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Q400, the non-heme iron of the photosystem II iron-quinone complex. A spectroscopic probe of quinone and inhibitor binding to the reaction center
Abstract
Q400, a high-potential electron acceptor associated with Photosystem II (PS II) of oxygenic photosynthesis, originally described by Ikegami and Katoh [3], has recently been identified by Petrouleas and Diner [8] as the non-heme iron of the iron-quinone complex of the PS II reaction center. This acceptor, which can function as the Fe(Ill)/Fe(II) redox couple with an Em.7, of 400 mV, demonstrates a pH-dependence of −60 mV/pH unit, indicative of a protonation reaction coupled to Fe(III) reduction.In this review, we describe the chemical and physical properties of the acceptor which led to its identification. Through a combination of optical, EPR and Mössbauer spectroscopy, we also show how the iron, unlike its bacterial reaction center homologue, is capable of redox chemistry involving the neighboring quinones, and how it serves as a sensitive spectroscopic probe, not only of its immediate coordination sphere, but of the sites at which quinones and inhibitors bind to the reaction center. A theoretical description of the Fe(III) EPR spectrum which accounts for the positions, amplitudes and energetics of the observed resonances is also presented.
... The DCMU inhibition of O 2 reduction may also be taken as an indication that the nonheme Fe 2+ is the O 2 binding site. DCMU binding shifts the redox potential of the Fe 3+ /Fe 2+ couple 120 mV to higher values, while other herbicides/inhibitors (atrazine and o-phenanthroline) induced much smaller shifts (42)(43)(44). Whether the DCMU inhibition of O 2 reduction reflects a perturbation of the electronic structure of the Fe 2+ (as manifest by the redox shift), minor structural changes, or both of these is unclear. These effects could be responsible for DCMU inhibiting bicarbonate binding and dissociation (45,46). ...
... This raises the possibility that in vivo the reduction of O 2 by Q •À A could be faster than reported here. The Fe 3+ /Fe 2+ couple has an E m of +430 mV at this pH (44). The Fe 2+ is located between the quinones Q A and Q B , but there is no evidence of a distinct redox role of the metal in the electron transfer process between the two quinones (49). ...
Significance
In Photosystem II (PSII), O 2 reduction by Q A • − is often discussed but has not been demonstrated. Here, we show in PSII membranes that Q A • − can reduce O 2 to superoxide, but only when bicarbonate is absent from its binding site on the nonheme Fe ²⁺ . Bicarbonate’s role in PSII was recently shown to involve a regulatory/protective redox-tuning mechanism linking PSII function to CO 2 concentration. A key aspect is the presence of stable Q A • − causing release of bicarbonate from its site on Fe ²⁺ . Here, we show that under these conditions, O 2 binds to the empty site on the Fe ²⁺ and is reduced by Q A • − . This unexpected reaction may be a further indication of cross-talk between the regulation of PSII and CO 2 fixation.
... A 60 ms flash was Fig. S1, Fig. S2, Fig. S3, and Table S1 in the Supporting Material). This relatively high value of double turnovers arises in part from the use of FeCN as an electron acceptor during a long dark adaptation, which oxidizes the nonheme in PSII (48). In turn, it serves as an endogenous electron acceptor, causing a higher double-turnover probability (49). ...
... Our studies confirm that outcome and reveal that this concentration directly increases the initial dark S 2 population, as evidenced by an increase in the double-hit parameter on the first 60-ms flash only. This outcome was identified as being due to oxidation of the nonheme iron (normally ferrous) (48), and was quantified as described next and verified by EPR spectroscopy (Fig. S4). ...
Photosynthetic O(2) production from water is catalyzed by a cluster of four manganese ions and a tyrosine residue that comprise the redox-active components of the water-oxidizing complex (WOC) of photosystem II (PSII) in all known oxygenic phototrophs. Knowledge of the oxidation states is indispensable for understanding the fundamental principles of catalysis by PSII and the catalytic mechanism of the WOC. Previous spectroscopic studies and redox titrations predicted the net oxidation state of the S(0) state to be (Mn(III))(3)Mn(IV). We have refined a previously developed photoassembly procedure that directly determines the number of oxidizing equivalents needed to assemble the Mn(4)Ca core of WOC during photoassembly, starting from free Mn(II) and the Mn-depleted apo-WOC complex. This experiment entails counting the number of light flashes required to produce the first O(2) molecules during photoassembly. Unlike spectroscopic methods, this process does not require reference to synthetic model complexes. We find the number of photoassembly intermediates required to reach the lowest oxidation state of the WOC, S(0), to be three, indicating a net oxidation state three equivalents above four Mn(II), formally (Mn(III))(3)Mn(II), whereas the O(2) releasing state, S(4), corresponds formally to (Mn(IV))(3)Mn(III). The results from this study have major implications for proposed mechanisms of photosynthetic water oxidation.
... The direct involvement of bicarbonate in binding to the iron is supported by several lines of evidence. For example, Mössbauer spectrum of Fe signal, indicative of the inner-coordination sphere of iron, was found to be significantly affected by the addition of formate, and it was fully restored upon the re-addition of bicarbonate (Diner and Petrouleas, 1987;Govindjee et al. 1997;Semin et al. 1990;van Rensen et al. 1999). Fourier transform infrared (FTIR) difference spectroscopy study, using 14C-bicarbonate, has further indicated that bicarbonate is a bidentate ligand of the NHI in PS II (Hienerwadel and Berthomieu 1995); in addition, the bicarbonate ion was shown to switch from Fifty Years of Research on the "Bicarbonate Effect" in Photosystem II a chelating to a monodentate binding mode when the iron is oxidized. ...
... The non-heme Fe 2+ complex might be involved in the photoprotection mechanism (Diner and Petrouleas, 1987;Muh et al., 2012). Notably, Brinkert et al. (2016) reported that the loss of the bicarbonate ligand from the non-heme Fe 2+ complex leads to an increase of 75 mV in E m (Q A ) in the presence of Q B ; this suggests that the loss of the bicarbonate ligand is responsible for the formation of the high-E m (Q A ) conformation. ...
