Chl) that compete with P680+ QA− charge recombination.
In order to study charge stabilization in photosystem II at low temperature we utilized a newly developed CCD-based absorption spectrometer providing minimal actinic fluence and reasonable time (100 ms) and spectral (0.5 nm) resolution while covering a large spectral window from 470 to 1,100 nm. This enabled us to follow the electron transfer reactions in photosystem II by monitoring the specific signals for QA−, Chl+ and Car+ simultaneously in the wavelength and time domain. We show that, as a result of inducing multiple turnovers in photosystem II core complexes from spinach, during the course of illumination with continuous green light the relative amounts of the donors Car+ and Chl+ depend on the number of photons absorbed. In a prolonged illumination Car+ is largely replaced by more stable donors.
There are multiple complementary and redundant mechanisms to provide protection against photo-oxidative damage, including non-photochemical quenching (NPQ). NPQ dissipates excess excitation energy as heat by using xanthophylls in combination with changes to the light-harvesting complex (LHC) antenna. The xanthophylls are oxygenated carotenoids that in addition to contributing to NPQ can quench singlet or triplet chlorophyll and are necessary for the assembly and stability of the antenna. We have genetically manipulated the expression of the epsilon -cyclase and beta -carotene hydroxylase carotenoid biosynthetic enzymes in Arabidopsis thaliana. The epsilon -cyclase overexpression confirmed that lut2 (lutein deficient) is a mutation in the epsilon -cyclase gene and demonstrated that lutein content carl be altered at the level of mRNA abundance with levels ranging from 0 to 180% of wild-type. Also, it is clear that lutein affects the induction and extent of NPQ, The deleterious effects of lutein deficiency on NPQ in Arabidopsis and Chlamydomonas are additive, no matter what the genetic background, whether npq1 (zeaxanthin deficient), aba1 or antisense beta -hydroxylase (xanthophyll cycle pool decreased). Additionally, increasing lutein content causes a marginal, but significant, increase in the rate of induction of NPQ despite a reduction in the xanthophyll cycle pool size.
Photosynthetic water oxidation is one of the most important biochemical processes on Earth. Oxygen-evolving photosynthetic organisms contain two types of photosynthetic reaction center, called photosystem I (PS I) and photosystem II (PS II). The PS II reaction center uses water as a source of electrons, light energy being used to oxidize water to provide protons and oxygen. How water is oxidized is a difficult problem that has already taken many years of work to understand, however, both the structure of oxygen evolving complex(OEC) and the mechanism of water oxidation still remain to be understood. In this review, we collected the current research results on this area and it will hopefully stimulate further work on the structure of oxygen evolving complex and the mechanism of water oxidation.
Carotenoids are tetraterpenoid pigments that are present in the chloroplasts and chromoplasts of the photosynthetic organisms. Carotenoids are divided into two classes: carotenes, composed of carbon and hydrogen and xanthophylls, containing additionally oxygen. In chloroplasts carotenoids play two key roles: they absorb light energy for use in the photosynthesis, and they are also important components of the antioxidant system of chloroplasts protecting photosynthetic apparatus from photodamage. In this chapter properties of carotenoids as effective nonenzymatic plant antioxidants are described. At the beginning the chemical structure of carotenoids in relation to their antioxidant properties is explained. Next, the photoprotective role of two all-. trans β-carotene molecules existing in PSII reaction center will be described. In the last part of the chapter the significance of xanthophylls in photoprotection will be discussed. Special attention is given to the role of carotenoids involved in several types of the xanthophyll cycle. Furthermore, the meaning of the xanthophyll cycles as antioxidant systems is discussed. Four essential mechanisms clarifying the protective role of xanthophyll cycles will be presented. Three of these mechanisms show an indirect and one a direct participation of xanthophylls in the photoprotection. The mechanisms based on indirect participation of pigments created as products of the light phase of xanthophyll cycle in quenching of overexcitation include: (I) aggregation-dependent LHCII quenching; (II) light-driven mechanisms in LHCII and (III) charge transfer quenching between Chl a and Zx. Selected results of research on the antioxidant properties of xanthophyll cycle pigments in model systems is also shown. Finally, the role of ascorbate as an antioxidant and as a reductant required to carry out de-epoxidation is also considered.
This chapter provides an overview of oxygenic photosynthesis, with primary focus on the oxygen-evolving complex (OEC) of photosystem II (PSII). The introduction includes a general discussion of photosynthesis and a brief overview of the metals involved. This is followed by an in-depth description of the metal centers within PSII, in which the structural and electronic characterizations of the OEC are reviewed in detail. The role of chloride in PSII and the proton exit pathway are discussed. Several mechanisms proposed for the O. O bond formation in the OEC are presented, along with a few detailed mechanisms for the complete catalytic cycle obtained from quantum mechanics/molecular mechanics and density functional theory calculations. Model complexes that are functional mimics of the OEC are also briefly discussed. General discussion on plastocyanin, iron-sulfur centers, [FeFe] and [NiFe] hydrogenases, and ribulose-1,5-bisphosphate carboxylase-oxygenase is included. The chapter concludes with the implications of natural photosynthesis on the development of an artificial photosynthetic framework.
IntroductionPrinciples of Resonance Raman SpectroscopyPrimary Processes in PhotosynthesisPhotosynthesis in PlantsThe Light-Harvesting System of PlantsProtection against Oxidative Stress: Light-Harvesting Regulation in PlantsRaman studies of LHCIICrystallographic Structure of LHCIIProperties of LHCII in CrystalRecent Developments and Perspectives
Introduction and overall organization of electron transfer pathwaysCyclic electron transfer in purple photosynthetic bacteriaCompleting the cycle - the cytochrome bc1 complexMembrane organization in purple bacteriaElectron transport in other anoxygenic bacteriaSpatial distribution of electron transport components in thylakoids of oxygenic photosynthetic organismsNoncyclic electron flow in oxygenic organismsThe structure and function of the cytochrome b5f complexPlastocyanin donates electrons to photosystem 1Ferredoxin and ferredoxin-NADP reductase complete the noncyclic electron transport chainPhotodamage and repair of photosystems 1 and 2Cyclic electron flow in photosystem 2The use of chlorophyll fluorescence to probe photosystem 2Fluorescence detection of nonphotochemical quenchingThe physical basis of variable fluorescence
A nutrient is defined as any substance that has nutritious qualities, i.e., that nourishes or promotes growth, and one that can be metabolized by an organism to give energy and build tissue. In this chapter a nutrient is assumed to be an amino acid, carotenoid, vitamin C, vitamin E, and other antioxidants such as polyphenols (Fig. 1 shows the structures of some important antioxidants discussed in chapter). While carotenoids, vitamin E, and vitamin C are often regarded as our most important dietary antioxidants, little is known of their possible interactions. The aim of this chapter is to discuss such interactions and to suggest how these may explain possible synergistic protective effects as well as how deleterious effects could arise. The carotenoids we consume, from our foods, food colorants, and possibly as dietary supplements, are thought to be antioxidants both by quenching singlet oxygen and by scavenging free radicals. This chapter concerns free radical reactions; readers interested in singlet oxygen may consult recent reviews (1,2) and the recent study of singlet oxygen quenching by carotenoids in liposomes (3).
