No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Identification of Q400, a high-potential electron acceptor of Photosystem II, with the iron of the quinone-iron acceptor complex
Abstract
Measurements of the area bounded by the variable fluorescence induction curve and the maximum fluorescence yield as a function of redox potential led I. Ikegami and S. Katoh ((1973) Plant Cell Physiol. 14, 829–836) to propose the existence of a high-potential electron acceptor, Q400 (Em7.8 = 360 mV), associated with Photosystem II (PS II). We have generated the oxidized form of this acceptor (Q+400) using ferricyanide and other oxidants in thylakoid membranes isolated from a mutant of Chlamydomonas reinhardtii lacking Photosystem I and the cytochrome complex. Q+400 was detected by a decrease in the extent of reduction of the primary quinone electron acceptor, QA, in a low-intensity light flash exciting PS II reaction centers only once. EPR measurements in the presence of Q+400 indicated the presence of new signals at g = 8, 6.4 and 5.5. These disappeared upon illumination at 200 K or upon reduction with ascorbate. Mössbauer absorption attributed to the Fe2+ of the QA-Fe2+ acceptor complex of PS II disappeared upon addition of ferricyanide due to the formation of Fe3+. The Fe2+ signal was restored by subsequent addition of ascorbate. All of these spectroscopic signals show similar pH-dependent (n = 1) midpoint potentials (approx. −60 mV / pH unit) and an Em7.5 = 370 mV. We assign the EPR signals to the Fe3+ state of the quinone-iron acceptor. Electron transfer to the Fe3+ is responsible for the decrease in QA reduction upon single-hit flash excitation. The properties of the redox couple are consistent with those of and we conclude that the iron of the QA-Fe acceptor complex is responsible for this species.
... Although the redox reaction of the non-heme iron is not directly involved in the electron transfer from Q A − to Q B , it can be preoxidized by an oxidant like ferricyanide and then be photoreduced through Q A − (Petrouleas and Diner 1986). An Fe 2+ /Fe 3+ FTIR difference spectrum is, thus, obtained upon single-flash illumination (Fig. 3) (Hienerwadel and Berthomieu 1995;Noguchi and Inoue 1995). ...
... Chemical redox titration using fluorescence, EPR, and FTIR detections have previously shown that the non-heme iron has the E m (Fe 2+ /Fe 3+ ) of ~+400 mV at pH 7 with pH dependence of about − 60 mV/pH in the range of pH 5.5 − 8.5 (Ikegami and Katoh 1973;Bowes et al. 1979;Petrouleas and Diner 1986;Deligiannakis et al. 1994;Noguchi and Inoue 1995). FTIR spectroelectrochemical measurements were performed to examine the effect of Mn depletion on the E m (Fe 2+ /Fe 3+ ) and the molecular mechanism of the pH dependence (Kato and Noguchi 2014;. ...
... Next, we investigated the mechanism of the pH dependence of E m (Fe 2+ /Fe 3+ ) using ATR-FTIR spectroelectrochemistry (Fig. 8a, b) . A linear pH dependence of E m (Fe 2+ /Fe 3+ ) was observed in the pH range of 5.0-8.5 (Fig. 8e), confirming the previous observations (Bowes et al. 1979;Petrouleas and Diner 1986). Further spectral analysis revealed that in addition to the deprotonation of D1-H215 at higher pH with a pK a of ∼5.6 in the Fe 3+ state (Fig. 8c), which is in agreement with the above ATR-FTIR result, carboxylate groups from some of the Glu/Asp residues located on the stromal side of PSII (Fig. 1b) were protonated at lower pH with a pK a of ∼5.7 in the Fe 2+ state (Fig. 8d). ...
Photosystem II (PSII) performs oxidation of water and reduction of plastoquinone through light-induced electron transfer. Electron transfer reactions at individual redox cofactors are controlled by their redox potentials, and the forward and backward electron flows in PSII are regulated by tuning them. It is, thus, crucial to accurately estimate the redox potentials of the cofactors and their shifts by environmental changes to understand the regulatory mechanisms in PSII. Fourier-transform infrared (FTIR) spectroelectrochemistry combined with a light-induced difference technique is a powerful method to investigate the mechanisms of the redox reactions in PSII. In this review, we introduce the methodology and the application of this method in the studies of the iron-quinone complex, which consists of two plastoquinone molecules, QA and QB, and the non-heme iron, on the electron-acceptor side of PSII. It is shown that FTIR spectroelectrochemistry is a useful method not only for estimating the redox potentials but also for detecting the reactions of nearby amino-acid residues coupled with the redox reactions.
... In bacterial RC this non-heme iron is coordinated by four histidines, and glutamate (Michel and Deisenhofer 1988), whereas the latter ligand in PS II is substituted by bicarbonate acting as a bidentate ligand (Hienerwadel and Berthomieu 1995;Ferreira et al. 2004). This difference in coordination might be responsible for the markedly lower redox potential of the couple Fe 3+ /Fe 2+ in PS II (Petrouleas and Diner 1986;Renger et al. 1987); its pH dependence, -60 mV/ pH unit between pH 6.1 and 8.5 indicates the deprotonation of D1His215, an iron ligand located at the Q B pocket (Berthomieu and Hienerwadel 2001). In PS II, non-heme iron can be oxidized from Fe 2+ to Fe 3+ in the dark by addition of potassium ferricyanide (Ikegami and Katoh 1973;Petrouleas and Diner 1986;. ...
... This difference in coordination might be responsible for the markedly lower redox potential of the couple Fe 3+ /Fe 2+ in PS II (Petrouleas and Diner 1986;Renger et al. 1987); its pH dependence, -60 mV/ pH unit between pH 6.1 and 8.5 indicates the deprotonation of D1His215, an iron ligand located at the Q B pocket (Berthomieu and Hienerwadel 2001). In PS II, non-heme iron can be oxidized from Fe 2+ to Fe 3+ in the dark by addition of potassium ferricyanide (Ikegami and Katoh 1973;Petrouleas and Diner 1986;. Under these conditions, after single excitation of PS II, a non-heme Fe 3+ is reduced by Q A in submillisecond time domain (Diner et al. 1991;Haumann and Junge 1994) followed by proton uptake probably by amino acid(s) at the stromal side of PS II (Bö gershausen and Junge 1995). ...
... In addition to these His ligands, other two His residues, D1-His272 and D2-His268, and a bicarbonate ion function as ligands to the non-heme iron [18,21,63]. Under physiological conditions, the non-heme iron is not involved in the electron transfer reaction from Q A to Q B [16,38,48], because of its high E m value of about +400 mV at pH 7.0 with a pH dependence of −60 mV/pH [8,25,44,49]. However, under oxidative conditions, e.g., in the presence of ferricyanide, the non-heme iron is oxidized to Fe 3+ and serves as an endogenous electron acceptor, and hence Fe 3+ is re-reduced to Fe 2+ by light illumination. ...
... Similarly, the value of β should depend on the oxidation state of Q B , i.e. be smaller if Q B is reduced. However, also this effect was neglected and only the possibility of a larger double hit in the first flash was included that can occur if the non-heme iron is in its oxidized Fe 3+ state [49,51,80]. The consequences of these limitations are discussed below in a separate section. ...
The oxygen-evolving complex (OEC) in photosystem II catalyzes the oxidation of water to molecular oxygen. Four decades ago, measurements of flash-induced oxygen evolution have shown that the OEC steps through oxidation states S0, S1, S2, S3 and S4 before O2 is released and the S0 state is reformed. The light-induced transitions between these states involve misses and double hits. While it is widely accepted that the miss parameter is S state dependent and may be further modulated by the oxidation state of the acceptor side, the traditional way of analyzing each flash-induced oxygen evolution pattern (FIOP) individually did not allow using enough free parameters to thoroughly test this proposal. Furthermore, this approach does not allow assessing whether the presently known recombination processes in photosystem II fully explain all measured oxygen yields during Si state lifetime measurements. Here we present a global fit program that simultaneously fits all flash-induced oxygen yields of a standard FIOP (2Hz flash frequency) and of 11-18 FIOPs each obtained while probing the S0, S2 and S3 state lifetimes in spinach thylakoids at neutral pH. This comprehensive data treatment demonstrates the presence of a very slow phase of S2 decay, in addition to the commonly discussed fast and slow reduction of S2 by YD and QB(-), respectively. Our data support previous suggestions that the S0→S1 and S1→S2 transitions involve low or no misses, while high misses occur in the S2→S3or S3→S0 transitions.
... Later, the redox midpoint potential of this component was shown to be pH-dependent, varying linearly from 450 to 350 mV between pH 6 and 8 ( Bowes et al. 1979). Electron paramagnetic resonance (EPR) and Mössbauer spectroscopy served to identify the component with the NHI (Petrouleas and Diner 1986). The pHdependence of the potential can be explained straightforwardly by coupling to a network of protonatable amino acid residues as shown by structure-based electrostatic calculations ( Ishikita and Knapp 2005). ...
Photosystem II (PSII), the light-driven water:plastoquinone (PQ) oxidoreductase of oxygenic photosynthesis, contains a nonheme iron (NHI) at its electron acceptor side. The NHI is situated between the two PQs QA and QB that serve as one-electron transmitter and substrate of the reductase part of PSII, respectively. Among the ligands of the NHI is a (bi)carbonate originating from CO2, the substrate of the dark reactions of oxygenic photosynthesis. Based on recent advances in the crystallography of PSII, we review the structure of the NHI in PSII and discuss ideas concerning its function and the role of bicarbonate along with a comparison to the reaction center of purple bacteria and other enzymes containing a mononuclear NHI site.
... Another potential explanation for the lack of a detectable Fe 2 Q 3 A EPR signal is the generation of an alternative state of the FeQ A electron acceptor complex, Fe 3 Q A ; this state has been generated in some studies in which certain exogenous acceptors were used, high pH values were employed, or PSII subunits were depleted (for example, see Ref. [68]). In this case, the oxidized Fe 3 can be reduced by Q 3 ...