Photo-induced charge separation, which is terminated by electron transfer from the primary quinone QA to the secondary quinone QB, provides the driving force for O2 evolution in photosystem II (PSII). However, the backward charge recombination using the same electron-transfer pathway leads to the triplet chlorophyll formation, generating harmful singlet-oxygen species. Here, we investigated the molecular mechanism of proton-mediated QA⋅– stabilization. Quantum mechanical/molecular mechanical (QM/MM) calculations show that in response to the loss of the bicarbonate ligand, a low-barrier H-bond forms between D2-His214 and QA⋅–. The migration of the proton from D2-His214 toward QA⋅– stabilizes QA⋅–. The release of the bicarbonate ligand from the binding Fe²⁺ site is an energetically uphill process, whereas the bidentate-to-monodentate reorientation is almost isoenergetic. These suggest that the bicarbonate protonation and decomposition may be a basis of the mechanism of photoprotection via QA⋅–/QAH⋅ stabilization, increasing the QA redox potential and activating a charge-recombination pathway that does not generate the harmful singlet oxygen.
... The potential of F X in type I reaction centres is about -700 mV [32]. The potential of nonheme Fe 2+ / Fe 3+ in type II reaction centre is over +400 mV [33], although this is not directly involved in electron transfer reactions. At the level of the quinone there is a more than 500 mV difference in the potential of the A 1 phylloquinone (about -800mV) in PSI [32] compared with that of Q A (-150 mV) in PSII [34], with most of the difference (about 500 mV) attributed to the electrostatic effect of the net negative charge on F X compared with the net positive charge on the nonheme Fe 2+ [35,36]. ...
One of the earliest events in the molecular evolution of photosynthesis is the structural and functional specialisation of type I (ferredoxin-reducing) and type II (quinone-reducing) reaction centres. In this opinion article we point out that the homodimeric type I reaction centre of heliobacteria has a calcium-binding site with striking structural similarities to the Mn4CaO5 cluster of photosystem II. These similarities indicate that most of the structural elements required to evolve water oxidation chemistry were present in the earliest reaction centres. We suggest that the divergence of type I and type II reaction centres was made possible by a drastic structural shift linked to a change in redox properties that coincided with or facilitated the origin of photosynthetic water oxidation.
... In this respect, transition metal centers are the most likely candidates. In principle, for PS II three sites have to be considered: i) the non heme iron located between QA and QB (Diner and Petrouleas 1988), ii) the heme iron of cytochrome b559 and iii) the manganese of the water oxidase. In order to analyze whether the manganese of the water oxidase is related to the SOD-activity of PS II, comparative studies were performed on the effect of TCNE.Figure 4 shows the oxygen evolution rate and the TCNE-stimulated photoreduction of cytochrome c (III) of PS II membrane fragments as a function of the TCNE-concentration. ...
In the present study the light induced formation of superoxide and intrinsic superoxide dismutase (SOD) activity in PS II membrane fragments and D1/D2/Cytb559-complexes from spinach have been analyzed by the use of ferricytochrome c (cyt c(III)) reduction and xanthine/xanthine oxidase as assay systems. The following results were obtained: 1.) Photoreduction of Cyt c (III) by PS II membrane fragments is induced by addition of sodium azide, tetracyane ethylene (TCNE) or carbonylcyanide-p-trifluoromethoxy-phenylhydrazone (FCCP) and after removal of the extrinsic polypeptides by a 1M CaCl2-treatment. This activity which is absent in control samples becomes completely inhibited by the addition of exogenous SOD. 2.) The TCNE induced cyt c(III) photoreduction by PS II membrane fragments was found to be characterized by a half maximal concentration of c1/2=10 μM TCNE. Simultaneously, TCNE inhibits the oxygen evolution rate of PS II membrane fragments with c1/2≈ 3 μM. 3.) The photoproduction of O2− is coupled with H+-uptake. This effect is diminished by the addition of the O2−-trap cyt c(III). 4.) D1/D2/Cytb559-complexes and PS II membrane fragments deprived of the extrinsic proteins and manganese exhibit no SOD-activity but are capable of producing O2− in the light if a PS II electron donor is added.
Based on these results the site(s) of light induced superoxide formation in PS II is (are) inferred to be located at the acceptor side. A part of the PS II donor side and Cyt b559 in its HP-form are proposed to provide an intrinsic superoxide dismutase (SOD) activity.
... Carboxylates induce a significant shift in the E m of the non-heme iron [20]. The ferrous state of the non-heme iron in PSII can be oxidised at an E m of + 400 mV [40], but if this occurs during electron transfer from Q A − to Q B has been disputed [41]. X-ray absorption spectroscopy has recently provided further evidence that a temporary structural change actually occurs rather than an oxidation of the non-heme iron [42]. ...
Chlamydomonas reinhardtii is a photoautotrophic green alga, which can be grown mixotrophically in acetate-supplemented media (TAP). We show that acetate has a direct effect on photosystem II (PSII). As a consequence, TAP-grown mixotrophic C. reinhardtii cultures are less susceptible to photoinhibition than photoautotrophic cultures when subjected to high light. Spin-trapping electron paramagnetic resonance spectroscopy showed that thylakoids from mixotrophic C. reinhardtii produced less (1)O2 than those from photoautotrophic cultures. The same was observed in vivo by measuring DanePy oxalate fluorescence quenching. Photoinhibition can be induced by the production of (1)O2 originating from charge recombination events in PSII, which are governed by the midpoint potentials (Em) of the quinone electron acceptors. Thermoluminescence indicated that the Em of the primary quinone acceptor (QA/QA(-)) of mixotrophic cells was stabilised while the Em of the secondary quinone acceptor (QB/QB(-)) was destabilised, therefore favouring direct non-radiative charge recombination events that do not lead to (1)O2 production. Acetate treatment of PSII-enriched membrane fragments from spinach led to the same thermoluminescence shifts as observed in C. reinhardtii, showing that acetate exhibits a direct effect on PSII independent from the metabolic state of a cell. A change in the environment of the non-heme iron of acetate-treated PSII particles was detected by low temperature EPR spectroscopy. We hypothesise that acetate replaces the bicarbonate associated to the non-heme iron and changes the environment of QA and QB affecting PSII charge recombination events and photoinhibition.