The exposure of plants to high light in excess of photosynthetic needs causes a reduction in photosynthetic capacity, which is known as photoinhibition. During photoinhibition reactive oxygen species are produced to an extent that leads to the destruction of carotenoids, chlorophyll, protein and to an increase in membrane lipid peroxidation. Plants have developed several strategies to sustain chloroplast functioning under high light conditions. In this review we summarize the latest knowledge about mechanisms for photoinhibition and photoprotective strategies such as: 1) chloroplast antioxidant systems (i.e. tocochromanols, water-water cycle); 2) the quenching of the triplet chlorophyll and reactive oxygen species by carotenoids.; 3) the reversible conversion of violxanthin to zeaxanthin in the light-harvesting complex (LHC) (xanthophyll cycles).
A complex redox titration pattern of cytochrome (Cyt) b559 in preparations of thylakoid membranes and photosystem (PS) II membrane fragments is commonly attributed to the presence of three conformational forms differing by a structure of the heme microenvironment. However, despite decades of research, structural determinants underlying differences between the redox forms of Cyt b559 have not been defined. In this work, we propose a different interpretation of redox heterogeneity in the native population of Cyt b559 assuming redox interaction between the Cyt b559 heme group and a nearby bound quinone (Q). The interacting quinone is supposed to be plastoquinone QC present in the unusual singly protonated form (QCH). The model successfully explains the unique redox properties of Cyt b559 and may provide a simple and effective mechanism of redox regulation of secondary electron transport in PS II. At the present time, the model of heme-quinone redox interaction can be considered as an alternative to the idea of conformational differences between the native redox forms of Cyt b559.
Photosystem II (PSII) uses light energy to oxidise water and reduce quinone. The water oxidation site is a Mn4Ca cluster located on the luminal side of the membrane protein complex, while the quinone reduction site is made up of two quinones (QA and QB) and a non-heme Fe2+ located on the stromal side of the membrane protein. In this thesis I worked on both oxidation and reduction functions of the enzyme. QA•- and QB•- are magnetically couple to the Fe2+ giving weak and complex EPR signals. The distorted octahedral Fe2+ has four histidines ligands and an exchangeable (bi)carbonate ligand. Formate can displace the exchangeable (bi)carbonate ligand, slowing electron transfer out of the PSII reaction centre. Here I report the formate-modified QB•- Fe2+ EPR signal, and this shows marked spectral changes and has a greatly enhanced intensity. I also discovered a second new EPR signal from formate-treated PSII that is attributed to formate-modified QA•- Fe2+ in the presence of a 2-electron reduced form of QB. In addition, I found that the native QA•- Fe2+ and QB•- Fe2+ EPR signals have a strong feature that had been previously missed because of overlapping signals (mainly the stable tyrosyl radical TyrD•). These previously unreported EPR signals should allow for the redox potential of this cofactor to be directly determined for the first time. I also observed that when QB•-Fe was formed; it was able to oxidise the iron slowly in the dark. This occurred in samples pumped to remove O2. This observation implies that at least in some centres, the QB•-/QBH2 couple has a higher potential then is often assumed and thus that the protein-bound semiquinone is thermodynamically less stable expected. It has yet to be determined if this represents a situation occurring in the majority of centres. Treatment of the system with dithionite generated a modified form of QA•-Fe2+ state and a change in the association of the proteins on gels. This indicates a redox induced modification of the protein, possibly structurally important cysteine bridge in PSII.On the water oxidation side of the enzyme, I studied the first step in the assembly of the Mn4Ca cluster looking at Mn2+ oxidation using kinetic EPR and high field EPR. Conditions were found for stabilising the first oxidised state and some discrepancies with the literature were observed. I also found that dithionite could be used to reduce the Mn4Ca, forming states that are formally equivalent to those that exist during the assembly of the enzyme.
Photosynthetic reaction centers (RCs) are nature's solar batteries. These nanometre-scale power producers are responsible for transducing the energy of sunlight into a form that can be used by biological systems, thereby powering virtually all of the biological activity on the planet. This chapter describes the structures and mechanisms of the different RCs that power biology, starting with the simplest and most heavily characterized system, the purple bacterial RC, before turning to the related Photosystem II RC. There then follows an account of charge separation in Photosystem I, and the chapter ends by outlining the homodimeric RCs from obligate anaerobic photosynthetic bacteria.
Electron transfer from the reduced tyrosine YD and cytochrome b559 (Cyt b559) to the S2 and S3 states of photosystem II was investigated at the temperature of 195 K. Electron transfer reactions were followed by measuring EPR signals of tyrosine YD·, oxidized Cyt b559 and the S2-state multiline signal. Long term incubation (∼90 days) at 195 K causes decay of the majority of S2 centers up to ∼40% of initial value, while in this time scale the intensity of YD· radical increases less than 10%. Samples advanced to S3 state demonstrates an increasing behavior of the S2-state multiline signal intensity in the beginning of incubation (∼20 days) and slow decay up to 40% of maximal amplitude during further incubation of the samples. Similarly to the S2 sample, small increase in YD· radical signal was observed during the S3 decay. However, in both types of samples prepared in S2 and S3 states after 90 days of incubation the signal of oxidized Cyt b559 is increased from 45%–50% up to 100% maximal intensity. The results obtained in this study support the conclusion of our early investigations which claimed the reduced Cyt b559 as electron source for the S2 and S3 states.
Siphonaxanthin and siphonein are two keto-carotenoids. Upon anthracene-sensitizing, triplet excitation dynamics of these two carotenoids were studied in n-hexane and in methanol, respectively, by ns flash photolysis spectroscopy. In n-hexane, bleaching of the ground state absorption (GSB) and the excitation triplet (3Car*) absorption were observed. In methanol, upon the decay of the 3Car*, the cation dehydrodimer of carotenoid, #[Car]2+, generated by the same rate, while an additional GSB generated synchronously, a polar solvent assisted and anthracene-sensitized mechanism was addressed based on the discussion. The environment-sensitive triplet excitation dynamics imply their potential role in photo-protection in vivo.
It has been shown by Khorobrykh et al. (Biochemistry (Moscow) 67:683-688, 2002); Yanykin et al. (Biochim Biophys Acta 1797:516-523, 2010); Khorobrykh et al. (Biochemistry 50:10658-10665, 2011) that Mn-depleted photosystem II (PSII) membrane fragments are characterized by an enhanced oxygen photoconsumption on the donor side of PSII which is accompanied with hydroperoxide formation and it was suggested that the events are related to the oxidative photoinhibition of PSII. Experimental confirmation of this suggestion is presented in this work. The degree of photoinhibition was determined by the loss of the capability of exogenous electron donors (Mn(2+) or sodium ascorbate) to the reactivation of electron transport [measured by the light-induced changes of chlorophyll fluorescence yield (∆F)] in Mn-depleted PSII membranes. The transition from anaerobic conditions to aerobic ones significantly activated photoinhibition of Mn-depleted PSII membranes both in the absence and in the presence of exogenous electron acceptor, ferricyanide. The photoinhibition of Mn-depleted PSII membranes was suppressed upon the addition of exogenous electron donors (Mn(2+), diphenylcarbazide, and ferrocyanide). The addition of superoxide dismutase did not affect the photoinhibition of Mn-depleted PSII membranes. It is concluded that the interaction of molecular oxygen (rather than superoxide anion radical formed on the acceptor side of PSII) with the oxidized components of the donor side of PSII reflects the involvement of O2 in the donor-side photoinhibition of Mn-depleted PSII membranes.