Photosystem II (PSII) is a multisubunit complex, which catalyzes the photo-induced oxidation of water and reduction of plastoquinone. Difference Fourier-transform infrared (FT-IR) spectroscopy can be used to obtain information about the structural changes accompanying oxidation of the redox-active tyrosines, D and Z, in PSII. The focus of our work is the assignment of the 1478 cm−1 vibration, which is observable in difference infrared spectra associated with these tyrosyl radicals. The first set of FT-IR experiments is performed with continuous illumination. Use of cyanobacterial strains, in which isotopomers of tyrosine have been incorporated, supports the assignment of a positive 1478/1477 cm−1 mode to the C–O stretching vibration of the tyrosyl radicals. In negative controls, the intensity of this spectral feature decreases. The negative controls involve the use of inhibitors or site-directed mutants, in which the oxidation of Z or D is eliminated, respectively. The assignment of the 1478/1477 cm−1 vibrational mode is also based on control EPR and fluorescence measurements, which demonstrate that no detectable Fe2+QA− signal is generated under FT-IR experimental conditions. Additionally, the difference infrared spectrum, associated with formation of the S2QA− state, argues against the assignment of the positive 1478 cm−1 line to the C–O vibration of QA−. In the second set of FT-IR experiments, single turnover flashes are employed, and infrared difference spectra are recorded as a function of time after photoexcitation. Comparison to kinetic transients generated in control EPR experiments shows that the decay of the 1477 cm−1 line precisely parallels the decay of the D⋅ EPR signal. Taken together, these two experimental approaches strongly support the assignment of a component of the 1478/1477 cm−1 vibrational lines to the C–O stretching modes of tyrosyl radicals in PSII. Possible reasons for the apparently contradictory results of Hienerwadel et al. (Biochemistry 35 (1996) 15447–15460 and Biochemistry 36 (1997) 14705–14711) are discussed.
... It is known from literature that the non-haem iron at the acceptor side is oxidized in the time range of minutes [24,28]. The pH-dependence of its redox potential (60 mV/pH-unit) implies the uptake of one proton per electron [32,33]. Accordingly, its rereduction upon the first flash in some tens of microseconds [34] causes extra proton uptake in a long-term dark-adapted sample, which is absent upon the following flashes. ...
By exposing dark-adapted Photosystem II core particles to a series of light flashes, we aimed at kinetic resolution of proton release during the four steps of water oxidation. The signal-to-noise ratio was improved by averaging under repetitive dark adaptation. The previously observed kinetic damping of pH-transients by particle aggregation was prevented by detergent. The complicating superimposition of protolytic events at the donor side (water oxidation) and at the acceptor side (quinone oxido-reduction) was unravelled by characterizing the rate constants of electron and proton transfer at the acceptor side (QA− · nH+ + DCBQ → QA + DCBQ− + nH+: k = 1.7 · 106 M−1 //2 DCBQ− + 2H+ → DCBQ + DCBQH2: k = 4 · 108 M−1 s−1). Contrasting with the pronounced period of four oscillations of the oxygen-evolving centre, the extent of proton release was practically constant. The apparent half-rise time of the stepped acidification was shortened upon lowering of the pH (250 μs at pH 7.5, 70 μs at pH 6.0 and 12 μs at pH 5.2). This kinetic behaviour was independent of the nature and the concentration of the added pH-indicator. We conclude that this reflects the protolysis of several electrostatically interacting acids at the surface of the protein in response to a new positive charge on YZ+, and persisting upon electron transfer from the manganese cluster to YZ+.
In photosynthetic reaction centers from purple bacteria (PbRCs) and photosystem II (PSII), the photoinduced charge separation is terminated by an electron transfer between the primary (QA) and secondary (QB) quinones. Here, we investigate the electron transfer route, calculating the superexchange coupling (HQA-QB) for electron transfer from QA to QB in the protein environment. HQA-QB is significantly larger in PbRC than in PSII. In superexchange electron tunneling, the electron transfer via unoccupied molecular orbitals of the nonheme Fe complex (QA → Fe → QB) is pronounced in PbRC, whereas the electron transfer via occupied molecular orbitals (Fe → QB followed by QA → Fe) is pronounced in PSII. The significantly large HQA-QB is caused by a water molecule that donates the H-bond to the ligand Glu-M234 in PbRC. The corresponding water molecule is absent in PSII due to the existence of D1-Tyr246. HQA-QB increases in response to the Ser-L223···QB H-bond formation caused by an extension of the H-bond network, which facilitates charge delocalization over the QB site. This explains the observed discrepancy in the QA-to-QB electron transfer between PbRC and PSII, despite their structural similarity.
The iron-quinone complex in photosystem II (PSII) consists of the two plastoquinone electron acceptors, QA and QB, and a non-heme iron connecting them. It has been suggested that nearby histidine residues play important roles in the electron and proton transfer reactions of the iron-quinone complex in PSII. In this study, we investigated the protonation/deprotonation reaction of D1-H215, which bridges the non-heme iron and QB, using attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. Flash-induced Fe2+/Fe3+ ATR-FTIR difference spectra were measured with PSII membranes in the pH range of 5.0-7.5. In the CN stretching region of histidine, the intensity of a negative peak at 1094 cm-1, which was assigned to the deprotonated anion form of D1-H215, increased as the pH increased. Singular-value decomposition analysis provided a component due to deprotonation of D1-H215 with a pKa of ∼5.5 in the Fe3+ state, whereas no component of histidine deprotonation was resolved in the Fe2+ state. This observation supports the previous proposal that D1-H215 is responsible for the proton release upon Fe2+ oxidation [Berthomieu, C., and Hienerwadel, R. (2001) Biochemistry 40, 4044-4052]. The pH dependence of the 13C isotope-edited bands of the bicarbonate ligand to the non-heme iron further showed that deprotonation of bicarbonate to carbonate does not take place at pH <8 in the Fe2+ or Fe3+ state. These results suggest that the putative mechanism of proton transfer to QBH- through D1-H215 and bicarbonate around Fe2+ functions throughout the physiological pH range.
The photosynthetic oxygen evolution is schematically described by the model of Kok et al. [1]. An excited PS II reaction center is able to extract electrons from the oxygen evolving complex (OEC) in a 4-step sequence. The evolution of 1 molecule of O2 requires cooperation of 4 oxidizing equivalents. The OEC contains 4 Mn atoms, which can successively be oxidized and thus accommodate positive charges.
Crystals of 57Fe enriched reaction centers have been investigated by Mössbauer spectroscopy. The cytochrome irons are in the low spin ferric state. The non-heme iron of the electron accepting side is partly ferrous high spin and partly ferrous low spin (or ferric high spin). Under the conditions of the experiment sodium ascorbate reduces only one cytochrome iron into the ferrous low spin state. Membrane bound proteins become flexible at higher temperatures than proteins with a hydrophilic surface. They are also less flexible, at least up to temperatures of about 250 K.
The origin of photosynthesis research in Greece can be traced to the early 1960s, and the first dedicated laboratory was established by George Akoyunoglou in the Nuclear Reseach Center (now National Center for Scientific Research) Demokritos, in Athens. More photosynthesis groups subsequently emerged, in Demokritos and in the universities. Research in Greece benefited greatly from the links of Greek scientists with laboratories and personalities, primarily in the USA and western Europe. The local research output is a proportional part of global research and, more or less, in tune with the shifting priorities of the latter. The list of references provided includes only a sample of publications: it is not inclusive.
Ikegami and Katoh (1) observed that oxidation of chloroplasts with ferri-cyanide doubled the area over the fluorescence induction curve in the presence of DCMU. The area over the curve is considered to reflect the concentration of electron acceptors available to photoreduction from P680 before the inhibition site for DCMU. The discovery by Ikegami and Katoh therefore indicated the presence of an additional acceptor beside QA. The existence of this acceptor has later been confirmed and it has been named Q400 from its redox potential at pH 7 (around 400mV) (1,2,3). from spectroscopic evidence Diner and Petrouleas (3) identified Q400 as tne oxidised form of the Fe-ion on the acceptor side in PSII. Recently, it was observed that the oxidation state of Q400 modulated the inhibition of electron transport by DCMU in spinach chloroplasts in a pH-dependent manner (4) suggesting direct interactions between the Fe-ion and the binding site for DCMU.
In photosystem II (PS II) as in the bacterial photosynthetic reaction center (RC), two quinones QA and QB constitute the primary and secondary electron acceptors. The three-dimensional structure of the bacterial RC showed that a non-heme iron (Fe) is located between QA and QB. This Fe is liganded to four histidine residues, the side-chain of a glutamate residue provides the fifth and sixth ligands (1). In PS II, the Fe is conserved and four histidine residues of the apoprotein could constitute its ligands. There is no equivalent in PS II of the glutamate present in the bacterial RC. A number of experiments showed that a bicarbonate ion interacts with the protein in the surounding of Fe and could possibly be a Fe ligand (2). This bicarbonate can be extracted or exchanged by formate; this procedure affects electron transfer between QA and QB (3). In PS II, the Fe can be oxidized by ferricyanide (4). In this study, we aimed to determine and characterize the Fe ligands in PS II by light-induced FTIR difference spectroscopy. Therefore, we monitored the photoreduction of Fe in PS II preparations where it is oxidized by ferricyanide in the dark. We used PS II membranes trypsinized at pH 6.2 where a very large fraction of Fe is accessible to ferricyanide. In the FTIR difference spectrum, we can follow the changes of interaction between Fe and the protein moeity (notably histidine residues) as well as IR signals from the bicarbonate ion.
The reaction center of Photosystem II contains an iron atom which is likely to be located between the acceptor-side quinones QA and QB, in analogy with the structure in photosynthetic purple bacteria. The iron, which is probably not directly involved in the electron transfer between the quinones, can be oxidized by strong oxidants, such as ferricyanide and high-potential semiquinones in plants (but not in purple bacteria) and may then be observed by EPR (1,2). This offers an additional possibility to probe the local environment of the primary iron-quinone acceptor other than that provided by the reduced complex, QA-Fe, which is EPR detectable in plants as well as in bacteria. Measurements, utilizing the sensitivity of EPR to changes in the ligand geometry around the Fe(III) ion, have detected distortions in the protein environment when quinone acceptor analogs or acceptor-side inhibitors bind (2,3). In this communication we report on EPR studies of the acceptor side in the cyanobacterium Anacystis nidulans which allow comparisons with the quinone-iron complex in plants.
There were four doublets in the Mossbauer spectrum of cucumber ( Cucumis sativus L.) photosystem II (PS II) particles. According to the value of isomer shift and quadrupole splitting, they represented oxidized cytochrome b559 (Cyt-b559), reduced Cyt-b559, Fe3+-quinone (Q) complex, and Fe2+-Q complex respectively. After water-stress, the electron transport rate between Q(A) (primary quinone electron acceptor of PS II)/Q(B)(secondary quinone electron acceptor of PS II) was affected and the absorption doublets of Fe2+ disappeared, suggesting that the reduced Cyt-b559 and Fe2+-Q complex had been oxidized. The results indicated that water-stress had changed the redox status of iron in the iron-quinone complex. Iron took part in electron transport through the change from a state of reduction to oxidation.