... There were no changes in the g = 1.95 region and the EPR signal from ferricyanide was too broad to detect any light-induced changes. The signal at g = 6 from oxidised non-haem iron [22] was not observed in any samples. In addition, no light-induced changes were seen in the g = 3 region of the EPR spectrum indicative of cytochrome b-559 reduction. ...
We have characterised the electron-transfer properties of the D1/D2/cytochrome b-559 complex using EPR spectrometry. The complex can transfer electrons to silicomolybdate and ferricyanide at cryogenic temperatures. In the presence of silicomolybdate or ferricyanide, two chlorophyll cation radicals were observed from P680+ (0.8 mT) and monomeric Chl (1.0 mT). Reduction of silicomolybdate was detected as a 2.7 mT signal at g = 1.942. A radical attributed to a tyrosine cation radical (D+/Z+) was also observed in a small percentage of centres.
Photosystem II (PSII) catalyses the oxidation of water through a four-step cycle of Si states (i = 0–4) at the Mn4CaO5 cluster1–3, during which an extra oxygen (O6) is incorporated at the S3 state to form a possible dioxygen4–7. Structural changes of the metal cluster and its environment during the S-state transitions have been studied on the microsecond timescale. Here we use pump-probe serial femtosecond crystallography to reveal the structural dynamics of PSII from nanoseconds to milliseconds after illumination with one flash (1F) or two flashes (2F). YZ, a tyrosine residue that connects the reaction centre P680 and the Mn4CaO5 cluster, showed structural changes on a nanosecond timescale, as did its surrounding amino acid residues and water molecules, reflecting the fast transfer of electrons and protons after flash illumination. Notably, one water molecule emerged in the vicinity of Glu189 of the D1 subunit of PSII (D1-E189), and was bound to the Ca²⁺ ion on a sub-microsecond timescale after 2F illumination. This water molecule disappeared later with the concomitant increase of O6, suggesting that it is the origin of O6. We also observed concerted movements of water molecules in the O1, O4 and Cl-1 channels and their surrounding amino acid residues to complete the sequence of electron transfer, proton release and substrate water delivery. These results provide crucial insights into the structural dynamics of PSII during S-state transitions as well as O–O bond formation.
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.
Photosystem II (PSII), the thylakoid pigment-protein complex of oxygenic photosynthetic organisms acts as the water:plastoquinone oxidoreductase. The binding site for plastoquinone on the PSII D1 protein (QB binding site) is a target for a large group of compounds which inhibit plastoquinone reduction and are extensively used as herbicides [1]. The binding of these herbicides to PSII not only inhibits electron flow through PSII but also affects the rate of the PSII photoinactivation (PSIIPI) and the metabolism of the D1 protein. The phenylurea-type herbicides have been found to retard PSIIPI in vivo and in vitro [2,3] while an acceleration of PSIIPI by phenol-type inhibitors has been shown in isolated PSII membranes from higher plants [3]. A similar acceleration has been recently observed also in the cells of the green alga Scenedesmus treated with the phenolic inhibitor bromonitrothymol (BNT)[4].
The main electron transfer events in PS2 are:
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Oxygen evolving photosystem two (PS2) particles isolated using Triton X-100 consist of a number of polypeptides. These include the polypeptides termed D1 and D2 and the two apoproteins of cytochrome b559.The proteins D1 and D2 which bind the primary electron transport components show considerable sequence homology with the purple bacterial reaction centre proteins L and M. Computer models have indicated that D1 and D2 each have five transmembrane helices, and that the binding regions for electron acceptors Qa and Qb reside in the loops between helices IV and V on the stromal side of the thylakoid membrane [1].
The herbicide binding domain of Photosystem 2 (PS2) has been localised to a region of the D1 polypeptide connecting the putative 4th and 5th membrane spanning alpha helices based on photoaffinity labelling studies, the locations of point mutations affecting herbicide binding, and homology with the purple bacterial reaction centre whose structure has been resolved. Although some commercial PS2 herbicides bind to the bacterial reaction centre, many other classes of PS2 herbicides, such as the phenylureas and the phenols, do not, indicating significant differences in architecture between PS2 and the bacterial reaction centre in the herbicide binding domain. Our studies are aimed at elucidating the tertiary structure of the herbicide binding domain of PS2 particularly with respect to phenylurea binding.
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.
Low-temperature steady-state emission properties have been analyzed of Photosystem II reaction center (RC) complexes isolated from spinach CP47-RC complexes after a short Triton X-100 treatment and stabilization in n-dodecyl beta,D-maltoside. Excitation spectra of the fluorescence anistropy were detected at the maximum of the single fluorescence band at 683.5 nm and at the vibrational subband of the same emission at 742 nm. The Q(y) transitions of the red-most absorbing pigment(s) showed positive anistropy with a value of about 0.22. The value is lower than that of the theoretical maximum (0.4) and is explained by a combination of (1) vibrational depolarization effects and (2) by assuming that the red-most absorbing pigments arise from the low-exciton component of P680, that the exciton coupling breaks upon excitation, and that the angle between the monomer Q(y) transitions of P680 is 48 +/- 10-degrees. The Q(x) transitions of pheophytin showed negative anisotropy. This result, combined with the results obtained with linear dichroism spectroscopy, suggests that the spatial organization of the Q(x) transitions of pheophytin matches the organization of the bacteriopheophytin residues in the bacterial reaction center. The spatial organization of the y-polarized transitions of pheophytin could be similar in both systems, although these transitions could also be tilted somewhat more towards the membrane plane in PS II. The data furthermore indicate that the accessory chlorophylls in PS II and in bacterial reaction centers have different average orientations, and suggest that at least some of the accessory chlorophylls in PS II have a pheophytin-like orientation.
This chapter provides an overview of oxygenic photosynthesis, with primary focus on the oxygen-evolving complex (OEC) of photosystem II (PSII). The introduction includes a general discussion of photosynthesis and a brief overview of the metals involved. This is followed by an in-depth description of the metal centers within PSII, in which the structural and electronic characterizations of the OEC are reviewed in detail. The role of chloride in PSII and the proton exit pathway are discussed. Several mechanisms proposed for the O. O bond formation in the OEC are presented, along with a few detailed mechanisms for the complete catalytic cycle obtained from quantum mechanics/molecular mechanics and density functional theory calculations. Model complexes that are functional mimics of the OEC are also briefly discussed. General discussion on plastocyanin, iron-sulfur centers, [FeFe] and [NiFe] hydrogenases, and ribulose-1,5-bisphosphate carboxylase-oxygenase is included. The chapter concludes with the implications of natural photosynthesis on the development of an artificial photosynthetic framework.