In sunflower leaves linear electron flow LEF = 4∙O2 evolution rate was measured at 20 ppm O2 in N2. PSII charge separation rate CSRII = aII∙PAD∙(Fm - F)/Fm, where aII is excitation partitioning to PSII, PAD is photon absorption density, Fm and F are maximum and actual fluorescence yields. Under 630 nm LED + 720 nm far-red light (FRL), LEF was equal to CSRII with aII = 0.51 to 0.58. After FRL was turned off, plastoquinol (PQH2) accumulated, but LEF decreased more than accountable by F increase, indicating PQH2-oxidizing cyclic electron flow in PSII (CEFII). CEFII was faster under conditions requiring more ATP, consistent with CEFII being coupled with proton translocation. We propose that PQH2 bound to the QC site is oxidized, one e(-) moving to P680(+), the other e(-) to Cyt b559. From Cyt b559 the e(-) reduces QB(∙-) at the QB site, forming PQH2. About 10-15% electrons may cycle, causing misses in the period-4 flash O2 evolution and lower quantum yield of photosynthesis under stress. We also measured concentration dependence of PQH2 oxidation by dioxygen, as indicated by post-illumination decrease of Chl fluorescence yield. After light was turned off, F rapidly decreased from Fm to 0.2Fv, but further decrease to F0 was slow and O2 concentration dependent. The rate constant of PQH2 oxidation, determined from this slow phase, was 0.054 s(-1) at 270 μM (21%) O2, decreasing with Km(O2) of 60 μM (4.6%) O2. This eliminates the interference of O2 in the measurements of CEFII.
Copyright © 2015. Published by Elsevier B.V.
We have earlier shown that all electron transfer reactions in Photosystem II are operational up to 800 nm at room temperature [Thapper et al. (2009), Plant Cell 21, 2391-2401]. This led us to suggest an alternative charge separation pathway for far-red excitation. Here we extend these studies to very low temperature (5 K). Illumination of photosystem II (PS II) with visible light at 5 K is known to result in oxidation of almost similar amounts of YZ and the Cyt b559/ChlZ/CarD2 pathway. This is reproduced here using laser flashes at 532 nm and we find the partition ratio between the two pathways to be 1:0.8 at 5 K (the partition ratio is here defined as (yield of YZ/CaMn4 oxidation):(yield of Cyt b559/ChlZ/CarD2 oxidation)). The result using far red laser flashes is very different. We find partition ratios of 1.8 at 730 nm; 2.7 at 740 nm and >2.7 at 750 nm. No photochemistry involving these pathways is observed above 750 nm at this temperature. Thus, far-red illumination preferentially oxidizes YZ while the Cyt b559/ChlZ/CarD2 pathway is hardly touched. We propose that the difference in the partition ratio between visible and far-red light at 5 K reflects the formation of different first stable charge pair. In visible light, the first stable charge pair is considered to be PD1+Qa-. In contrast, we propose that the electron hole is residing on the ChlD1 molecule after illumination by far red at light 5 K resulting in the first stable charge pair being ChlD1+QA-. ChlD1 is much closer to YZ (11.3 Å) than to any component in Cyt b559/ChlZ/CarD2 pathway (closest distance is ChlD1 - CarD2 is 28.8 Å). This would then explain that far-red illumination preferentially drives efficient electron transfer from YZ. We also discuss mechanisms to account for the absorption of the far-red light and the existence of a hitherto unobserved charge transfer states. The involvement of two or more of the porphyrin molecules in the core of the Photosystem II reaction center is proposed.
Photosystem II (PSII) is a pigment-protein complex of thylakoid membrane of higher plants, algae, and cyanobacteria where light energy is used for oxidation of water and reduction of plastoquinone. Light-dependent reactions (generation of excited states of pigments, electron transfer, water oxidation) taking place in PSII can lead to the formation of reactive oxygen species. In this review attention is focused on the problem of interaction of molecular oxygen with the donor site of PSII, where after the removal of manganese from the water-oxidizing complex illumination induces formation of long-lived states (P680(+•) and TyrZ(•)) capable of oxidizing surrounding organic molecules to form radicals.
Light-driven electron transport in liposome-bound photosystem II (PS-II) particles between water and ferricyanide was monitored by bare platinum electrode oxymetry. The modification of the experimental system with the exogenous quinones α-tocopherol quinone ( α-TQ) or plastoquinone (PQ) resulted in a pronounced effect on photosynthetic oxygen evolution. The presence of α-tocopherolquinone ( α-TQ) in PS-II samples decreased the rate of red light-induced oxygen evolution but increased the rate of green light-induced oxygen evolution. Blue light applied to the assay system in which oxygen evolution was saturated by red light resulted in a further increase of the oxygen signal. These findings are interpreted in terms of a cyclic electron transport around PS-II, regulated by an excitation state of β-carotene in the reaction centre of PS-II. A mechanism is postulated according to which energetic coupling of β-carotene in the reaction centre of PS-II and that of other antenna carotenoid pigments is regulated by the portion of the xanthophyll violaxanthin, which is under control of the xanthophyll cycle.
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.
If Norflurazon-treated mustard (Sinapis alba L.) seedlings are grown in low-fluence-rate white light, accumulation of carotenoids is completely inhibited, while levels of chlorophyll (Chl) a and b are comparable to those of control seedlings. Measurements of fluorescence yield and oxygen evolution indicate that carotenoid-free, green cotyledons are unable to perform leephotosynthesis in vivo. When thylakoid membranes were prepared and electron transport was measured in vitro, only PSI but not PSII activity was detected. Solubilization of the photosystems from thylakoid membranes and separation by sucrose-gradient centrifugation confirmed that PSII is absent in carotenoid-free seedlings, while PSI is present. Western blot analysis for representative proteins of the four photosynthetic complexes showed that subunits 1 and 2 of PSI, the Rieske-iron sulfur-protein, the α-subunit of the CF1 moiety of the ATP-synthase complex, cytochrome b
559 and the lumenal 33-kDa protein of the water-splitting apparatus of PSII are present in comparable amounts in Norflurazon-treated and control plants, while the amounts of Chl-binding proteins of PSII (the major light-harvesting Chl-a/b-binding protein of the antenna complex and the 51- and 44-kDa Chl-a-binding proteins) and two components of the PSII reaction center, (the D1 and D2 protein) are substantially reduced. The data indicate that accumulation of PSII polypeptides is either not inhibited or not completely inhibited in carotenoid-free mustard seedlings, but that assembly of a functional PSII complex does not occur. If Norflurazon-treated seedlings are transferred to water, lutein accumulates rapidly and reaches about 80% of the level detectable in control plants, while the level of other carotenoids is still less than 1%. The accumulation kinetics for lutein are similar to the kinetics for the appearance of PSII activity. This indicates that the availibility of lutein rather than that of other carotenoids might be rate-limiting for the appearance of PSII activity.