Current concepts on Photosystem II reaction center (PSII RC) function are strongly influenced by the elucidation of the three-dimensional molecular structure of RC of purple bacteria1, and the recognized homology of the two types of RC regarding their core proteins and chromophores2,3. This led to the proposal, in almost all of the published models (e.g. ref. 2 & 29), of charge separation in PSII RC along only one branch of the chromophore system, as was found in bacteria1. This concept, however, did not give a satisfactory answer to the following questions: how is the high redox potential needed to oxidize water, achieved; what is the role of the other branch of chromophores (B); what is the role of the ubiquitous cytochrome b-559; what is the origin of efficient thermal energy dissipation in the RC; how to explain a number of anomalies concerning the overlapping of the acceptor and donor side of the RC? I have proposed recently4,5 a model for the function of PSII RC designed to incorporate several phenomena related to the variable yield of chlorophyll fluorescence during photosynthetic induction. This alternative model seems to give an answer to most of the above mentioned questions because, in it, the same currently accepted and most probable structure of PSII RC is involved in charge separation that includes all the chromophores present, and a dual role is assigned to the QB binding site.
Iron is not only the biologically most important transition metal, but, by fortunate circumstances, one of its isotopes, 57Fe, is also the most useful Mössbauer nucleus. Accordingly, nuclear gamma resonance, commonly known as the Mössbauer effect, has become a significant tool in the study of iron proteins. It complements earlier methods, in particular magnetic resonance techniques, and a comparison of the approaches is therefore in order. Distinctive features of the Mössbauer effect are
1.
the sensitivity to all 57Fe regardless of the oxidation and spin state of the iron as long as the atoms are bound in a solid, and
2.
the fact that, for a given sample, the experimentalist has control over temperature and magnetic field only.
The theoretical description of paramagnetic states and spin transitions was stimulated by the advent of EPR, and most of the formalism developed for EPR can readily be adapted to Mössbauer spectroscopy.
Synthesis and characterization of manganese (II, III) dimer with O-propionyl oxime of ortho-functionalised paranaphthoquinone-phthiocol(I), are described. The structural features within the inner coordination sphere are consistent with Mn(III) and Mn(II) centres, together with one of the independent ligands are coordinated in catecholate (CAT), and semiquinone (SQ), forms respectively. The thermochromic behaviour of complex and magnetic susceptibility data indicate effective charge transfers between metal and ligand. The latter studies are interpreted in terms of Heisenberg's isotropic model. Here both ferromagnetic (J=42 cm-1) and antiferromagnetic (J=-27 cm-1) exchanges occur at lower and higher temperatures with effective spin pairs (S=2,2) and (S=5/2, 3/2) respectively. The electron transport via SQ form of ligand results in such effective spin pairs at variable temperatures. The room temperature and glassy sample EPR spectra exhibit the Class-II mixed-valence nature of complex, indicating localized electron interaction with Mn(II) centre. The six hyperfine spin-allowed transitions are flanked by ten satellite lines. The line positions of allowed and forbidden doublets are computed with coupling constant A =100 G. The zero field splitting and quadrupole splittings are estimated as 220 G and 0.55 G respectively. In cyclic voltammograms, irreversible oxidation peaks at +1.195 V and+0.65 V together with reduction peak at+0.45 V are due to Mn (III) → Mn (IV) oxidation and Mn(II) (SQ)2/ Mn(III) (CAT)2 redox species in dimer respectively.
Mossbauer spectroscopy was applied, for the first time, to study the interaction of copper ions with the non-heme iron and the heme iron of cytochrome bssg in photosystem II thylakoids isolated from a Chlamydomonas reinhardtii photosystem I minus mutant. We showed that copper ions oxidize the heme iron and change its low spin state into a high spin state. This is probably due to deprotonation of the histidine coordinating the heme. We also found that copper preserves the non-heme iron in a low spin ferrous state, enhancing the covalence of iron bonds as compared to the untreated sample. We suggest that a disruption of hydrogen bonds stabilizing the quinone-iron complex by Cu 2+ is the mechanism responsible for a new arrangement of the binding site of the non-heme iron leading to its more "tense" structure. Such a diamagnetic state of the non-heme iron induced by copper results in a magnetic decoupling of iron from the primary quinone acceptor. These results indicate that Cu does not cause removal of the non-heme iron from its binding site. The observed Cu2+ action on the non-heme iron and cytochrome bsss is similar to that previously observed for α-tocopherol quinone.
Photosystem II (PS II) is a pigment-protein complex in thylakoid membranes from all oxygenic photosynthetic organisms (cyanobacteria and photosynthetic eukaryotes). It catalyzes the light-induced reduction of plastoquinone by water through a number of redox reactions. The electron transport chain in PS II is composed of various protein-bound components, which are held in close proximity and suitable orientation with respect to each other by the protein environment, so that a rapid and efficient electron transport is feasible. The PS II complex consists of at least five integral membrane proteins in the thylakoid (together forming the PS II “core complex”), in addition to several peripheral proteins. Many of these polypeptides interact with one or more components of the electron transport chain or with light-harvesting pigments, such that these protein ligands can fulfill a specific function in PS II.
This chapter discusses the primary reactions of photosystems I and II of algae and higher plants. In photosynthetic organisms, the “primary reactions” fulfil the objective of converting the energy of light into a primary form of chemical energy which lasts for a time compatible with ordinary biochemical processes—that is, milliseconds. In these reactions, a rather large fraction, approximately 40%, of the photon energy is stored as chemical free energy. The primary reactions can be viewed from two major perspectives. Firstly, from a photochemical point of view: pigment molecules are excited to their lowest excited singlet state, which reacts in an electron transfer reaction, the first step of a process of charge separation. Secondly, from a biochemical point of view the reactions take place in highly organized complexes, the reaction centres, which are made up of several classes of molecules that cooperate in fulfilling complementary roles, such as: architectural support, light absorption, energy transfer and electron transfer. All oxygenic organisms, ranging from cyanobacteria to algae and higher plants, contain photosystem I (PS I) and PS II reaction centres, with only minor variations in spite of their large taxonomic and ecological diversity.
Charge distribution in complexes containing quinone ligands coordinated with electroactive transition metal ions is determined by the relative energies of metal and quinone electronic levels. Intramolecular metal-quinone electron transfer may result from variations in the form of the complex which change the order of metal and quinone orbital energies. Iron complexes have been prepared with 3,5-di-t-butyl-l,2-benzoquinone (DBBQ). The structures of the complexes have been determined using crystallographic methods, the charge on the metal and the charge distribution in the molecule have been studied using spectroscopic techniques, and magnetic coupling between paramagnetic semiquinone ligands and the high spin ferric center has been investigated using variable temperature magnetic susceptibility measurements. Reactions carried out between 3,5-di-t-butylcatechol and ammonia in the presence of the divalent metal ions of Fe, Mn, and Co lead to neutral complexes of form ML2, where L is a tridentate ligand resulting from Schiff base condensation of two catecholate ligands. This ligand may exist in charges ranging from +1 to -3, and spectroscopic, structural and magnetic methods of analysis have been used to determine charge distribution in the complexes. EPR spectra indicate that complexes containing the more oxidized form of the ligand have surprisingly localized electronic structures.
It is known that inactivation of the Mn4CaO5 cluster, the catalytic center of water oxidation in photosystem II (PSII), induces a positive shift of the redox potential (Em) of the primary quinone electron acceptor QA by ~+150 mV, resulting in suppression of the electron transfer from QA to the secondary quinone acceptor QB. Although this Em(QA(-)/QA) shift has been argued in relevance to to photoprotection of PSII, its molecular mechanism is still enigmatic from a structural viewpoint because QA is ~40 Å distant from the Mn4CaO5 cluster. In this work, we have investigated the influence of Mn depletion on the Em of the non-heme iron, which is located between QA and QB, and its surrounding structure. Electrochemical measurements in combination with Fourier transform infrared (FTIR) spectroscopy revealed that Mn depletion shifts the Em(Fe(2+)/Fe(3+)) by +18 mV, which is ~8 times smaller than the shift of the Em(QA(-)/QA). Comparison of the Fe(2+)/Fe(3+) FTIR difference spectra between intact and Mn-depleted PSII samples showed that Mn depletion altered the pKa's of a His ligand to the non-heme iron, most probably D1-His215 interacting QB, and a carboxylate group, possibly D1-Glu244, coupled with the non-heme iron. It was further shown that Mn depletion influences the C≡N vibration of bromoxynil bound to the QB site, indicative of the modification of the QB binding site. Based on these results, we discuss the mechanism of a long-range interaction between the donor and acceptor sides of PSII.
The possibility to determine the difference spectra Δεi+1jλ of each univalent redox step Si→Si+1(i=0,...3) of the water-oxidizing enzyme system was analyzed by theoretical calculations and by measurements of 320 nm absorption changes induced by a train of saturating laser flashes (FWHM:7 ns) in PS II membrane fragments. It was found: a) Lipophilic quinones complicate the experimental determination of optical changes due the Si-state transitions because they lead to an additional binary oscillation probably caused by a reductant-induced oxidation of the Fe2+ at the PS II acceptor side. b) In principle, a proper separation can be achieved at sufficiently high K3[Fe(CN)6] concentrations. c) An unequivocal deconvolution into the difference spectra Δεi+1jλ of flash train-induced optical changes which are exclusively due to Si-state transitions is impossible unless the Kok parameters α, β and [Si]0 can be determined by an independent method.
Measurements of the oxygen yield induced by a flash train reveals, that in thylakoids and PS II membrane fragments Si is the stable state of dark adapted samples even at alkaline pH (up to pH=9). However, in PS II membrane fragments at pH>7.7 the misses probability α markedly increases, in contrast to the properties of intact thylakoids. Based on these data the possibility is discussed that an equilibrium exists of two types of S2-states with different properties.
Thermoluminescence (TL) from autotrophically and photoheterotrophically cultivated Chlamydobotrys stellata was measured. Strong TL was emitted at 30°C after acetatenutrition of the alga. DCMU enhanced this band, as also did ferricyanide. It also appeared after poisoning of the alga with NH2OH or ANT-2p. These observations suggest that an alternative donor to photosystem II and not the water-splitting system is responsible for the TL + 30 band.