Photosynthesis involves the capture of the Sun's energy into biochemical energy that sustains all life on the Earth. It is a massive process that has radically changed the composition of the atmosphere. Its scale can be judged from the fact that man's entire fossil fuel use from 1860 to 1988 was equivalent to only 2 years’ global photosynthesis. Although photosynthesis occurs in a wide range of organisms (bacteria, cyanobacteria, algae, and plants), and about a third of it occurs in the oceans, this discussion concentrates on plants.
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.
Effects of photoinhibition at 0 °C on the PS II acceptor side have been analyzed by comparative studies in isolated thylakoids, PS II membrane fragments and PS II core complexes from spinach under conditions where degradation of polypeptide(s) D1(D2) is highly retarded. The following results were obtained by measurements of the transient fluorescence quantum and oxygen yield, respectively, induced by a train of short flashes in dark-adapted samples: (a) in the control the decay of the fluorescence quantum yield is very rapid after the first flash, if the dark incubation was performed in the presence of 300 μM K3[Fe(CN)6]; whereas, a characteristic binary oscillation was observed in the presence of 100 μM phenyl-p-benzoquinone with a very fast relaxation after the even flashes (2nd, 4th. . . ) of the sequence; (b) illumination of the samples in the presence of K3[Fe(CN)6] for only 5 min with white light (180 W m-2) largely eliminates the very fast fluorescence decay after the first flash due to QA- reoxidation by preoxidized endogenous non-heme Fe3+, while a smaller effect arises on the relaxation kinetics of the fluorescence transients induced by the subsequent flashes; (c) the extent of the normalized variable fluorescence due to the second (and subsequent) flash(es) declines in all sample types with a biphasic time dependence at longer illumination. The decay times of the fast (6–9 min) and the slow degradation component (60–75 min) are practically independent of the absence or presence of K3[Fe(CN)6] and of anaerobic and aerobic conditions during the photo-inhibitory treatment, while the relative extent of the fast decay component is higher under anaerobic conditions. (d) The relaxation kinetics of the variable fluorescence induced by the second (and subsequent) flash(es) become retarded due to photoinhibition, and (e) the oscillation pattern of the oxygen yield caused by a flash train is not drastically changed due to photoinhibition.
Based on these findings, it is concluded that photoinhibition modifies the reaction pattern of the PS II acceptor side prior to protein degradation. The endogenous high spin Fe2+ located between QA and QB is shown to become highly susceptible to modification by photoinhibition in the presence of K3[Fe(CN)6] (and other exogenous acceptors), while the rate constant of QA- reoxidation by QB(QB-) and other acceptors (except the special reaction via Fe3+) is markedly less affected by a short photoinhibition. The equilibrium constant between QA- and QB(QB-) is not drastically changed as reflected by the damping parameters of the oscillation pattern of oxygen evolution.
The functional properties of a purified homogeneous spinach PS II-core complex with high oxygen evolution capacity (Haag et al. 1990a) were investigated in detail by measuring thermoluminescence and oscillation patterns of flash induced oxygen evolution and fluorescence quantum yield changes. The following results were obtained:a)
Depending on the illumination conditions the PS II-core complexes exhibit several thermoluminescence bands corresponding to the A band, Q band and Zv band in PS II membrane fragments. The lifetime of the Q band (Tmax=10°C) was determined to be 8s at T=10°C. No B band corresponding to S2QB− or S3QB− recombination could be detected.
b)
The flash induced transient fluorescence quantum yield changes exhibit a multiphasi relaxation kinetics shich reflect the reoxidation of Q
A−. In control samples without exogenous acceptors this process is markedly slower than in PS II membrane fragments. The reaction becomes significantly retarded by addition of 10 μM DCMU. After dark incubation in the presence of K3[Fe(CN)6
c)
Excitation of dark-adapted samples with a train of short saturating flashes gives rise to a typical pattern dominated by a high O2 yield due to the third flash and a highly damped period four oscillation. The decay of redox states S2 and S3 are dominated by short life times of 4.3 s and 1.5 s, respectively, at 20°C.
The results of the present study reveal that in purified homogeneous PS II-core complexes with high oxygen evolution isolated from higher plants by β-dodecylmaltoside solubilization the thermodynamic properties and the kinetic parameters of the redox groups leading to electron transfer from water to QA are well preserved. The most obvious phenomenon is a severe modification of the QB binding site. The implications of this finding are discussed.
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.
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.
Site directed mutagenesis studies of reaction centers (RCs) have so far focused on the L- and M-subunits, because they bind all the cofactors involved in the light-induced charge separation processes. However, the H-subunit gene, puh4, of the RC has also been isolated and sequenced from Rb. sphaemides, Rb. Capsularus, and Rps. Viridis, and site-directed mutagenesis studies can be expected in the near future. This chapter addresses significant advances made through the mutagenesis of the L- and M-subunits in understanding the roles of key residues in specific functions of the RC. The effects of the mutations have been dramatic and unexpected including the heterodimer mutants substitution of BphA by Bchl and, by opposite extremes of functional disturbance, the mutation of Asp1213 and of GIuM234. Part of the unexpectedness of these and many other mutations is the capacity of the RC to accommodate radical changes to assemble and, to a large extent, to function.