The general aim of this investigation is to determine whether carotenoid cation radicals can be produced, and stabilized, electrochemically. Hence, the authors have undertaken a detailed study of the electrooxidation of various carotenoids (..beta..-carotene (I), ..beta..-apo-8'-carotenal (II), and canthaxanthin (III) using the techniques of cyclic voltammetry, controlled-potential electrolysis (cpe) in conjunction with optical spectroscopy, and EPR spectroscopy coupled with in situ electrolysis. They report the successful generation of carotenoid cation radicals via electrochemical oxidation and, furthermore, the stabilization of these radicals for several minutes in CH/sub 2/Cl/sub 2/ and C/sub 2/H/sub 4/Cl/sub 2/ solvents.
Photosystem-II-enriched membrane fragments obtained by detergent solubilisation of thylakoid membranes were found to contain almost exclusively the high redox potential form of cytochrome b-559 (Em,6.0 = + 353 mV), provided that the haem was maintained in the reduced state during the isolation procedure. A reducing potential was required during the isolation due to the instability of the oxidised form of the high-potential couple. Additional detergent treatment of such preparations converted all of the cytochrome to a low potential form (Em,6.0 of around + 100 mV). Dissociation of the 23 kDa extrinsic polypeptide, bound at the lumenal side of Photosystem II, had no effect on the redox state of the cytochrome provided that calcium remained in association with the Photosystem II complex. Removal of the 33 kDa extrinsic protein, in addition to the 23 kDa, resulted in the conversion of the haem to an intermediate (Em,6.0 = + 169 mV) redox form, independent of the presence of calcium. Considering that these preparations are derived from the granal regions of the thylakoid membranes, the data suggest that, in vivo, Photosystem II complexes in these regions contain only the high redox potential form of the cytochrome. The data further suggest that, in addition to the 33 kDa protein, ligation of calcium rather than the 23 kDa polypeptide is required for the stabilisation of this form of cytochrome b-559.
The radical cations and anions of the photosynthetic polyenes: all-trans-β-carotene, 15,15′-cis-β-carotene and all-trans-lycopene have been produced and characterised in hexane, cyclohexane and methanol (anions only) using pulse radiolysis techniques. Both types of radicals are believed to be monomeric and have intense absorptions in the near infrared, their maximum extinctions being in excess of 105 dm3 mol–1 cm–1. The cationic and anionic band maxima are well separated, the cationic maxima lying about 150 nm to the red of the anionic maxima.
Cyclic voltammogram (CV) and simultaneous electrochemical and electron paramagnetic resonance (SEEPR) measurements have been carried out on the oxygenated carotenoids: echinenone, canthaxanthin, isozeaxanthin and rhodoxanthin in dichloromethane. The CV displays are markedly different from that of β-carotene. Comproportionation constants, deduced from EPR spin concentration measurements of electrochemically oxidized dihydroxy β-carotene and several keto carotenoids vary by four orders of magnitude. ΔHpp values deduced from the SEEPR spectra of the cation radicals are in the range 13.2 to 14.5 G and the g-factors are 2.0027 ± 0.0002. These EPR parameters are in accordance with a polyene π-cation radical structure. Theoretical CVs are calculated using DigiSim, a CV simulation program, and the proposed mechanism involves three electrode and two homogeneous reactions.
The oxidation process involving the transfer of two electrons for {beta}-carotene is confirmed by bulk electrolysis in a CH{sub 2}Cl{sub 2} solvent and the observation of {Delta}E = 42 mV from cyclic voltammetric measurements. A similar process is also found to occur for {beta}-apo-8{prime}-carotenal and canthaxanthin. An additional cathodic peak between 0.2 0.5 relative to SCE is shown to be dependent on the initial formation of dications followed by the loss of H{sup +} as evidenced by a large isotope effect and most likely due to the reduction of a carotenoid cation. EPR evidence exists for the formation of radical cations by the reaction of diffusing carotenoid dictations with neutral carotenoids. The rate of formation is consistent with the differences in the diffusion coefficients of the carotenoids deduced by chronocoulometric measurements, being fastest for canthaxanthin.
Light excitation of chloroplasts at low temperature produces absorption changes (ΔA) with a large positive peak at 990 nm and a bleaching around 480 nm. ΔA at 990 nm rises with at 20–77 K and remains largely stable. This signal is not observed when Photosystem II (PS II) photochemistry is blocked by reduction of the primary plastoquinone. It is observed also in purified PS II particles, in which case it could be shown that during a sequence of short flashes, the absorption at 990 nm rises in parallel with plastoquinone reduction measured at 320 nm. In chloroplasts the light-induced 990-nm ΔA at 77 K is increased under oxidizing conditions (addition of ferricyanide) and upon addition of 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT2p). At 21°C, flash excitation of chloroplasts or of PS II particles induces only a very small ΔA at 990 nm, even when this is measured with a 100-ns time resolution or when the material is preilluminated. In both materials, however, a large flash-induced ΔA takes place when various lipophilic anions are added. After a flash the signal rises in less than 100 μs and its decay varies with experimental conditions; the decay is strongly accelerated by benzidine. The difference spectrum measured in PS II particles includes a broad peak around 990 nm and a bleaching around 490 nm. These absorption changes are attributed to a carotenoid radical cation formed at the PS II reaction center. It is estimated that in the presence of lipophilic anions at room temperature, one cation can be formed by a single flash in 80% of the reaction centers. At cryogenic temperature approx. 8% of the PS II reaction centers can oxidize a carotenoid after a single flash.