Inhibition of electron flow from H2O to methylviologen by 3-(3′4′ dichlorophenyl)-1,1 dimethyl urea (DCMU), yields a biphasic curve — an initial high sensitivity phase and a subsequent low sensitivity phase. The two phases of electron flow have a different pH dependence and differ in the light intensity required for saturation.Preincubation of chloroplasts with ferricyanide causes an inhibition of the high sensitivity phase, but has no effect on the low sensitivity phase. The extent of inhibition increases as the redox potential during preincubation becomes more positive. Tris-treatment, contrary to preincubation with ferricyanide, affects, to a much greater extent, the low sensitivity phase.Trypsin digestion of chloroplasts is known to block electron flow between Q
A and Q
B, allowing electron flow to ferricyanide, in a DCMU insensitive reaction. We have found that in trypsinated chloroplasts, electron flow becomes progressively inhibited by DCMU with increase in pH, and that DCMU acts as a competitive inhibitor with respect to [H+]. The sensitivity to DCMU rises when a more negative redox potential is maintained during trypsin treatment. Under these conditions, only the high sensitivity, but not the low sensitivity phase is inhibited by DCMU.The above results indicate the existence of two types of electron transport chains. One type, in which electron flow is more sensitive to DCMU contains, presumably Fe in a Q
A Fe complex and is affected by its oxidation state, i.e., when Fe is reduced, it allows electron flow to Q
B in a DCMU sensitive step; and a second type, in which electron transport is less sensitive to DCMU, where Fe is either absent or, if present in its oxidized state, is inaccessible to reducing agents.
In the last few years our knowledge of the structure and function of Photosystem II in oxygen-evolving organisms has increased significantly. The biochemical isolation and characterization of essential protein components and the comparative analysis from purple photosynthetic bacteria (Deisenhofer, Epp, Miki, Huber and Michel (1984) J Mol Biol 180: 385-398) have led to a more concise picture of Photosystem II organization. Thus, it is now generally accepted that the so-called D1 and D2 intrinsic proteins bind the primary reactants and the reducing-side components. Simultaneously, the nature and reaction kinetics of the major electron transfer components have been further clarified. For example, the radicals giving rise to the different forms of EPR Signal II have recently been assigned to oxidized tyrosine residues on the D1 and D2 proteins, while the so-called Q400 component has been assigned to the ferric form of the acceptor-side iron. The primary charge-separation has been meaured to take place in about 3 ps. However, despite all recent major efforts, the location of the manganese ions and the water-oxidation mechanism still remain largely unknown. Other topics which lately have received much attention include the organization of Photosystem II in the thylakoid membrane and the role of lipids and ionic cofactors like bicarbonate, calcium and chloride. This article attempts to give an overall update in this rapidly expanding field.
A study was made of the fluorescence induction curves from gently-broken spinach chloroplasts inhibited with DCMU. It was found that there were four kinetically different phases associated with such curves of which only the fastest did not appear to follow exponential kinetics. A comparison of the effects of various concentrations of DCMU on the rate of oxygen evolution and on the fluorescence induction curve did not support the hypothesis that any of the kinetic phases was simply an artefact caused by incomplete inhibition of electron transport. It was also found that 5 min of dark incubation did not maximally oxidize the electron acceptors to photosystem 2 since some acceptors were only oxidized following far-red illumination, suggesting a heterogeneity among these acceptors with respect to their re-oxidation properties. Investigation of the effect of the Q400 oxidation state on the fluorescence induction curve revealed that it only influenced the slowest kinetic phase and that Q400 did not seem to be associated with the other phases.
It was found that DCMU had a differential effect at two concentration ranges on variable fluorescence kinetics in isolated chloroplasts. The increase in fluorescence rate at low concentrations of DCMU was abolished by preincubation of chloroplasts with ferricyanide or formate, treatments which were shown to convert Fe in the PS II reaction center (i.e., the FeQA complex) into a non-oxidizable form, but it was not affected by Tris treatment. Increase in fluorescence kinetics (at the initial linear rate) at high concentrations of DCMU was found to be abolished by Tris treatment but it was only marginally affected by ferricyanide or formate treatments. The effect of Tris could be abolished by addition of hydroquinone-ascorbate, which restored electron flow to the pool of secondary acceptors.
Contrary to the effect of DCMU, no such differential concentration dependence of the variable fluorescence kinetics was found for atrazine.
The increase in fluorescence kinetics (at the initial linear rate) at a low concentration rate of DCMU is presumably restricted to units which contain an oxidizable Fe in the FeQA complex. Increase in fluorescence kinetics (at the initial linear rate) at high DCMU concentration is probably related to the effect of DCMU at the QB site.
The protonophoric uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP), 2,3,4,5,6-pentachlorophenol (PCP) and 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole (TTFB) inhibited the Hill reaction with K3[Fe(CN)6] (but not with SiMo) in chloroplast and cyanobacterial membranes (the I50 values were approx. 1-2, 4-6 and 0.04-0.10 μM, respectively). The inhibition is due to oxidation of the uncouplers on the Photosystem II donor side (ADRY effect) and their subsequent reduction on the acceptor side, ie. to the formation of a cyclic electron transfer chain around Photosystem II involving the uncouplers as redox carriers. The relative amplitude of nanosecond chlorophyll fluorescence in chloroplasts was increased by DCMU or HQNO and did not change upon addition of uncouplers, DBMIB or DNP-INT; the HQNO effect was not removed by the uncouplers. The uncouplers did not inhibit the electron transfer from reduced TMPD or duroquinol to methylviologen which is driven by Photosystem I. These data show that CCCP, PCP and TTFB oxidized on the Photosystem II donor side are reduced by the membrane pool of plastoquinone (Qp) which is also the electron donor for K3 [Fe(CN)6] in the Hill reaction as deduced from the data obtained in the presence of inhibitors. Inhibition of the Hill reaction by the uncouplers was maximum at the pH values corresponding to the pK of these compounds. It is suggested that the tested uncouplers serve as proton donors, and not merely as electron donors on the oxidizing side of Photosystem II.
A series of four high-spin ferrous complexes of polydentate Schiff base ligands has been prepared and studied with IR, X-ray absorption and Mössbauer spectroscopy and variable-temperature magnetic susceptibility. The ligands include potentially tridentate and hexadentate Schiff bases with N2O and N4O2 donor sets, respectively, and result from the condensation of 5-nitrosalicylaldehyde with 2- (aminoethyl)pyridine or tetramines. The results obtained provide evidence that the four complexes described herein are mononuclear high-spin iron(II) species at room temperature. The crystal and molecular structure of [Fe(5NO2-salaep)2] (1) has been determined. 1 crystallizes in the orthorhombic system, space group Pbcn with Z=4 and a=14.756(3), b=9.682(3), c=18.632(4) Å. The structure was solved by the heavy-atom method and refined to conventional agreement indices R=0.042 and Rw=0.043 with 2845 unique reflections for which I>3σ. The structure of 1 consists of [Fe(5NO2-salaep)2] complex molecules stacked through π interactions involving the salicylaldimine rings of adjacent molecules to afford ribbons along the [001] direction. The central iron atom of each molecule is triply coordinated to two 5NO2-salaep ligands, affording a distorted coordination octahedron. The Mössbauer spectroscopy is consistent with an increase of the distortion of the iron(II) ligand environment in the series from Fe11(5NO2,-salaep)2] (1) to Fe11[5NO2-sal-N(1,5,9,13)] (4). The variable temperature magnetic susceptibility evidences appreciable zero-field splitting of the iron(II) ground state in [Fe11(5NO2-salaep)2] (1) and Fe11[5NO2-sal-N(1,5,8,12)] (3). 1 exhibits several properties required to afford a modelling of the iron center of the ‘ferroquinone complex’ of photosystem 2. Fe11[5NO2-sal-N(1,4,7,10)] (2) exhibits a thermally induced 5T2g ↔ 1A1g spin conversion with unprecedented features: iron(II) center in a N4O2 ligand environment, spin conversion of discontinuous nature occurring in two steps separated by a 30 K broad spin equilibrium domain in which c. 50% of high-spin and low-spin molecules coexist.
We have examined the orientation dependence of two EPR signals associated with the iron of the ferroquinone complex of Photosystem II. The first signal with g values (4.09, 3.95 and approx. 2.0) results from the reversible interaction of the ferrous ion with NO (Petrouleas et al. (1990) Biochim. Biophys. Acta 1015, 131–140). Studies of oriented spinach BBY membranes treated with NO show that gx lies on the membrane plane, gy is oriented at 30° and gz is oriented at 60° with respect to the membrane plane. The latter, most likely, indicates the approximate direction of the Fe-NO bond. Since NO displaces CO2/HCO−3 upon binding (Diner et al. (1990) Biochim. Biophys. Acta 1015, 141–149), the latter physiological ligand is probably well above the membrane plane containing the iron. This places CO2/HCO−3 at an approximately homologous position with the bacterial glutamate ligand (Deisenhofer and Michel (1989) EMBO J. 8, 2149–2169); it is not known, however, whether CO2/HCO−3 binds as a monodentate or bidentate ligand. The second signal with characteristic g values in the region 5.6–8.1 results from the oxidized non-heme iron (Fe3+) [3]. The orientation results show that the gx = 8.1 and gz = 5.6 resonances have both maxima in the plane of the membrane. The gy axis is, therefore, perpendicular to the plane of the membrane and runs along the homologous direction of the bacterial twofold symmetry axis. Based on the strong effect that molecules bound at the QB binding site have on the x-y plane anisotropy () but not on the axial field strength, D, and assuming basic similarities with the bacterial reaction center, it is suggested that, the x-y plane is defined by the two oxygen ligands and the two nitrogens from the histidine imidazoles which have contacts with the QA and QB site. The gz-axis lies then along the N-Fe-N direction defined by the nitrogen ligands from the other two histidines. An unusual finding is that the process of membrane orientation (slow drying of the membranes) induces an axial signal, gxy = 6, in a fraction of centers. The axial plane of the latter is perpendicular to the membrane in agreement with the orientation results of the more rhombic signals. A possible arrangement of the nitrogen and oxygen ligands in the equatorial plane (vertical to the membrane) as to impose pseudo-C3 symmetry is discussed.
It is well known that two photosystems, I and II, are needed to transfer electrons from H2O to NADP+ in oxygenic photosynthesis. Each photosystem consists of several components: (a) the light-harvesting antenna (L-HA) system, (b) the reaction center (RC) complex, and (c) the polypeptides and other co-factors involved in electron and proton transport. First, we present a mini review on the heterogeneity which has been identified with the electron acceptor side of Photosystem II (PS II) including (a) L-HA system: the PS IIα and PS IIβ units, (b) RC complex containing electron acceptor Q1 or Q2; and (c) electron acceptor complex: QA (having two different redox potentials QL and QH) and QB (QB-type; Q'B type; and non-QB type); additional components such as iron (Q-400), U (Em,7=−450 mV) and Q-318 (or Aq) are also mentioned. Furthermore, we summarize the current ideas on the so-called inactive (those that transfer electrons to the plastoquinone pool rather slowly) and active reaction centers. Second, we discuss the bearing of the first section on the ratio of the PS II reaction center (RC-II) and the PS I reaction center (RC-I). Third, we review recent results that relate the inactive and active RC-II, obtained by the use of quinones DMQ and DCBQ, with the fluorescence transient at room temperature and in heated spinach and soybean thylakoids. These data show that inactive RC-II can be easily monitored by the OID phase of fluorescence transient and that heating converts active into inactive centers.