The reactions of two Co(III)-tetramine (N4) complexes, cis-alpha-Dichloro(triethylenetetramine)cobalt(III) chloride = [Co(III)Cl2(trien)]Cl (1) and aquabromo[(2R, 5R, 8R, 11R)-2,5,8,11-tetraethyl-1,4,7,10-tetraazacyclododecane] cobalt(III) bromide = [Co(III)Br(Et4[12]-ane N4)(H2O)]Br2 (2) with 3,5-di-t-butylcatechol (H2dbc) have been examined by visible, NMR and ESR spectroscopies to elucidate the mechanism of the activation and the oxygenation of catechol by the complex formation. The reactions proceeded in a mixed solvent (H2O:MeOH = 1:1) at 50-degrees-C in the presence and absence of dioxygen. NMR showed that the complexes react with H2dbc and dioxygen, and yield oxidatively cleavaged products. ESR measurements detected a Co(III)-semiquinone (dbsq) radical and a high-spin Co(II) complex, regardless of the presence of dioxygen. Especially in the case of 1, the magnetic moment of the complex isolated from the anaerobic reaction solution, exhibited that there exists an antiferromagnetic spin-coupling between the high-spin (S = 3/2) Co(II) ion and the semiquinone radical in the complex. These results show that two species detected by ESR are in equilibrium with a high-spin Co(II)-dbsq binary complex formed by intramolecular electron transfer. It is conclusively clarified that the one-electron-transferred intermediate [Co(II)(N4)dbsq]+ is the activated intermediate, which introduces dioxygen into the semiquinone moiety.
By using the hindered tris(pyrazolyl)borate ligand HB(3,5-iPr2pz)3, (hydrotris(3,5-diisopropyl-1-pyrazolyl)-borate), a series of hydroxo complexes of first-row divalent metal ions (Mn (1), Fe (2), Co (3), Ni (4), Cu (5), Zn (6)) was synthesized. X-ray crystallography was applied to 1-5, establishing that all these hydroxo complexes have a dinuclear structure solely bridged with a bis(hydroxo) unit. The structure of 6 was characterized by spectroscopy, which indicates that 6 is monomeric. All these hydroxo complexes were found to react with CO2, even atmospheric CO2, to afford mu-carbonato dinuclear complexes of Mn (7), Fe (8), Co (9), Ni (10), Cu (11), and Zn (12). The molecular structures of the complexes 8-12 were determined. A variety of coordination modes of the carbonate group was seen. In 10 and 11, the carbonate group is bound to both metal centers bidentately in a symmetric fashion, while in 8 and 9, the carbonate coordination modes are described as an unsymmetric bidentate. The carbonate group in 12 is coordinated to one zinc ion bidentately, but it is bound to the other zinc ion unidentately. From IR data, the coordination mode of the carbonate group in 7 was suggested to be similar to those found in 8 and 9. Thus, the order of the coordination distortions of the carbonate groups in this series of mu-carbonato dinuclear complexes is as follows: Zn > Mn almost-equal-to Fe almost-equal-to Co > Ni almost-equal-to Cu. On the other hand, the reactivities of the hydroxo complexes toward CO2 fixation were found to be ordered Zn > Cu > Ni almost-equal-to Co > Mn > Fe. It is noteworthy that the order of the CO2 fixation capabilities of the hydroxo complexes does not fit with the order of activities known for metal-substituted carbonic anhydrases. The order of activities for CO2 hydration by the carbonic anhydrases is Zn > Co >> Ni almost-equal-to Mn > Cu almost-equal-to 0. Thus, the order is correlated mostly with the coordination distortions of the carbonate group in the mu-carbonate complexes but not the reactivities of the hydroxo complexes toward CO2.
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.
We have investigated the EPR characteristics of native QB and QB analogues in higher plant PS II. We show that, as in cyanobacteria, an interaction between QA and QB iron-semiquinones (QA−-Fe2+-QB−) is observed which gives an EPR signal near g=1.6. Bicarbonate binding close to the non-haem iron is required to observe this interaction. The EPR signal of QB semiquinone is weak and difficult to distinguish from that of QA. The appearance of the g=1.6 signal from QA−-Fe2+-QB− after 77 K illumination is a better marker for the presence of QB semiquinone. The yield of QB semiquinone in plant PS II is lower than found in cyanobacteria. The brominated quinones DBMIB, TBTQ and bromanil were used as QB analogues to increase the yield of QA−-Fe2+-QB−. These analogues act by forming a stable semiquinone at the QB site and not by covalent binding.
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 magnetic susceptibility of the non-heme iron in plant photosystem II was first measured by a highly sensitive magnetometer equipped with a pulsed magnetic field. From the temperature dependence of the paramagnetic susceptibility of the non-heme ferrous iron with S=2, the zero-field interaction parameters D and E were determined to be 5.5±1.0 and 0.75±0.75 cm-1, respectively. These values are almost the same as those observed in a purple photosynthetic bacterium Rhodobacter sphaeroides, suggesting that the structure of the acceptor side of plant photosystem II is also similar to that of the purple photosynthetic bacterium.
The role of chloride in photosystem II (PSII) is unclear. Several monovalent anions compete for the Cl- site(s) in PSII, and some even support activity. NO2- has been reported to be an activator in Cl--depleted PSII membranes. In this paper, we report a detailed investigation of the chemistry of NO2- with PSII. NO2- is shown to be inhibitory to PSII activity, and the effects on the donor side as well as the acceptor side are characterized using steady-state O2-evolution assays, electron paramagnetic resonance (EPR) spectroscopy, electron-transfer assays, and flash-induced polarographic O2 yield measurements. Enzyme kinetics analysis shows multiple sites of NO2- inhibition in PSII with significant inhibition of oxygen evolution at concentrations of NO2- below 5 mM. By EPR spectroscopy, the yield of the S2 state remains unchanged up to a concentration of 15 mM NO2-. However, the S2 state g = 4.1 signal is favored over the g = 2 multiline signal with increasing NO2- concentration. This could indicate competition of NO2- for the Cl- site at higher concentrations of NO2-. In addition to the donor-side chemistry, there is clear evidence of an acceptor-side effect of NO2-. The g = 1.9 Fe(II)-QA-• signal is replaced by a broad g = 1.6 signal in the presence of NO2-. Additionally, a g = 1.8 Fe(II)-Q-• signal is present in the dark, indicating the formation of a NO2--bound Fe(II)-QB-• species in the dark. Electron-transfer assays suggest that the inhibitory effect of NO2- on the activity of PSII is largely due to the donor side chemistry of NO2-. UV-visible spectroscopy and flash-induced polarographic O2 yield measurements indicate that NO2- is oxidized by the oxygen-evolving complex (OEC) in the higher S states, contributing to the donor-side inhibition by NO2-.