Phenolic herbicides were added to suspensions of spinach chloroplasts or to oxygen-evolving Photosystem II membranes. Flash absorption spectroscopy at 21°C around 1000 nm reveals that these chemicals lead to a flash-induced absorption increase attributed to the radical-cation of a carotenoid. The herbicides studied can be arranged in the following order of decreasing efficiency for the reported effect: i-dinoseb, bromonitrothymol, trinitrophenol, ioxynil, dinitroorthocresol, 2,4-dinitrophenol. A similar effect was not observed with atrazine, DCMU or o-phenanthroline. For a given herbicide concentration, the amount of flash-induced carotenoid cation increases sharply when the pH is lowered below 5.5. A similar effect does not take place with other molecules which induce the formation of a carotenoid cation: tetraphenylboron, FCCP, 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT-2p). The previous effects are observed in both oxygen-evolving Photosystem II and in preparations in which oxygen evolution is inhibited with alkaline Tris. In untreated material, the carotenoid cation is formed with a half-time of 10–35 μs. After Tris treatment, this half-time is a little longer at low than at high pH. These results indicate the existence of a specific site where phenolic inhibitors interact in the oxygen-evolving site of Photosystem II
The conversion of cytochrome (Cyt) b-559 from the low-potential (LP) to the high-potential (HP) form under conditions for photoactivation of O2 evolution (reconstitution of the Mn cluster) was investigated using Photosystem II (PS II) membranes that had been depleted of the Mn cluster by treatment with Tris. Illumination of the PS II membranes with continuous or flashing light in the presence of 0.1 mM Mn2+ reactivated O2 evolution and increased the level of the HP form of Cyt b-559 with a concomitant decrease in the level of the LP form. When illumination was achieved with flashing light, the restoration of the HP form occurred after two flashes, while reactivation of O2 evolution required more than six flashes. It was also found that the HP form could be restored when the PS II membranes were illuminated in the presence of artificial electron donors instead of Mn2+. NH2OH (10–100 μM), 1,5-diphenylcarbazide (50–100 μM) and semicarbazide (0.5–1 mM) were effective in restoring the HP form. These observations suggest that, under photoactivation conditions, not the reconstitution of the Mn cluster but electron donation by Mn2+ to PS II is responsible for the restoration of the HP form. The restoration of the HP form by illumination in the presence of Mn2+ was not affected by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) but it was completely suppressed by artificial electron acceptors which bind to the QB site and reoxidize QA−, namely, 2,5-dibromo-3-methyl-6-isopropyl-1,4-benzoquinone (DBMIB) and 2,6-dichloro-1,4-benzoquinone. These results suggest that some redox reaction(s) at the acceptor side of PS II, which probably involves QA−, occurs during the course of the restoration of the HP form.
The effect of photoactivation (the assembly of the Mn cluster involved in oxygen evolution) in Photosystem II (PS II), on the redox midpoint potential of the primary quinone electron acceptor, QA, has been investigated. Measurements of the redox state of QA were performed using chlorophyll fluorescence. Cells of Scenedesmus obliquus were grown in the dark to obtain PS II lacking the oxygen-evolving complex. Growth in the light leads to photoactivation. The midpoint potential of QA was shifted, upon photoactivation, from + 110 mV to −80 mV. In cells of a low-fluorescence mutant, LF1, that is unable to assemble the oxygen-evolving complex but that has an otherwise normal PS II, the higher potential form of QA was found. NH2OH treatment of spinach PS II, which releases the Mn and thus inactivates the oxygen-evolving complex, causes an upshift of the redox potential of QA (Krieger and Weis (1992) Photosynthetica 27, 89–98). Oxygen evolution can be reconstituted by incubation in the light in the presence of MnCl2 and CaCl2. Such photoactivation caused the midpoint potential of QA to be shifted back from around +55 mV to lower potentials (−80 mV), typical for active PS II. The above results indicate that the state of the donor side of PS II has a direct influence on the properties of the acceptor side. It is suggested that the change from the high- to the low-potential form of QA may represent a mechanism for protection of PS II during the assembly of the O2-evolving enzyme.
Illumination of isolated Photosystem II reaction centres in the presence of the electron acceptors, silicomolybdate (SiMo) or 3,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), leads to selective photooxidation and irreversible photobleaching of β-carotene. No such effect is observed in the absence of the electron acceptors and it is dependent on the ability of the reaction centres to carry out charge separation. Flash absorption studies indicate that prior to the irreversible photobleaching, β-carotene is photooxidised by electron transfer to P680+. The rate of photobleaching of β-carotene is faster when SiMo is used as the acceptor and occurs both in the presence and absence of oxygen. However, with DBMIB present photobleaching is more clearly observed when oxygen is present. It is argued that when oxygen is absent, photoreduced DBMIB can rapidly rereduce P680+ by an electron transfer cycle involving cytochrome b-559, while in aerobic conditions the cycle is partially inhibited by oxygen acting as an electron acceptor. When Mn(II) is added as an electron donor to P680+, no photobleaching of β-carotene occurs. The kinetics of photobleaching shows two phases, with 50% loss of the total β-carotene pool occurring rapidly. Coupled with the loss of β-carotene is a photobleaching of accessory chlorophyll which absorbs at 670 nm. Therefore our results indicate that, when the Photosystem II reaction centre is photoactivated under conditions in which P680+ can photoaccumulate, there is a secondary oxidation of β-carotene and accessory chlorophyll which leads to irreversible photobleaching. No such photobleaching occurs if P680+ is rapidly reduced by an exogenous electron donor or by a quinone dependent cyclic flow of electrons around PSII. We discuss the physiological role of β-carotene oxidation and cyclic electron transport in the function of PSII in vivo.
1.1. We have investigated the role of plastoquinone and β-carotene in Photosystem II by examining their effects when added back to freeze-dried chloroplasts that had been extracted with heptane.2.2. Under our conditions, extraction removed about 99.5% of the β-carotene and 90% of the plastoquinone. Photochemical activities associated with Photosystem II were reduced to a low level and 85–90% of the cytochrome b-559HP (E′o = +0.37 V) was converted into a low-potential form.3.3. Plastoquinone alone restored the potential of the cytochrome to the original high value. The amount of cytochrome restored by plastoquinone was as great as by the heptane extract, and β-carotene was either ineffective or slightly inhibitory. Plastoquinone allowed a substantial rate of 2,6-dichlorophenolindophenol (DCIP) photoreduction, but a greater rate was observed when β-carotene was also present (in agreement with previous work). β-Carotene improved the efficiency of light utilisation and protected the chloroplasts from photoinactivation.4.4. The Photosystem II-linked oxidation of cytochrome b-559HP and reduction of C550 (P546) at 77 °K could only be observed when both β-carotene and plastoquinone were present. A small effect with β-carotene alone could be attributed to unextracted plastoquinone.5.5. The results support a model in which C550 (depending on β-carotene) indicates the redox state of the primary acceptor but is not essential for electron flow; β-carotene is needed to maintain the optimum conformation of membrane components for light utilisation and cytochrome photooxidation.
CO2 depletion leads to an approximately 10-fold increase in the light-induced EPR signal at g = 1.82, attributed to the QA− · Fe2+ complex, in Photosystem II-enriched thylakoid membrane fragments. Upon reconstitution with HCO3−the signal decreases to the size in control samples. The split pheophytin− signal is broader in control or reconstituted than in CO2-depleted samples. It is concluded that HCO2− strongly influences the localization and conformation of the QA− · Fe+ complex. The QA− · Fe2+ and split pheophytirr− EPR signals from triazine-resistant Brassica napus were virtually identical to those from triazine-susceptible samples, indicating that the change in the 32-kDa azidoatrazine-binding protein does not lead to a confonnational change of the Qa− · Fe2+ complex.