The action of low pH treatment (pH 3.6) known to release Ca2+ from the oxygen-evolving complex in photosystem II (PSII) membranes and to induce Ca2+-reversible inhibition of electron transport at the acceptor side of PSII in thylakoid membranes (TM) was compared in PSII membranes and TM. The rate of the inactivation of electron transport by low pH was four times higher in TM than in PSII membranes. Ferricyanide accelerated the inactivation of PSII membranes but decreased it in the case of TM. Low pH treatment also greatly modified the fluorescence induction kinetics in both preparations, but significant differences have been found in the fluorescence induction kinetics of treated TM and PSII membranes. Calcium restored the electron transport activity and fluorescence induction kinetics in PSII membranes and TM, whereas diphenylcarbazide restored these functions only in PSII membranes. The reactivation of Ca-depleted PSII membranes was more effective in the dark, whereas the reactivation of TM required weak light. In the case of PSII membranes subjected to low pH citrate buffer, maximal reactivation was observed at 60 mM Ca2+ but for TM about 10 mM Ca2+ was required and 60 mM fully inhibited electron transport in TM during reactivation. These results indicate that the Ca-dependent inactivation of the acceptor side of PSII in TM after low pH treatment cannot be explained by the extraction of Ca2+ from the oxygen-evolving complex. It is rather suggested that the Ca2+ involved in this inhibition is bound to the acceptor side of the photosystem near to the QA-non-heme iron binding site and may participate in the binding of a polypeptide of the PSII light antenna complex to the PSII reaction center.
The process of photosynthetic water oxidation has been investigated by using a new type of water oxidation inhibitor, the alkyl hydrazones. Acetone hydrazone (AceH), (CHâ)âCNNHâ, inhibits water oxidation by a mechanism that is analogous to that of NHâOH. This involves binding to the water-oxidizing complex (WOC), followed by photoreversible reduction of manganese (loss of the Sâ â Sâ reaction). At higher AceH concentrations the Sâ state is reduced in the dark and Mn is released, albeit to a lesser extent than with NH²OH. Following extraction of Mn, AceH is able to donate electrons rapidly to the reaction center tyrosine radical Z{sup +} (¹â¶Â¹Tyr-Dâ protein), more slowly to a reaction center radical C{sup +}, and not at all to the dark-stable tyrosine radical D{sup +} (¹â¶Â°Tyr-Dâ protein) which must be sequestered in an inaccessible site. Unexpectedly, Clâ» was found not to interfere or compete with AceH for binding to the WOC in the Sâ state, in contrast to the reported rate of binding of N,N-dimethylhydroxylamine (CHâ)âNOH. The authors interpret the latter behavior as due to ionic screening of the thylakoid membrane, rather than a specific Cl site involved in water oxidation. AceH appears not to bind to the acceptor side of PSII as evidenced by normal EPR signals both for Q{sub A}â»Fe(II), the primary electron acceptor, and for the oxidized Fe(III) acceptor (Qâââ species), in contrast to that observed with NHâOH. AceH can be oxidized in solution by a variety of oxidants including Mn(III) to form a reactive diazo intermediate, (CHâ)âCNN, which reacts with carbonyl compounds. Oxidation to this diazo intermediate is postulated to be responsible for inhibition of the WOC.
Dipolar interactions of tyrosine D+ (Y+D) radical with the Mn-cluster in oxygen-evolving center (OEC) and with Fe2+ ion on the acceptor side of Photosystem II was studied by a novel electron spin echo method employing selective hole burning in EPR spectra with subsequent detection of the hole broadening. The mechanism of hole broadening is analyzed by fluctuation of the local dipolar field at the site of Y+D caused by a random spin flip of these transition metal ions. Experimental data was analyzed using a simple theory (Dzuba, S.A., Kodera, Y., Hara, H. and Kawamori, A. (1993) J. Magn. Res. A 102, 257–260). Based on this theory, the distance from Y+D to Fe2+ ion has been determined to be less than 52 Å in Mn-depleted PS II. The distance from Y+D to the spin center of the Mn-cluster in OEC was estimated to be 28 Å in S2 state and 30 Å in S1 state. Furthermore, the distribution of the distance was estimated to be in the range 4–6 Å.
We report here the pH dependence of the kinetics of the decay of variable chlorophyll a fluorescence after one or two actinic flashes in the absence or the presence of DCMU (3-(3,4-dichlorophenyl)-1,1-dimethyl-urea) in HCO2−-depleted or anion-inhibited spinach thylakoid membranes. All the reported effects of HCO3− removal are reversed by the addition of 5 mM HCO3−. The initial first-order component for the oxidation of QA− (the reduced primary plastoquinone acceptor of Photosystem II (PS II) by QB (the secondary plastoquinone acceptor) was reversibly inhibited in a pH-dependent manner in HCO3−-depleted membranes. After a single actinic flash, the half-time of QA− decay was 630 μs (amplitude, 29%) at pH 6.5 which changed to a value of 320 μs (amplitude, 66%) at pH 7.75. The rate and amplitude at pH 7.75 were approximately the same as found in the restored and control membranes which were pH independent over the same pH range. A similar observation was made after the second actinic flash. Thus, at alkaline pH HCO3−-depleted membranes behave as control membranes with respect to electron flow from QA to QB or to QB−. The time (t50) at which the [QA−] is 50% of the maximum [QA−] during the back reaction between QA− and the S2 state of the oxygen-evolving complex, in the presence of 5 μM DCMU, was increased from 1.3 s in control and restored samples to 5.3 s in HCO3−-depleted samples below pH 7.0, but was unaffected above pH 7.5 (2.3–2.9 s in all cases). Furthermore, a new pathway of QA− with a half-time of less than 100 μs was present at pH 8.0 in the presence of DCMU, in approx. one-third of the PS II centers in HCO3−-depleted membranes. The apparent equilibrium for the sharing of an electron between QA and QB is estimated to decrease by a factor of 4 at pH 6.0 in treated membranes (Kapp ≈ 16) as compared to the restored or control membranes (Kapp ≈ 62); there was no difference in Kapp at pH 7.75. Estimates of the operating redox potential for the QB/QB− couple from the results presented here indicated that the pH dependence of this parameter is greatly reduced in treated membranes (−60 mV at pH 6.0 to −72 mV at pH 7.75) as compared to restored or control membranes (−25 mV at pH 6.0 to −72 mV at pH 7.75). We iscuss our results in the context of a model that envisages HCO3− to act as a proton donor to the protein dissociable group believed to participate in the protonation of QB−. Finally, the possibility of HCO3− being a ligand to Fe2+ in the QA-Fe-QB complex of the PS II reaction center is also discussed.
The temperature dependence of S-state transition kinetics was measured by spectroscopy in the ultraviolet region with O2-evolving Photosystem II particles from a thermophilic cyanobacterium. By proper selection of ferricyanide concentration and the measuring wavelength, the absorption changes due to S-state transitions were explicitly measured with minimized superposition of binary absorption changes due to acceptor side reactions. The half-times of S1 → S2, S2 → S3 and S3 → S0 transitions were 60, 60 and 800 μs, respectively, at 50°C, the optimal temperature for oxygen evolution by the particles, but were slowed down to 70, 120–150 and 1300 μs at 25°C, and 106, 300 and 5500 μs at 1°C, respectively. In the whole range of 1–50°C the Arrhenius plots showed no break or discontinuity for S1 → S2 and S2 → S3 transitions, with apparent activation energies of 9.6 and 26.8 kJ/mol, respectively. The Arrhenius plot for S3 → S0 transition, however, was composed of two straight lines with a clear break at 16°C, and the apparent activation energies above and below the break temperature were 15.5 and 59.4 kJ/mol, respectively. The implications of these data and especially of the break temperature for the S3 → S0 transition were discussed.
The effects of vitamin K1 (phylloquinone) addition on the turnover of the FeS centers in photosysten, I photochemistry were studied in diethyl ether-extracted spinach photosystem I particles. Reconstitution of one Molecule of vitamin K1/reaction center was sufficient to suppress the charge recombination between the oxidized reaction center chlorophyll P700+ and the reduced electron acceptor intermediate chlorophyll a and to restore the turnover of FeS centers in the ether-extracted particles. This strongly suggests that reconstituted vitamin K1, functions the primary electron acceptor A1 and exhibits a redox midpoint potential low enough to reduce the FeS centers. The quinone binding site in photosystem I, which enables vitamin K1 to show such a low redox potential, seems to be more hydrophobic than those in reaction centers of photosystem II or purple bacteria.
The hydroxyparaquinone complex bis(phthiocolato)bis(pyridine)manganese(II) has been synthesized and characterized spectrally and by X-ray crystallography.
In the companion paper (Petrouleas, V., Deligiannalcis, Y. and Diner, B.A. (1994) Biochim. Biophys. Acta 1188, 260–270) we examined the effects of a number of carboxylate anions on the EPR signals associated with the iron quinone complex, the ligand field parameters of the Fe3+, and the redox properties of the iron. In this paper, we show that three representative anions, glycolate, glyoxylate, and oxalate, compete with NO, formate and bicarbonate for binding to the iron. Furthermore, the bound anions affect diversely the electron transfer rate. Glycolate has an extreme effect, similar to what is observed with high levels of formate, and is characterised by a dissociation constant, Kd, ∼ 0.5–0.7 mM. Oxalate gives a marked slowing of the rate of Q−A oxidation on all flashes but preserves a marked oscillation of the rate of period two. Glyoxylate appears to have an intermediate effect. These results offer new information on the stereochemistry of the binding of dissociable ligands to the non-heme iron of PS II and a tool for probing the redox chemistry of the iron and the electron transfer properties of the iron-quinone complex.
We have examined the effects of a number of carboxylate anions on the iron-quinone complex of Photosystem II (PS II). Typical effects are the following. In the state Q−AFe2+ oxalate enhances significantly the g = 1.84 EPR resonance while, for example, glycolate and glyoxylate suppress it. The anions have variable effects on the iron midpoint potential. Formate and oxalate raise significantly the Em of the iron. Glycolate lowers the Em significantly and the Em shows a weak pH dependence. In the presence of glycolate the native plastosemiquinone () can oxidise the iron. Glyoxylate also lowers the Em, but the Em shows a greater pH dependence than with glycolate but still weaker than the −60 mV/pH unit of the untreated iron. The Fe3+ EPR spectra are characterised by small but distinct shifts, while in addition an unusual resonance at close to g = 4.3 is observed. These as well as the temperature dependence of the spectra are analysed by a spin-Hamiltonian model. Comparison with competition studies in the companion paper indicates that the anions bind as iron ligands displacing bicarbonate.