α-Fe(2)O(3) nanoparticles were synthesized by a low temperature solution combustion method. The structural, magnetic and luminescence properties were studied. Powder X-ray diffraction (PXRD) pattern of α-Fe(2)O(3) exhibits pure rhombohedral structure. SEM micrographs reveal the dumbbell shaped particles. The EPR spectrum shows an intense resonance signal at g≈5.61 corresponding to isolated Fe(3+) ions situated in axially distorted sites, whereas the g≈2.30 is due to Fe(3+) ions coupled by exchange interaction. Raman studies show A(1g) (225cm(-1)) and E(g) (293 and 409cm(-1)) phonon modes. The absorption at 300nm results from the ligand to metal charge transfer transitions whereas the 540nm peak is mainly due to the (6)A(1)+(6)A(1)→(4)T(1)(4G)+(4)T(1)(4G) excitation of an Fe(3+)-Fe(3+) pair. A prominent TL glow peak was observed at 140°C at heating rate of 5°Cs(-1). The trapping parameters namely activation energy (E), frequency factor (s) and order of kinetics (b) were evaluated and discussed.
Oxidation of the reduced primary electron acceptor, QA−, of Photosystem II (PS II) in formate-treated spinach thylakoids, was inhibited more after the second than after the first actinic flash. This indicates a slowing of electron flow on the acceptor side of PS II from QA− to QB−, the semiquinone form of the secondary plastoquinone acceptor, formed by electron transfer after the first flash. A hypothesis of electron transfer on the acceptor side of PS II is proposed to accommodate the bicarbonate-reversible formate/formic acid inhibition of electron transfer after single turnover flashes. We suggest that the large inhibition in QA− oxidation after the second flash reflects a blockage of the proton uptake that stabilizes QB−. Kinetics of onset of inhibition following formate addition were followed by measuring the chlorophyll a fluorescence yield, reflecting the concentration of QA−, 1 ms after the second actinic flash as a function of time after the addition of formate. The apparent rate constants for binding and unbinding, and the dissociation constant of formate were determined in the pH range from 5.5 to 7.5. The rate of onset of inhibition following formate addition, reflecting formate or formic acid binding, was highly dependent on the medium pH. Measurements on the initial binding rate, when one of the two (HCO2−/HCOOH) equilibrium species was kept constant and the other varied, suggested that formic acid is the binding species. This conclusion was consistent with the observed pH dependence of formate binding.
Quite a range of spectroscopic techniques has been applied to the characterization of photosystem II (PSII) mutants. These have been applied to whole cells, thylakoids, core complexes, and reaction centers. This chapter emphasizes those techniques that have most contributed to mutant characterization. They include measurement of the variation of the chlorophyll fluorescence yield, thermoluminescence, ultraviolet/visible (UV/Vis) optical spectroscopy, Fourier transform infrared spectroscopy (FFIR), and the magnetic resonance techniques of electron paramagnetic resonance (EPR), electron spin echo envelope modulation (ESEEM) and cw and pulsed electron nuclear double resonance (ENDOR). Other spectroscopic techniques that have been applied to the study of structure and function of PSII include X-ray absorption spectroscopy, which has been extensively employed by several groups to examine the structure and changes in oxidation state of the Mn cluster during the Kok-Joliot cycle of advance of the S-states.
Water oxidation by plants and cyanobacteria is performed via a light-driven cycle of five intermediates called S states (S(0)-S(4)) at the water oxidizing center (WOC) in photosystem II (PSII). The information about misses, i.e., the probabilities that the S-state transitions failed to advance, is crucial for detailed analysis of various spectroscopic data in investigations of the water oxidation mechanism. In this study, we have determined the miss probabilities of the individual S-state transitions using light-induced Fourier transform infrared (FTIR) difference spectroscopy. The extent of S-state transitions in the WOC upon each saturating flash was monitored by detecting the flow of electrons from the WOC to ferricyanide, an exogenous electron acceptor, using the CN stretching bands of ferricyanide and ferrocyanide. Simulation of the oscillation pattern of the flash-number dependence of the signal amplitude provided the miss probabilities for the S(0) → S(1), S(1) → S(2), S(2) → S(3), and S(3) → S(0) transitions (α(0)-α(3), respectively) without any assumption about fitting parameters. The results for PSII preparations from Thermosynechococcus elongatus and spinach showed a general tendency of misses in the order, α(0) ≤ α(1) < α(2) < α(3), indicating that a more oxidized WOC has a higher miss probability. A very similar result observed for the Y(D)-less mutant (D2-Y160F) of T. elongatus confirmed that Y(D) does not affect the estimated misses. It was further shown that NO(3)(-) treatment specifically increased α(3), consistent with inactivation of the S(3) state reported previously. These results demonstrate the usefulness of this FTIR method for estimating individual miss probabilities in the S-state cycle in elucidation of the molecular mechanism of photosynthetic water oxidation.
The iron quinone-complex of the reaction centers of photosystem II and the purple non-sulphur photosynthetic bacteria contains two quinones, QA and QB connected in series with respect to electron transfer, and separated by a non-heme iron coordinated by amino acid residues. It is the site of inhibition of many of the common photosynthetic herbicides, which act by displacing QB from the center. The complex is responsible for reducing QB to QBH2 in two successive one-electron photo acts. OBH2 dissociates from the center, to be replaced by a new QB molecule and reduces the following membrane-bound electron-transfer complex (cytochrome b6/for b/c1). The energetic, kinetics and mechanism of complex function are reviewed here. Recent crystallographic, spectroscopic and molecular biological evidence has produced a considerable quantity of structural information on this complex. These data have given a less formal and more molecular view of how the complex functions. They have also revealed fundamental differences between the photo system II and bacterial complexes, particularly with respect to the coordination of the iron and its chemistry. The comparative anatomy of the complexes is reviewed and its implications for function discussed.