Cytochrome b-559, which is normally reduced in the dark, was oxidized by preillumination in the presence of N-methyl-phenazonium methosulfate with low intensity far-red light. The average half-time for the photoreduction of oxidized cytochrome b-559 by a long actinic flash ranged from 90 to 110 ms. In the presence of 0.25 μM 3-(3,4-dichlorophenyl)-1,1-dimethylurea the half-time for the photoreduction increased to 230 ms although the extent of the absorbance increase was unchanged. Under similar conditions inhibition of electron transport by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and the increase in the chlorophyll fluorescence show that a large fraction of the Photosystem II reaction centers are blocked. These results are consistent with the concept that electrons are shared between different photosynthetic units by a common pool of plastoquinone and imply that the principle pathway for the reduction of cytochrome b-559 by Photosystem II occurs through plastoquinone. In the presence of the uncoupler gramicidin which stimulates non-cyclic electron transport, the rate of photoreduction of cytochrome b-559 is slower (), from which it is inferred that cytochrome b-559 competes with cytochrome f for electrons out of this pool. Comparison of cytochrome b-559 photoreduction and electron transport rates using untreated and KCN-treated chloroplasts indicate that, under conditions of basal electron transport from water to ferricyanide, approximately one-fifth of the electrons from Photosystem II go through cytochrome b-559 to ferricyanide. Further support for this pathway is provided by a comparison of the effect of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (dibromothymoquinone) on the rates of reduction of cytochrome b-559 and ferricyanide.
Electron paramagnetic resonance (EPR) and optical absorbance difference spectra and kinetics upon illumination by saturating flashes and continuous light of spinach chloroplasts frozen under various conditions were measured between 10 and 180 K. 1. At 100 K illumination with continuous light caused an EPR signal which decayed during the light in about 30 ms. This change is probably due to the reduction of P+-680, the oxidized primary electron donor of Photosystem II, by a secondary electron donor, cytochrome b-559. Flash illumination yielded the previously observed rapid (2 ms) transient. This transient has been ascribed to a back-reaction of the two primary reagents of Photosystem II (Malkin, R. and Bearden, A.J. (1975) Biochim. Biophys. Acta 396, 250-259; Visser, J.W.M. (1975) Thesis, Leiden). 2. Between 10 and 40 K, illumination with continuous light showed a transient which decayed in about 500 ms. The extent decreased with increasing temperature. However, the half time appeared to be temperature independent. This signal was also attributed to P+-680. 3. At 180 K it appeared to be impossible to observe the 2 and 30 ms components in dark frozen chloroplasts. However, they could be observed again if two short saturating flashes were given shortly before freezing. These changes seem to be dependent on the S-state of the reaction center. 4. After oxidizing the sample with ferricyanide (Eh = 540 mV), the light induced absorbance difference spectrum showed a bleaching near 676 nm. This change is ascribed to the irreversible oxidation of a dimeric chlorophyll molecule which acts as electron donor to P+-680 under these conditions. 5. Titration curves of the irreversible light-induced absorbance change at 676 nm and the irreversible light-induced EPR change near g = 2.00 provide strong evidence that these two changes reflect the same compound. Finally, a model is given to explain the observed reactions of Photosystem II at 10-180 K. The model involves three different ultimate and one intermediate electron donor to P+-680 at these temperatures.
Cytochrome b559 (cyt b559) is an intrinsic and essential component of the photosystem II (PSII) protein complex, but its function, stoichiometry, and electron-transfer kinetics in the physiological system are not well-defined. In this study, we have used flash-detection optical spectroscopy to measure the kinetics and yields of photooxidation and dark reduction of cyt b559 in untreated, O2-evolving PSII-enriched membranes at room temperature. The dark redox states of cyt b559 and the primary electron acceptor, QA, were determined over the pH range 5.0-8.5. Both the fraction of dark-oxidized cyt b559 and dark-reduced QA increased with increasing acidity. Consistent with these results, an acid-induced drop in pH from 8.5 to 4.9 in a dark-adapted sample caused the oxidation of cyt b559, indicating a shift in the redox state during the dark reequilibration. As expected from the dark redox state of cyt b559, the rate and extent of photooxidation of cyt b559 during continuous illumination decreased toward more acidic pH values. After a single, saturating flash, the rate of photooxidation of cyt b559 was of the same order of magnitude as the rate of S2QA- charge recombination. In untreated PSII samples at pH 8.0 with 42% of cyt b559 oxidized and 15% of QA reduced in the dark, 4.7% of one copy of cyt b559 was photooxidized after one flash with a t1/2 of 540 +/- 90 ms. On the basis of our previous work [Buser, C. A., Thompson, L. K., Diner, B. A., & Brudvig, G. W (1990) Biochemistry 29, 8977] and the data presented here, we conclude that Sn+1, YZ., and P680+ are in redox equilibrium and cyt b559 (and YD) are oxidized via P680+. After a period of illumination sufficient to fully reduce the plastoquinone pool, we also observed the pH-dependent dark reduction of photooxidized cyt b559, where the rate of reduction decreased with decreasing pH and was not observed at pH < 6.4. To determine the direct source of reductant to oxidized cyt b559, we studied the dark reduction of cyt b559 and the reduction of the PQ pool as a function of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) concentration. We find that DCMU inhibits the reduction of cyt b559 under conditions where the plastoquinone pool and QA are reduced. We conclude that QB-. (H+) or QBH2 is the most likely source of the electron required for the reduction of oxidized cyt b559.(ABSTRACT TRUNCATED AT 400 WORDS)
Electron paramagnetic resonance (EPR) analyses (g = 2 region) and optical spectrophotometric analyses of P680+ were made of NH2OH-extracted photosystem II (PSII) membranes after various durations of weak-light photoinhibition, in order to identify the sites of damage responsible for the observed kinetic components of the loss of electron transport [Blubaugh, D.J., & Cheniae, G.M. (1990) Biochemistry 29, 5109-5118]. The EPR spectra, recorded in the presence of K3Fe(CN)6, gave evidence for rapid (t1/2 = 2-3 min) and slow (t1/2 = 3-4) losses of formation of the tyrosyl radicals YZ+ and YD+, respectively, and the rapid appearance (t1/2 = 0.8 min) of a 12-G-wide signal, centered at g = 2.004, which persisted at 4 degrees C in subsequent darkness in rather constant abundance (approximately 1/2 spin per PSII). This latter EPR signal is correlated with quenching of the variable chlorophyll a fluorescence yield and is tentatively attributed to a carotenoid (Car) cation. Exogenous reductants (NH2OH greater than or equal to NH2NH2 greater than DPC much greater than Mn2+) were observed to reduce the quencher, but did not reverse other photoinhibition effects. An additional 10-G-wide signal, tentatively attributed to a chlorophyll (Chl) cation, is observed during illumination of photoinhibited membranes and rapidly decays following illumination. The amplitude of formation of the oxidized primary electron donor, P680+, was unaffected throughout 120 min of photoinhibition, indicating no impairment of charge separation from P680, via pheophytin (Pheo), to the first stable electron acceptor, QA. However, a 4-microsecond decay of P680+, reflecting YZ----P680+, was rapidly (t1/2 = 0.8 min) replaced by an 80-140 microsecond decay, presumably reflecting QA-/P680+ back-reaction. Photoinhibition caused no discernible decoupling of the antenna chlorophyll from the reaction center complex. We conclude that the order of susceptibility of PSII components to photodamage when O2 evolution is impaired is Chl/Car greater than YZ greater than YD much greater than P680, Pheo, QA.