The state of iron in a purified oxygen-evolving core complex from Phormidium laminosum was characterized using Mössbauer spectroscopy at 77 K. Mössbauer spectra of the sample, as prepared, were fitted by two quadrupole pairs corresponding to Fe of high spin (2+) and low spin (2+). The signal of the high-spin (2+) species disappeared in the presence of the oxidant K3Fe(CN)6. After dialysis of the oxidized sample and addition of the reductant Na2S2O4, the spectra recovered the original shape, and the signals corresponding to the high-spin (2+) and low-spin (2+) species reappeared. Furthermore, light excitation of the sample at 200 K, previously oxidized with K3Fe(CN)6 at 4°C, induced the accumulation of the high-spin (2+) species. Based on Mössbauer and optical spectroscopy, we ascribe the high-spin and low-spin species to the Fe of the iron-quinone complex and to both cytochrome b-559 and c-549, respectively. The Mössbauer results also indicate that in this cyanobacterium the Fe of the iron-quinone complex can undergo redox changes induced either chemically or by light. Moreover, we observe approximately one cytochrome b-559 per reaction center in this preparation.
We have studied the EPR signal in PS II from Phormidium laminosum with a g-value of 1.66, which we assign to an interaction between the semiquinones of Qa and Qb and the non-heme iron. 77 K illumination of samples from dark-poised redox titrations show the rise of the signal has a midpoint potential (Em) of about +60 mV, and it is lost with an Em of about −10 mV. Under the same conditions, the rise of the g = 1.9 signal from Q·−a-Fe2+ in the dark was found to be about +10-mV. The g = 1.66 signal can also be formed with a high yield by first illuminating dark-adapted PS II particles at 293 K, followed by a short period of darkness at 273 K and subsequent illumination at 77 K. We have measured the effect on signal yield of varying the period of darkness following 293 K illumination. Over 60% of the maximum signal size is seen after 1 min darkness, and increases further over 2 h. In these samples a signal attributed to Q·−b-Fe2+ is seen prior to 77 K illumination. Confirmation of the presence of Q·t-b was obtained by reductant-linked oxidation of the non-haem iron using phenyl-para-benzoquinone (PPBQ). Samples treated with the Qb-analogue tribromotoluquinone (TBTQ) give a modified EPR signal. We propose (i) that Qb is preserved in PS II preparations from P. laminosum; (ii) that Qb-semiquinone can be readily formed and trapped by freezing; and (iii) the g = 1.66 signal arises from a coupling between the primary and secondary plastosemiquinones and the non-haem iron.
The effects of ferricyanide on Photosystem II reactions have been investigated by measurements of microsecond and millisecond prompt fluorescence and microsecond-delayed fluorescence in dark-adapted chloroplasts: (1) Titrations using ferri-ferrocyanide mixtures on: (a) the fast phase of the increase in fluorescence yield observed during a xenon flash, and (b) the normalised area above the millisecond fluorescence induction curve for chloroplasts inhibited by DCMU, showed a pH dependent mid point potential of 400 mV at pH 7.0 which varied by approx. -60 mV/pH unit between pH 6 and 8.5. (2) A saturating laser flash induced a fluorescence increase (as monitored by a weak measuring beam) of only 50% of that reached following a second flash in chloroplasts preincubated with ferricyanide and inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) prior to illumination. In the absence of ferricyanide, the fluorescence level reached after a single flash was initially close to that measured after a second flash (although the level subsequently declined). (3) The initial amplitude of the microsecond-delayed fluorescence excited by a single laser flash was diminished in chloroplasts dark-adapted with ferricyanide. In the presence of DCMU and ferricyanide, the amplitude was also diminished for the first flash of a series, but subsequently enhanced above the level obtained in chloroplasts in the presence of DCMU alone. (4) The above effects were not seen if DCMU was added to the chloroplasts before ferricyanide, or if the period of incubation with ferricyanide was much less than 4 min. (5) These results suggested the presence of a second acceptor Q2, with Em7 = 400 mV and n = 1, before the DCMU block in Photosystem II. There is 0.35--1 equivalent of the acceptor per reaction centre, and its reduction occurs within less than 5 mus. The role of the acceptor in double turnovers of the photochemistry during a single flash and its likely operating redox potential are discussed.
Pulse-labeling of wild-type and a Photosystem II mutant strain of Chlamydomonas reinhardtii was carried out in the presence or absence of inhibitors of either cytoplasmic or chloroplast ribosomes, and their thylakoid membrane polypeptides were analyzed by polyacrylamide gel electrophoresis. A pulse-chase study was also done on the wild-type strain in the presence of anisomycin, an inhibitor of protein synthesis on cytoplasmic ribosomes. The following results were obtained: the Photosystem II reaction center is mainly composed of integral membrane proteins synthesized within the chloroplast. Several of the proteins of the Photosystem II reaction center are post-translationally modified, after they have been inserted in the thylakoid membrane.
Photoreduction of the intermediary electron acceptor, pheophytin (Pheo), in photosystem II reaction centers of spinach chloroplasts or subchloroplast particles (TSF-II and TSF-IIa) at 220 K and redox potential E(h) = -450 mV produces an EPR doublet centered at g = 2.00 with a splitting of 52 G at 7 K in addition to a narrow signal attributed to Pheo([unk]) (g = 2.0033, DeltaH approximately 13 G). The doublet is eliminated after extraction of lyophilized TSF-II with hexane containing 0.13-0.16% methanol but is restored by reconstitution with plastoquinone A (alone or with beta-carotene) although not with vitamin K(1). TSF-II and TSF-IIa are found to contain approximately 2 nonheme Fe atoms per reaction center. Incubation with 0.55 M LiClO(4) plus 2.5 mM o-phenanthroline (but not with 0.55 M LiClO(4) alone) decreases this value to approximately 0.6 and completely eliminates the EPR doublet, but photoreduction of Pheo is not significantly affected. Partial restoration of the doublet (about 25%) was achieved by subsequent incubation with 0.2 mM Fe(2+), but not with either Mn(2+) or Mg(2+). The Fe removal results in the development of a photoinduced EPR signal (g = 2.0044 +/- 0.0003, DeltaH = 9.2 +/- 0.5 G) at E(h) = 50 mV, which is not observed after extraction with 0.16% methanol in hexane. It is ascribed to plastosemiquinone no longer coupled to Fe in photosystem II reaction centers. The results show that a complex of plastoquinone and Fe can act as the stable "primary" electron acceptor in photosystem II reaction centers and that the interaction of its singly reduced form with the reduced intermediary acceptor, Pheo([unk]), is responsible for the EPR doublet.
Preincubation of isolated chloroplasts with ferricyanide, prior to addition of DCMU, unmasks a high-potential electron acceptor (Q400) in Photosystem II that acts as an additional quencher and prolongs the fluorescence induction curve in the presence of DCMU (Ikegami, I. and Katoh, S. (1973) Plant Cell Physiol. 14, 829–836). This study confirms that Q400 is endogenous to Photosystem II and is not a bound ferricyanide, and several new characteristics of this high potential acceptor are established. (a) It is accessible to ferricyanide even in the presence of DCMU. The rate of oxidation, however, is very slow, consistent with access only via QA. Accessibility may be enhanced by magnesium, reminiscent of the oxidation of Q−A by ferricyanide. (b) Oxidation of Q400 drastically suppresses the binding of DCMU at neutral and alkaline pH. Below pH 6, however, DCMU binding is essentially normal. The pH dependence of DCMU binding is consistent with the known pH dependence of the redox midpoint potential of Q400. (c) Binding of many other inhibitors of QA-to-QB electron transfer is much less affected or even completely unaffected. These results have implications for current notions of herbicide binding and may also bear on the origin of slow phases of fluorescence induction in the presence of DCMU.
The light-induced rise of chlorophyll fluorescence in the presence of DCMU was measured in intact chloroplasts (class A), mildly shocked chloroplasts (class D) and ruptured chloroplasts (class C). Kinetic analysis of the rise curves revealed: 1. Class A chloroplasts at 3·10−6 M DCMU show a biphasic rise in variable fluorescence (Fv) with the slow phase comprising only 7% of Fv but 45% of the total area over the induction curve. 2. The slow rise component in class A chloroplasts is inhibited with increasing concentrations of DCMU. Only about 2% slow Fv and 10% slow area component persist 3·10−4 M DCMU. 3. By mild osmotic shock of class A chloroplasts the slow rise phase is substantially suppressed already at 3·10−6 M DCMU. 4. In class C chloroplasts, with grana structure damaged, a slow component is observed which cannot be eliminated by high DCMU concentrations. The relative contribution of this slow phase increases with decreasing size of thylakoid fragments. It is concluded that the properties of the slow fluorescence rise component, and consequently the apparent PS II heterogeneity, are decisively influenced by the degree of chloroplast and grana integrity. The slow Fv appears to reflect a PS II reaction with an unusually low affinity to DCMU.
When dark-adapted spinach chloroplasts are illuminated by an oversaturating laser pulse (total duration less than 150 ns) a double advancement in the S-states (S1 → S3) is observed, as revealed by the relatively high oxygen yield measured after a second flash. The oxygen evolved on the second (Y2) and the third (Y3) flashes of a series has been measured as a function of the energy of a first laser pulse; the half-saturating energy for Y2 is about 7-times higher than that for Y3. This high energy requirement for Y2 shows that double photoreactions occur during the course of the first laser pulse. Experiments with double laser pulses show that for a fraction of the centers, the turnover time is limited by a submicrosecond dark step, which could conceivably be the reduction of photooxidized chlorophyll by a secondary donor. Under the same experimental conditions, Chlorella cells do not undergo double photoreactions upon illumination by a laser pulse. The absence of such efficient double photoreactions has already been reported (Jursinic, P. (1981) Biochim. Biophys. Acta 635, 38–52) using Chlorella or pea chloroplasts. In the presence of a saturating concentration of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (20 μM DCMU), chloroplasts are able to evolve oxygen with a maximum on the second flash. The inhibition is 89% for the second and close to 99% for the third flash. These results indicate that two electron-acceptot sites are accessible during the course of a laser pulse. The probability of charge stabilization for the second photoreaction always remains low in chloroplasts and is close to zero for Chlorella cells.