Depletion of bicarbonate (carbon dioxide) from oxygenic cells or organelles not only causes cessation of carbon dioxide fixation, but also a strong decrease in the activity of photosystem II; the photosystem II activity can be restored by readdition of bicarbonate. Effects of bicarbonate exist on both the acceptor as well as on the donor side of photosystem II. The influence on the acceptor side is located between the primary and secondary quinone electron acceptor of photosystem II, and can be demonstrated in intact cells or leaves as well as in isolated thylakoids and reaction center preparations. At physiological pH, bicarbonate ions are suggested to form hydrogen bonds to several amino acids on both D1 and D2 proteins, the reaction center subunits of photosystem II, as well as to form ligands to the non-heme iron between the D1 and D2 proteins. Bicarbonate, at physiological pH, has an important role in the water-plastoquinone oxido-reductase: on the one hand it may stabilize, by conformational means, the reaction center protein of photosystem II that allows efficient electron flow and protonation of certain amino acids near the secondary quinone electron acceptor of photosystem II; and, on the other hand, it akppears to play a significant role in the assembly or functioning of the manganese complex at the donor side. Functional roles of bicarbonate in vivo, including protection against photoinhibition, are also discussed.
The fast (up to 1 s) chlorophyll (Chl) a fluorescence induction (FI) curve, measured under saturating continuous light, has a photochemical phase, the O-J rise, related mainly to the reduction of Q(A), the primary electron acceptor plastoquinone of Photosystem II (PSII); here, the fluorescence rise depends strongly on the number of photons absorbed. This is followed by a thermal phase, the J-I-P rise, which disappears at subfreezing temperatures. According to the mainstream interpretation of the fast FI, the variable fluorescence originates from PSII antenna, and the oxidized Q(A) is the most important quencher influencing the O-J-I-P curve. As the reaction centers of PSII are gradually closed by the photochemical reduction of Q(A), Chl fluorescence, F, rises from the O level (the minimal level) to the P level (the peak); yet, the relationship between F and [Q(A) (-)] is not linear, due to the presence of other quenchers and modifiers. Several alternative theories have been proposed, which give different interpretations of the O-J-I-P transient. The main idea in these alternative theories is that in saturating light, Q(A) is almost completely reduced already at the end of the photochemical phase O-J, but the fluorescence yield is lower than its maximum value due to the presence of either a second quencher besides Q(A), or there is an another process quenching the fluorescence; in the second quencher hypothesis, this quencher is consumed (or the process of quenching the fluorescence is reversed) during the thermal phase J-I-P. In this review, we discuss these theories. Based on our critical examination, that includes pros and cons of each theory, as well mathematical modeling, we conclude that the mainstream interpretation of the O-J-I-P transient is the most credible one, as none of the alternative ideas provide adequate explanation or experimental proof for the almost complete reduction of Q(A) at the end of the O-J phase, and for the origin of the fluorescence rise during the thermal phase. However, we suggest that some of the factors influencing the fluorescence yield that have been proposed in these newer theories, as e.g., the membrane potential ΔΨ, as suggested by Vredenberg and his associates, can potentially contribute to modulate the O-J-I-P transient in parallel with the reduction of Q(A), through changes at the PSII antenna and/or at the reaction center, or, possibly, through the control of the oxidation-reduction of the PQ-pool, including proton transfer into the lumen, as suggested by Rubin and his associates. We present in this review our personal perspective mainly on our understanding of the thermal phase, the J-I-P rise during Chl a FI in plants and algae.
Earlier studies have demonstrated that NO binds to the non-heme iron of the PS II ferroquinone complex in competition with the physiological ligand CO2/HCO−3 (Petrouleas, V. and Diner, B.A. (1990) Biochim. Biophys. Acta 1015, 131–140; Diner, B.A. and Petrouleas, V. (1990) Biochim. Biophys. Acta 1015, 141–149). We examine in this paper the effect of cyanide, also a potential iron chelator. Competition experiments involving CN− and NO show that 50 mM CN− at pH 6.5 eliminates the EPR signal at g = 4 arising from the Fe2+-NO adduct. Illumination of CN−-treated PS II preparations under conditions which induce single charge separation produces a new EPR signal at g = 1.98. The temperature and power dependence indicate that this signal is directly or indirectly associated with a transition metal species. The signal is produced with undiminished intensity upon illumination in the presence of hydroxylamine as an exogenous electron donor and of DCMU, indicating that it originates from an acceptor side species prior to the DCMU block. Upon successive one-electron charge separations the g = 1.98 signal oscillates with period of two. These results strongly suggest that the g = 1.98 signal originates from the state Q−AFe2+. An approximate titration of the g = 1.98 signal development as a function of the total cyanide concentration at pH 6.5 indicates a kd of 50–80 mM, significantly higher than the kd for cyanide-NO competition, estimated to be in the range of 10 mM. It is likely that, while displacement of NO requires the binding of one cyanide molecule, development of the modified Q−AFe2+ signal at g = 1.98 requires the binding of more than one cyanide molecules. The kinetics of the fluorescence relaxation following saturating flashes show only subtle differences over the concentration range at which cyanide displaces NO and probably bicarbonate as well. Treatment with higher concentrations of cyanide at pH 6.5 causes an inversion in the phase of the oscillation pattern that characterizes the decay of the fluorescence yield. The latter effect becomes more pronounced at higher pH levels. The absence of a slowing of the fluorescence relaxation in the presence of cyanide may indicate that CN− can fulfill the same role as bicarbonate, perhaps in promoting proton transfer coupled to interquinone electron transfer. Only the relative rates of the two interquinone electron transfer reactions are reversed.
The herbicide binding site of the D1 protein in Photosystem II from spinach was modeled on the basis of the homologous L subunit of the photosynthetic reaction center of the bacterium Rhodopseudomonas viridis (Deisenhofer, J., Epp, O., Miki, K., Huber, R. and Michel, H. (1985) Nature 318, 618–624), and on the assignment of functional amino acid residues occurring in these two protein subunits. The overall structure of the L subunit with α-helices D, DE and E was assumed to be conserved in the D1 protein, although the loops connecting helices DE, E and D, DE were enlarged by 3 and 14 amino acid residues, respectively. Protein data bank searches for appropriate loop structures were performed, but none was found to be compatible with experimental data. The binding positions of some herbicides and of the natural substrate in the spinach Photosystem II are proposed and discussed in terms of different binding modes, and interpreted on the basis of data obtained from mutant D1 proteins.