Kinetic analyses were made of the effects of weak-light photoinhibition on the capacity of NH2OH-extracted photosystem II membranes to photooxidize the exogenous electron donors Mn2+, diphenylcarbazide, and I- or to assemble functional water-oxidizing complexes during photoactivation. The loss of capacity for photooxidation of the donors showed two first-order components (half-times of 2-3 min and 1-4 h) with relative amplitudes dependent on the donor, suggesting two photodamageable sites of electron donation (sites 1 and 2, respectively), a conclusion confirmed by analyses of velocity curves of electron donation by each donor. All of the donors appear to be oxidized preferentially by site 1 both at saturating and at limiting light intensity; however, the contribution by site 2 was nearly comparable in saturating light. Loss of photoactivation also exhibited biphasic kinetics, with components having half-times of approximately 0.8 and 3.2 min. The major component (t1/2 = 3.2 min) corresponded to loss of site 1; essentially no photoactivation was observed after its loss. From these and other analyses, we conclude (1) the relative contributions of site 1 and site 2 to the photooxidation of various exogenous electron donors is determined largely by the rates of equilibration of the donors with the two sites, and (2) only site 1 contributes to photoactivation of the water-oxidizing complex. Site 1 is attributed to tyrosine Z of the reaction center's D1 polypeptide. The molecular identity of site 2 is unknown but may be tyrosine D of the D2 polypeptide.
The photochemistry in photosystem II of spinach has been characterized by electron paramagnetic resonance (EPR) spectroscopy in the temperature range of 77-235 K, and the yields of the photooxidized species have been determined by integration of their EPR signals. In samples treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a single stable charge separation occurred throughout the temperature range studied as reflected by the constant yield of the Fe(II)-QA-EPR signal. Three distinct electron donation pathways were observed, however. Below 100 K, one molecule of cytochrome b559 was photooxidized per reaction center. Between 100 and 200 K, cytochrome b559 and the S1 state competed for electron donation to P680+. Photooxidation of the S1 state occurred via two intermediates: the g = 4.1 EPR signal species first reported by Casey and Sauer [Casey, J. L., & Sauer, K. (1984) Biochim. Biophys. Acta 767, 21-28] was photooxidized between 100 and 160 K, and upon being warmed to 200 K in the dark, this EPR signal yielded the multiline EPR signal associated with the S2-state. Only the S1 state donated electrons to P680+ at 200 K or above, giving rise to the light-induced S2-state multiline EPR signal. These results demonstrate that the maximum S2-state multiline EPR signal accounts for 100% of the reaction center concentration. In samples where electron donation from cytochrome b559 was prevented by chemical oxidation, illumination at 77 K produced a radical, probably a chlorophyll cation, which accounted for 95% of the reaction center concentration. This electron donor competed with the S1 state for electron donation to P680+ below 100 K.(ABSTRACT TRUNCATED AT 250 WORDS)
Although cytochrome b-559 is an integral component of the photosystem II complex (PSII), its function is unknown. Because cytochrome b-559 has been shown to be both photooxidized and photoreduced in PSII, one of several proposals is that it mediates cyclic electron transfer around PSII, possibly as a protective mechanism. We have used electron paramagnetic resonance spectroscopy to investigate the pathway of photooxidation of cytochrome b-559 in PSII and have shown that it proceeds via photooxidation of chlorophyll. We propose that this photooxidation of chlorophyll is the first step in the photoinhibition of PSII. The unique susceptibility of PSII to photoinhibition is probably due to the fact that it is the only reaction center in photosynthesis which generates an oxidant with a reduction potential high enough to oxidize chlorophyll. We propose that the function of cytochrome b-559 is to mediate cyclic electron transfer to rereduce photooxidized chlorophyll and protect PSII from photoinhibition. We also suggest that the chlorophyll(s) which are susceptible to photooxidation are analogous to the monomer chlorophylls found in the bacterial photosynthetic reaction center complex.
The EPR characteristics of oxygen evolving particles prepared from Phormidium laminosum are described. These particles are enriched in Photosystem II allowing EPR investigation of signals which were previously small or masked by those from Photosystem I in other preparations. EPR signals from a Signal II species and high potential cytochrome beta-559 appear as they are photooxidised at cryogenic temperatures by Photosystem II. The Signal II species is a donor close to the Photosystem II reaction centre and may represent part of the charge accumulation system of water oxidation. An EPR signal from an iron-sulphur centre which may represent an unidentified component of photosynthetic electron transport is also described. The properties of the oxygen evolving particles show that the preparation is superior to chloroplasts or unfractionated alga membranes for the study of Photosystem II with a functional water oxidation system.
A Fourier-transform infrared (FTIR) spectrum of the radical cation of beta-carotene photoinduced in photosystem II (PSII) membranes was obtained at 80K under oxidizing conditions, by utilizing the light-induced FTIR difference technique. Formation of the beta-carotene cation was monitored with the electronic absorption band at 993 nm. An FTIR spectrum of a chemically-generated beta-carotene cation in chloroform was also measured and compared with the spectrum of PSII. Since the FTIR bands of carotenoid cation have characteristic features with strong intensities, they can be useful markers in studying the reaction of carotenoid in PSII.