Tris-washed chloroplasts were submitted to saturating short flashes, and then rapidly mixed with dichlorophenyldimethylurea (DCMU). The amount of singly reduced secondary acceptor was estimated from the DCMU-induced increase in fluorescence, caused by the reverse electron flow from secondary to primary acceptor. The back-transfer from the singly reduced secondary acceptor to the primary acceptor Q induced by DCMU addition affects only a part (60%) of the variable fluorescence (ΔFmax). As previously shown, the quenchers involved in this phenomenon, ‘B-type’ quenchers, are different from those controlling the complementary part of the fluorescence, the non-B-type. In this report, we show that at pH 8.5 in the B-type systems, there exist two kinds of secondary electron acceptors: B, a two-electron acceptor, the corresponding Q accounting for 40% of the variable fluorescence; B′, a one-electron acceptor, the corresponding Q accounting for 20% of the variable fluorescence. The lifetimes of B− and B′− in the absence of DCMU are 40 and 1 s, respectively. The primary acceptors of the B and B′ systems can be considered as corresponding to the Q1s defined previously (Joliot, P. and Joliot, A. (1981) in Proceedings of the 5th International Congress on Photosynthesis (Akoynoglou, G., ed.), pp. 885–899, Balaban International Science Services, Philadelphia). The B′ centers seems to be equivalent to the Qβ centers as defined by other workers (Van Gorkom, H.J., Thielen, A.P.G.M. and Gorren, A.C.F. (1982) in The Function of Quinones in Energy Conserving Systems (Trumpower, B.L., ed.), Academic Press, New York, in the press).
Incubation of PS II membranes with herbicides results in changes in EPR signals arising from reaction centre components. Dinoseb, a phenolic herbicide which binds to the reaction centre polypeptide, changes the width and form of the EPR signal arising from photoreduced Q−AFe. o-Phenanthroline slightly broadens the Q−AFe signal. These effects are attributed to changes in the interaction between the semi-quinone and the iron. DCMU, which binds to the 32 kDa protein, has virtually no effect on the width of the Q−AFe signal but does give rise to an increase in its amplitude. This could result from a change in redox state of an interacting component. Herbicide effects can also be seen when Q−AFe is chemically reduced and these seen to be reflected by changes in splitting and amplitude of the split pheophytin− signal. Dinoseb also results in the loss of ‘Signal II dark’, the conversion of reduced high-potential cytochrome b559 to its oxidized low-potential form and the presence of transiently photooxidized carotenoid after a flash at 25°C; these effects indicate that dinoseb may also act as an ADRY reagent.
K4W(CN)8 · 2 H2O was prepared by the reduction of WO (in potassium cyanide medium) by KBH4. The reduction proceeds on the slow, drop-by-drop addition of concentrated acetic acid. The synthesis is simple and efficient because the reduction and complexation are accomplished at the same time.Darstellung von K4W(CN)8 · 2 H2OK4W(CN)8 · 2 H2O ist hergestellt worden durch Reduktion von WO mit KBH4 in Gegenwart von KCN. Die Reduktion findet whrend langsamer Zugabe von Essigsure statt. Reduktion und Komplexbildung finden gleichzeitig statt, daher wird eine gute Ausbeute erhalten.
The stoichiometry of chlorophyll/Photosystem II was determined in pea thylakoids. The concentration of Photosystem II was determined by the absorption change at 325 nm. When the 325 nm measurement was made on the first flash in the presence of ferricyanide, the Photosystem II absorption change was found to increase by up to 100% of the same measurement made in the absence of ferricyanide. The increase in absorption change in the presence of various amounts of ferricyanide was found to correlate well with the increase in area above the Chl a fluorescence induction curve. Also, the dark recovery of both the 325 nm absorption change and the area above the Chl a fluorescence curve are similar and in the order of several minutes. Absorption changes made under repetitive flash excitation showed no increase in signal with the addition of ferricyanide. We conclude that there are two acceptors, Qa and Q400, for each active oxygen-evolving complex and only Qa is involved in active electron transport to Photosystem I.
Redox titration of the electrochromic carotenoid band shift, detected at 50 μs after a saturating actinic flash, in spinach chloroplasts, shows that only one electron acceptor in Photosystem II participates in a transmembrane primary electron transfer. This species, the primary quinone acceptor, Q, shows only one midpoint potential (Em,7.5) of approx. 0 V and is undoubtedly equivalent to the fluorescence quencher, QH. A second titration wave is observed at low potential () and at greater than 3 ms after a saturating actinic flash. This wave has an action spectrum different from that of Photosystem II centers containing Q and could arise from a secondary but not primary electron transfer. A low-potential fluorescence quencher is observed in chloroplasts which largely disappears in a single saturating flash at − 185 mV and which does not participate in a transmembrane electron transfer. This low-potential quencher (probably equivalent to fluorescence quencher, QL) and Q are altogether different species. Redox titration of C550 shows that if electron acceptor Qβ is indeed characterized by an Em,7 of + 120 mV, then this acceptor does not give rise to a C550 signal upon reduction and does not participate in a transmembrane electron transfer. This titration also shows that C550 is not associated with QL.
Thylakoid membrane protein phosphorylation affects photochemical reactions of Photosystem II. Incubation of thylakoids in the light with ATP leads to: (1) an increase in the amplitude of three components (4–6, 25–45 and 280–300 μs) of delayed light emission after a single flash without any change in their kinetics; (2) a reduction of the flash-dependent binary oscillations of chlorophyll a fluorescence yield associated with electron transfer from the primary quinone acceptor, Q, to the secondary quinone acceptor, B; (3) an increase in the ratio resulting from an increase in stability of the semiquinone anion during dark adaptation; and (4) no change in the redox state of the plastoquinone pool as determined by flash-induced photooxidation of the Photosystem I reaction center, P-700. All the above observations are reversible upon dephosphorylation of the thylakoid membranes. These data are explained by a protein phosphorylation-induced stabilization of the bound semiquinone anion, B−. It is proposed that this increased stability may be due to an alteration in the accessibility of an endogenous reductant to B, or to an increase in dissipative cycling of charge around Photosystem II.
57Fe Mössbauer spectroscopy measurements on reaction centers differing in ubiquinone content, detergent, oxidation state, or the presence of o-phenanthroline all show a single quadrupole doublet of similar splitting (ΔEQ), center shift (δ) and temperature dependence. The results are indicative of high-spin Fe2+ with an approximately invariant first coordination sphere. A crystal field model with strong electron delocalization can account for the temperature dependence of ΔEQ, but further data are needed to achieve a unique parameterization.
Detailed absorbance difference spectra are reported for the Photosystem II acceptor Q, the secondary donor Z, and the donor involved in photosynthetic oxygen evolution which we call M. The spectra of Z and Q could be resolved by analysis of flash-induced kinetics of prompt and delayed fluorescence, EPR signal IIf and absorbance changes in Tris-washed system II preparations in the presence of ferricyanide and 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU). The spectrum of Z oxidation consists mainly of positive bands at 260, 300 and 390–450 nm on which a chlorophyll a band shift around 438 nm is superimposed, and is largely pH-independent as is also the case for the spectrum of Q reduction. The re-reduction of Z+ occurred in the millisecond time range, and could be explained by a competition between back reaction with Q− (120 ms at pH 6.0) and reduction by ferrocyanide. When the Tris treatment is omitted the preparations evolve oxygen, and the photoreduction of Q (with DCMU present) is accompanied by the oxidation of M. The Q spectrum being known, the spectrum of the oxidation of M could be determined as well. It consists of a broad, asymmetric increase peaking near 305 nm and of a Chl a band shift, which is about the same as that accompanying Z in Tris-washed system II. Comparison with spectra of model compounds suggests that Z is a bound plastoquinol which is oxidized to the semiquinone cation and that the oxidation of M is an Mn(III) → Mn(IV) transition.
A study of signals, light-induced at 77 K in O2-evolving Photosystem II (PS II) membranes showed that the EPR signal that has been attributed to the semiquinone-iron form of the primary quinone acceptor, Q−AFe, at g = 1.82 was usually accompanied by a broad signal at g = 1.90. In some preparations, the usual g = 1.82 signal was almost completely absent, while the intensity of the g = 1.90 signal was significantly increased. The g = 1.90 signal is attributed to a second EPR form of the primary semiquinone-iron acceptor of PS II on the basis of the following evidence. (1) The signal is chemically and photochemically induced under the same conditions as the usual g = 1.82 signal. (2) The extent of the signal induced by the addition of chemical reducing agents is the same as that photochemically induced by illumination at 77 K. (3) When the g = 1.82 signal is absent and instead the g = 1.90 signal is present, illumination at 200 K of a sample containing a reducing agent results in formation of the characteristic split pheophytin− signal, which is thought to arise from an interaction between the photoreduced pheophytin acceptor and the semiquinone-iron complex. (4) Both the g = 1.82 and g = 1.90 signals disappear when illumination is given at room temperature in the presence of a reducing agent. This is thought to be due to a reduction of the semiquinone to the nonparamagnetic quinol form. (5) Both the g = 1.90 and g = 1.82 signals are affected by herbicides which block electron transfer between the primary and secondary quinone acceptors. It was found that increasing the pH results in an increase of the g = 1.90 form, while lowering the pH favours the g = 1.82 form. The change from the g = 1.82 form to the g = 1.90 form is accompanied by a splitting change in the split pheophytin− signal from approx. 42 to approx. 50 G. Results using chloroplasts suggest that the g = 1.90 signal could represent the form present in vivo.
Detergent-treatment of higher plant thylakoids with Triton X-100 at pH 6.3 has been used to purify a PS2 fraction with very high rates of oxygen evolution (1000 μmol.mg chl−1.h−1). A photosynthetic unit size of about 300 chlorophyll (chl) molecules has been determined by optical methods, suggesting an average turnover time for PS2 of about 2 ms. The donor system for P680+ is particularly well preserved in the preparation, as judged by P680+ reduction kinetics, the detection by EPR of Signal IILT and the presence of the high potential form of cytochrome b-559 (at a ratio of 1:1 with the reaction centre).
1.1. The kinetics of prompt and delayed fluorescence of isolated chloroplasts or algae have been monitored after flash preillumination (in the time-range extending from 0.4 s after the flash). A rapid mixing with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) may take place after the last flash.2.2. 1 s after the mixing with DCMU, the prompt fluorescence displays binary oscillations with the number of preilluminating flashes, similarly to the observation of Velthuys, B.R. and Amesz, J. ((1974) Biochim. Biophys. Acta 333, 85–94), with chloroplasts to which an artificial Photosystem II donor was added. These oscillations are due to a back-transfer of electrons from the secondary acceptor, B, to the primary acceptor, Q, caused by DCMU. At longer times after the mixing, charge recombination takes place to a variable extent according to the charge storage state Si on the donor side, yielding the oscillatory pattern observed by Wollman, F.A. ((1978) Biochim. Biophys. Acta 503, 263–273).3.3. Shifting the pH from 6 to 8 causes an acceleration of the DCMU-induced back-transfer to Q and an about 2-fold increase in the amplitude of the fluorescence oscillations. The rate of the DCMU-induced rise of fluorescence is sensitive to the pH during the mixing, whereas the amplitude of the oscillations depends on the pH during the preillumination. Even under optimal conditions, the oscillations account only for a fraction of the total variable fluorescence.4.4. The delayed light emitted by isolated chloroplasts in the 100 ms—seconds range oscillates weakly (periodicity 4) with the number of preilluminating flashes. Mixing with DCMU after the preillumination causes a delayed light stimulation which varies with the flash number. The enhancement factor oscillates with periodicities of both 2 and 4. The amplitude of the period-2 contribution varies with the amount of B oxidized in the dark, while that of the period-4 contribution depends on the extent of this type of oscillation in the control experiment.5.5. The period-2 oscillations of the DCMU-stimulation of delayed light behave similarly to the fluorescence oscillations. It is shown that they are not due to a modulation of luminescence by the fluorescence yield, but rather to the variations of the amount of Q− as a substrate.6.6. It is concluded that in the absence of DCMU, the reduced secondary acceptor B− is not the main source of electrons involved in radiative recombination of functional centers in the time-range we have studied. Possible models are discussed.
The primary events of photosynthetic light reactions excel in efficiency and speed. They occur in highly organized protein-pigment aggregates that have been well characterized, both chemically and functionally. The recent structural characterization at the atomic level of some important components of the primary photosynthetic processes in bacteria helps our understanding of the underlying physical principles.
Simultaneous measurements of hydroxylamine photo-oxidation and fluorescence induction were performed in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). The results provide a justification for the common use of fluorescence data to estimate the concentration of active System II centers in the presence of inhibitors.The addition of DCMU to dark-adapted chloroplasts under special conditions induces a large increase of the initial yield of fluorescence. A reversible inactivation of part of the System II centers is responsible for this effect. Similar data were obtained with other classical inhibitors of oxygen evolution.
In thylakoid membranes incubated in the dark with ferricyanide, an auxiliary acceptor (Q400) associated with Photosystem II becomes oxidized. It has been reported that, based on oxygen flash-yield data, electron flow to Q400 did not occur in ‘bicarbonate-depleted’ (formate-pretreated) samples. Contrary to this earlier report, we find, based on oxygen flash-yield data and chlorophyll a fluorescence-transient measurements, that Q400 is active as an electron acceptor in formate-pretreated samples. It is concluded that the effect of formate pretreatment is on the flow of electrons between Q, B and the plastoquinone pool and not the flow to Q400. We also believe that another auxiliary acceptor of Photosystem II exists under conditions of formate pretreatment and pH larger than 7.0. This belief is based on increased double advancement in the oxygen flash-yield pattern and increased area above the chlorophyll a fluorescence-rise curve. The double advancement in the oxygen pattern shows a second-order dependence on flash intensity. These effects are eliminated by bicarbonate addition or shifts to lower values of pH such as 6.8. This new acceptor is believed to be different from Q400.
In order to determine the major site of bicarbonate action in the electron transport complex of Photosystem II, the following experimental techniques were used: electron spin resonance measurements of Signal IIvf, measurements of chlorophyll a fluorescence yield rise and decay kinetics, and delayed light emission decay. From data obtained using these experimental techniques the following conclusions were made: (1) absence of bicarbonate causes a reversible inactivation of up to 40% of Photosystem II reaction center activity; (2) there is no significant effect of bicarbonate on electron flow from the charge accumulating S state to Z; (3) there is no significant effect of bicarbonate on electron flow from Z to P-680+; (4) electron flow from Q-- to the intersystem electron transport pool is inhibited by from 4- to 6-fold under bicarbonate depletion conditions.
ESR studies at approximately 10 °K on the reaction centre complex of the photosynthetic bacterium Rhodopseudomonas spheroides (strain R26), have revealed bacteriochlorophyll triplet states and a component which has an ESR absorption centred at g = 1.82. The triplet-state bacteriochlorophyll is induced only in the light and is only detectable when the reaction-centre bacteriochlorophyll and its primary electron acceptor are reduced; the ESR triplet state signals are composed of both ESR absorption and ESR emission bands. The oxidation-reduction properties of the g = 1.82 component and its flash-induced kinetic behavior in relation to that of P870 are those expected for the primary electron acceptor in bacterial photosynthesis.
1. Spinach chloroplasts, but not whole Chlorella cells, show an acceleration of the Photosystem II turnover time when excited by non-saturating flashes (exciting 25 % of centers) or when excited by saturating flashes for 85–95 % inhibition by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Following dark adaptation, the turnover is accelerated after a non-saturating flash, preceded by none or several saturating flashes, and primarily after a first saturating flash for 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibition. A rapid phase ( approx. 0.75 s) is observed for the deactivation of State S2 in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea.2. These accelerated relaxations suggest that centers of Photosystem II are interconnected at the level of the primary electron transfer and compete for primary oxidizing equivalents in a saturating flash. The model in best agreement with the experimental data consists of a paired interconnection of centers.3. Under the conditions mentioned above, an accelerated turnover may be observed following a flash for centers in S0, S1 or S2 prior to the flash. This acceleration is interpreted in terms of a shift of the rate-limiting steps of Photosystem II turnover from the acceptor to the donor side.
The conditions for steady-state Signal IIf formation in response to single turnover flashes in Tris-treated, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-inhibited chloroplasts have been investigated. DCMU inhibits Signal IIf generation as the photoinactive state, Z P680 Q-A, accumulates. Potassium ferricyanide relieves this inhibition so that Signal IIf can be fully developed on each flash in a flash series. The effectiveness of ferricyanide in stimulating Signal IIf formation is dependent on its concentration, the flash repetition rate, and the salt composition of the chloroplast suspension. There are two models in the literature for Q-A oxidation under these inhibitory conditions: direct oxidation of Q-A by exogenous acceptors like ferricyanide or oxidation of Q-A by an endogenous acceptor, AH, which has a midpoint potential of approximately 400 mV. It is found that the direct exogenous acceptor model accounts well for these data, whereas the AH model does not explain several of these results. The apparent rate constant for the direct oxidation of Q-A by ferricyanide at various concentrations of salt has been calculated from our electron paramagnetic resonance (EPR) data and compared with the corresponding rate constant determined by S. Itoh from fluorescence data (Biochim, Biophys. Acta 504, 324-340, 1978); good agreement is found for the two different experimental approaches.
A test of the 'iron-wire' hypothesis for the role of Fe2+ in promoting the electron transfer between the primary (Q1) and secondary (Q2) quinones in bacterial reaction centers of Rhodopseudomonas sphaeroides strain R-26.1 has been conducted. Kinetics of this step, P+Q-1Q2----P+Q1Q-2, and of recombination with the oxidized donor, P+Q-1----PQ1 and P+Q-2----PQ2, were followed optically at 4 degrees C in normal iron-containing reaction centers and in reaction centers having 58% Mn2+, replacing Fe2+. This significant replacement is accomplished biosynthetically by control of the growth conditions, and so should preserve the native interactions between the cofactors. There are no significant differences observed in the recombination kinetics of the two types of reaction centers. The electron transfer between the quinones was observed to show apparent biphasic kinetics with major components of approx. 170 microseconds and 1.5 ms at 4 degrees C and pH = 7.5. There is no statistically significant difference observed between the two types of reaction centers. This major change in the electronic structure of the metal and the unaltered kinetics discount the likelihood of any direct orbital participation of the metal in the electron transfer between the quinones.
Inhibitors of carbonic anhydrase were tested for their effects on Photosystem II (PS II) activity in chloroplasts. We find that formate inhibition of PS II turnover rates increases as the pH of the reaction medium is lowered. Bicarbonate ions can inhibit PS II turnover rates. The relative potency of the anionic inhibitors N-3, I-, OA-c, and Cl- is the same for both carbonic anhydrase and PS II. The inhibitory effect of acetazolamide on PS II increases as light intensity decreases, indicating a lowering of quantum yields in the presence of the inhibitor. Imidazole inhibition of PS II increases with pH in a manner suggesting that the unprotonated form of the compound is inhibitory. Formate, bicarbonate, acetazolamide, and imidazole all inhibit DCMU-insensitive, silicomolybdate-supported oxygen evolution, indicating that the site(s) of action of the inhibitors is at, or before, the primary stable PS II electron acceptor, Q. This inhibitory effect of low levels of HCO-3 along with the known enhancement by HCO-3 of quinone-mediated electron flow suggests an antagonistic control effect on PS II photochemistry. We conclude that the responses of PS II to anions (formate, bicarbonate), acetazolamide, and imidazole are analogous to the responses shown by carbonic anhydrase. These findings suggest that the enzyme carbonic anhydrase may provide a model system to gain insight into the "bicarbonate-effect" associated with PS II in chloroplasts.
The characteristics of double hitting in Photosystem II charge separation and oxygen evolution in algae and chloroplasts were investigated with saturating excitation flashes of 3 microseconds, 300 ns and 5 ns duration. Two types of double hitting or advancement in S-states were found to occur in oxygen evolution: a non-photochemical type found even with 5 ns flashes and a photochemical type seen only with microsecond-long flashes, which have extensive tails. The non-photochemical type, occurring with a probability of about 3%, is sensitive to the physiological condition of the sample, and is only present in algae or chloroplast samples that have been freshly prepared. In chloroplasts incubated with ferricyanide, a 3-fold increase in double advancement of S-states is observed with xenon-flash illumination but not with 300 ns or 5 ns laser illumination. However, double turnovers in Photosystem II reaction center charge separation are large with xenon flash or 300 ns laser illumination but not with 5 ns laser illumination. This indicates that quite different kinetic processes are involved in double advancement in S-states for oxygen evolution and double turnovers in charge separation. Various models of the Photosystem II reaction center are discussed. Also, based on experiments with chloroplasts incubated with ferricyanide, an unique solution to the oxygen S-state distribution in the dark suggested by Thibault (Thibault, P. (1978) C.R. Acad. Sci. Paris 287, 725-728) can be rejected.
Highly active photosystem-II particles were rapidly isolated using detergents and obtained in good yield from a mutant of the green alga Chlamydomonas reinhardtii. The particles are completely devoid of reaction centers of photosystem I, and of the secondary electron acceptor to photosystem II. They show: (a) a specific activity (ΔA of C550/unit chlorophyll) 4–7-times that of the starting material and of spinach chloroplasts; (b) an antenna size of 40 to 50 chlorophyll molecules containing little light-harvesting chlorophyll a/b complex (chlorophyll a/chlorophyll b= 4–6.4); (c) a ratio of variable to dark-adapted fluorescence yield of up to 3.
Further treatment of these particles by ion-exchange chromatography largely removes five proteins and further decreases the antenna size with little loss in primary photoactivity.