Saturation-recovery and progressive microwave power saturation EPR spectroscopies have been used to probe the location of the chlorophyllZ+ (ChlZ+) radical species in Mn-depleted photosystems II (PSII). The spin-lattice relaxation transients of ChlZ+ were non-single-exponential due to a dipole-dipole interaction with one of the other paramagnetic centers in PSII. Measurements on CN(-)-treated, Mn-depleted PSII membrane samples, in which the non-heme Fe(II) is converted into its low-spin, diamagnetic form, confirmed that the non-heme Fe(II) caused the dipolar relaxation enhancement of ChlZ+. The saturation-recovery EPR data were fit to a dipolar model [Hirsh, D. J., Beck, W. F., Innes, J. B., & Brudvig, G. W. (1992) Biochemistry 31, 532] which takes into account the isotropic (scalar) and orientation-dependent (dipolar) contributions to the spin-lattice relaxation of the radical. The temperature dependence of the dipolar rate constants of ChlZ+ was identical to the temperature dependencies recently observed for the stable tyrosine radical, YD., and the special pair bacteriochlorophyll radical, (BChla)2+, in PSII and in reaction centers from Rhodobacter sphaeroides, respectively. Because the non-heme Fe(II) is known to cause a dipolar relaxation enhancement of the radicals in both of the latter cases, this result provides further evidence that the non-heme Fe(II) causes the dipolar relaxation enhancement of ChlZ+ and, moreover, demonstrates that the magnetic properties of the non-heme Fe(II) in PSII and in reaction centers from Rhodobacter sphaeroides are very similar. By using the known Fe(II)-(BChla)2+ distance for calibration, we estimate the Fe(II)-ChlZ+ distance to be 39.5 +/- 2.5 A.(ABSTRACT TRUNCATED AT 250 WORDS)
Electron nuclear double resonance (ENDOR) and special triple (ST) resonance spectroscopies have been used to study the cation radicals of the primary donor, P680, and two secondary donor chlorophylls (Chl) in photosystem 2 (PS2). Two different preparations were employed, Tris-washed PS2 membranes and PS2 reaction centers (D1-D2-I-Cytb559 complex). One secondary donor Chl a cation radical, Chl1.+, was generated in the Tris-washed preparation, while the P680.+ radical cation and a further Chl a cation radical, Chl2.+, were produced in the reaction center preparation. The ENDOR spectrum of the primary donor radical cation of photosystem 1 (P700.+) is also presented for comparison. Hyperfine coupling constants for methyl groups have been measured for all three PS2 radical species and assigned by comparison with previously published spectra of Chl a radicals in vitro. Electron spin densities were calculated from these hyperfine couplings. Comparison of ENDOR spectral features with those of Chla.+ in vitro indicates similar values for Chl1.+ and Chl2.+ radicals but an apparent reduction in unpaired electron spin density for P680.+. It has been proposed from the more detailed studies of purple bacterial reaction centers that such a reduction in spin density can be interpreted as a delocalization over two Chl a molecules. Our calculations therefore suggest that P680.+ is a weakly coupled chlorophyll pair with 82% of the unpaired electron spin located on one chlorophyll of the pair at 15 K. Environmental or geometrical changes to the chlorin ring structure to give a novel monomeric primary donor are also possible.(ABSTRACT TRUNCATED AT 250 WORDS)
Inhibitors of the phytoene desaturase in carotene biosynthesis were tested in the enhanced rapid turnover of the D1 protein of photosystem II in high light exposure of Chlamydomonas reinhardtii cells. After 1 h high light on heterotrophically grown cells in the presence of norflurazon or fluridone, photosynthesis activity in vivo and PS II activity in vitro is lost. The D1 protein has disappeared. PS I activity is not affected, nor is the D2 protein. It is concluded that beta-carotene is essential for the assembly of the D1 protein into functional photosystem II. It is suspected that bleaching of beta-carotene in the reaction center of PS II by high light destabilizes the structure and triggers the degradation of the D1 protein.
Although fluorescence is widely used to study photosynthetic systems, the mechanisms that affect the fluorescence in photosystem II (PSII) are not completely understood. The aim of this study is to define the low-temperature steady-state fluorescence quenching of redox-active centers that function on the electron donor side of PSII. The redox states of the electron donors and acceptors were systematically varied by using a combination of pretreatments and illumination to produce and trap, at low temperature, a specific charge-separated state. Electron paramagnetic resonance spectroscopy and fluorescence intensity measurements were carried out on the same samples to obtain a correlation between the redox state and the fluorescence. It was found that illumination of PSII at temperatures between 85 and 260 K induced a fluorescence quenching state in two phases. At 85 K, where the fast phase was most prominent, only one electron-transfer pathway is active on the donor side of PSII. This pathway involves electron donation to the primary electron donor in PSII, P680, from cytochrome b559 and a redox-active chlorophyll molecule, ChlZ. Oxidized ChlZ was found to be a potent quencher of chlorophyll fluorescence with 15% of oxidized ChlZ sufficient to quench 70% of the fluorescence intensity. This implies that neighboring PSII reaction centers are energetically connected, allowing oxidized ChlZ in a few centers to quench most of the fluorescence. The presence of a well-defined quencher in PSII may make it possible to study the connectivity between antenna systems in different sample preparations. The other redox-active components on the donor side of PSII studied were the O2-evolving complex, the redox-active tyrosines (YZ and YD), and cytochrome b559. No significant changes in fluorescence intensity could be attributed to changes in the redox state of these components. The fast phase of fluorescence quenching is attributed to the rapid photooxidation of ChlZ, and the slow phase is attributed to multiple turnovers providing for further oxidation of ChlZ and irreversible photoinhibition. Significant photoinhibition only occurred at Chl concentrations below 0.7 mg/mL and above 150 K. The reversible oxidation of ChlZ in intact systems may function as a photoprotection mechanism under high-light conditions and account for a portion of the nonphotochemical fluorescence quenching.
Chlorophyll Z (ChlZ) is a redox-active chlorophyll (Chl) which is photooxidized by low-temperature (<100 K) illumination of photosystem II (PSII) to form a cation radical, ChlZ+. This cofactor has been proposed to be an "accessory" Chl in the PSII reaction center and is expected to be buried in the transmembrane region of the PSII complex, but the location of ChlZ is unknown. A series of single-replacement site-directed mutants of PSII were made in which each of two potentially Chl-ligating histidines, D1-H118 or D2-H117, was substituted with amino acids which varied in their ability to coordinate Chl. Assays of the wild-type and mutant strains showed parallel phenotypes for the D1-118 and D2-117 mutants: noncoordinating or poorly coordinating residues at either position decreased photosynthetic competence and impaired assembly of PSII complexes. Only the mutants substituted with glutamine (D1-H118Q and D2-H117Q) had phenotypes comparable to the wild-type strain. The ChlZ+ cation was characterized by low-temperature electron paramagnetic resonance (EPR), near-infrared (IR) absorbance, and resonance Raman (RR) spectroscopies in wild-type, H118Q, and H117Q PSII core complexes. The quantum yield of ChlZ+ formation is the same (approximately 2.5% per saturating flash at 77 K) for wild-type, H118Q, and H117Q, indicating that its efficiency of photooxidation is unchanged by the mutations. Similarly, the EPR and near-IR absorbance spectra of ChlZ+ are insensitive to the mutations made at D1-118 and D2-117. In contrast, the RR signature of ChlZ+ in H118Q PSII, obtained by selective near-IR excitation into the ChlZ+ cation absorbance band, is significantly altered relative to wild-type PSII while the RR spectrum of ChlZ+ in the H117Q mutant remains identical to wild-type. Shifts in the RR spectrum of ChlZ+ in H118Q reflect a change in the structure of the Chl ring, most likely due to a perturbation of the core size and/or extent of doming caused by a change in the axial ligand to Mg(II). Thus, we conclude that the axial ligand to ChlZ is H118 of the D1 polypeptide. Furthermore, we propose that H117 of the D2 polypeptide is the ligand to a homologous redox-inactive accessory Chl which we term ChlD. The Chl Z and D terminology reflects the 2-fold structural symmetry of PSII which is apparent in the redox-active tyrosines, YZ and YD, and the active/inactive branch homology of the D1/D2 polypeptides with the L/M polypeptides of the bacterial reaction center.
On illuminating chloroplasts with “short-wavelength” monochromatic light that supports oxygen evolution, spectral evidence was obtained for a new photoreactive chloroplast component, provisionally designated C550, which shows a reversible decrease of absorbance with a maximum at 550 mμ. The light-induced absorbance changes in C550 have been separated from those due to cytochromes in the same spectral region.
The light-induced decrease of absorbance in C550 appears to be independent of temperature, persisting even at -189° and is therefore likely to be linked to the primary light reaction associated with oxygen evolution in photosynthesis.