By studying the electron paramagnetic resonance (EPR) signals of QA/−-Fe2+TBTQ− and the oxidised non-haem iron we have found that detergent solubilisation of BBY Photosystem II (PS II) preparations using standard methods, involving either the detergents n-octyl β-d-glucopyranoside (OGP) or n-heptyl β-d-thioglucoside (HTG) at pH 6.0, results in loss of bicarbonate binding. New preparations including a dodecyl maltoside (DM) prepared CP47, CP43, D1, D2, cytochrome b-559 complex are described which at pH 7.5 retain native bicarbonate binding. These preparations provide a new system for studies on the ‘bicarbonate effect’ because bicarbonate depletion can now be achieved without displacement by another anion. They are also a more suitable starting material for the isolation of QA retaining D1/D2 reaction centres because the detrimental changes to the QA binding region are avoided.
Electron paramagnetic resonance (EPR) spectra of the reduced quinone-iron acceptor complex in reaction centers were measured in a variety of environments and compared with spectra calculated from a theoretical model. Spectra were obtained at microwave frequencies of 1, 9, and 35 GHz and at temperatures from 1.4 to 30 K. The spectra are characterized by a broad absorption peak centered at g = 1.8 with wings extending from g approximately equal to 5 to g less than 0.8. The peak is split with the low-field component increasing in amplitude with temperature. The theoretical model is based on a spin Hamiltonian, in which the reduced quinone, Q-, interacts magnetically with Fe2+. In this model the ground manifold of the interacting Q-Fe2+ system has two lowest doublets that are separated by approximately 3 K. Both perturbation analyses and exact numerical calculations were used to show how the observed spectrum arises from these two doublets. The following spin Hamiltonian parameters optimized the agreement between simulated and observed spectra: the electronic g tensor gFe, x = 2.16, gFe, y = 2.27, gFez = 2.04, the crystal field parameters D = 7.60 K and E/D = 0.25, and the antiferromagnetic magnetic interaction tensor, Jx = -0.13 K, Jy = -0.58 K, Jz = -0.58 K. The model accounts well for the g value (1.8) of the broad peak, the observed splitting of the peak, the high and low g value wings, and the observed temperature dependence of the shape of the spectra. The structural implications of the value of the magnetic interaction, J, and the influence of the environment on the spin Hamiltonian parameters are discussed. The similarity of spectra and relaxation times observed from the primary and secondary acceptor complexes Q-AFe2+ and Fe2+Q-B leads to the conclusion that the Fe2+ is approximately equidistant from QA and QB.
The 32 kDa herbicide and QBbinding peptide (D-1 protein) and its homologous 34 kDa peptide (D-2 protein) are integral membrane subunits of photosystem II. A model for their folding through the thylakoid membrane in five transmembrane α-helices is proposed from the comparison of amino acid sequence and hydropathy index plot homologies with subunits of the bacterial system. Following recent data on the X-ray structure of a bacterial photosystem the binding niche for QBis interpreted on the basis of the amino acid changes found in the 32 kDa peptide in herbicide tolerant higher plants and algae. © 1986, Verlag der Zeitschrift für Naturforschung. All rights reserved.
The folding through the membrane of the plastoquinone and herbicide binding protein subunits of photosystem II and the topology of the binding niche for plastoquinone and herbicides is described. The model is based on the homology in amino acid sequence and folding prediction from the hydropathy analysis of the D-1 and D-2 subunits of photosystem II to the reaction center polypeptides L and M of the bacterial reaction center. It incorporates the amino acid changes in the D-1 polypeptide in herbicide tolerant plants and those indicated by chemical tagging to be involved in QB binding. It proposes hom ologous amino acids in the D-1/D-2 polypeptides to those indicated by the X-ray structure of the bacterial reaction center to be involved in Fe-, quinone- and reaction center chlorophyll-binding. The different chemical compounds known to interfere with QB function are grouped into two families depending on their orientation in the QB binding niche.
This chapter discusses the mechanism involved in electron transfer on the acceptor side of photosystem II (PSII). Cytochrome b6 (cyt b6) is included in a protein complex that also contains cytochrome f (cyt f) and a FeS protein. It has been proposed that the reduction of cyt b6 by the plastoquinone (PQ) pool is necessarily coupled with the reduction of a photosystem I donor, probably cyt f. This concept is also used in Mitchell's Q-cycle. This hypothesis implies that cyt f must be oxidized prior to the reduction of cyt b6. Under strongly oxidizing conditions, that is, in the presence of ferricyanide, both the PQ pool and cyt f are totally oxidized. Therefore, the reduction of cyt b6 is limited by the electron transfer from the PSII centers to the cyt b6–f complex. The reduction of cyt b6 in the presence of ferricyanide requires an illumination by two successive flashes, which shows that the formation of the doubly-reduced secondary acceptor BH2 is a necessary step in this reduction.
According to the generally accepted model of O2 evolution (1, 2), the O2 centers, each associated with a photosysfcem II unit, act independently of each other. Driven by 4 successive photoacts, the O2 enzyme cycles through 5 oxidation states:
This paper points out that the orientations of the porphyrins, bacteriochlorophyll and bacteriopheophytin, in the reaction centers of Rhodopseudomonas viridis, as shown by the new X-ray determined structure, have a peculiar orientation towards each other: electron donors are broadside toward the acceptors and acceptors are edgeon toward donors. Vibronic coupling which is the mechanism of converting free-energy loss in electron transport to vibrational energy is examined as a possible explanation. Preliminary calculations do not support this as an explanation of the orientations but suggest strongly that the non-heme iron atom has the function of promoting vibronic coupling in the electron transfer from bacteriopheophytin to menaquinone. It is further suggested that the system of electron transport from the special pair of bacteriochlorophyll to the bacteriopheophytin is arranged to keep virbonic coupling to a minimum to match the very small electronic free-energy loss in this region.
Integrated intensities of EPR lines and the simulation of powder shapes in the presence of large anisotropy are discussed for field-sweep spectra. It is pointed out that the shape function, normalized by integration over the magnetic field, must be multiplied by a factor which in the case equals the inverse of the g-value. This factor seemingly has been omitted in previous calculations of intensities and shapes. As a consequence, the integrated intensity of an isotropic line is proportional to its g-value and not to g2. The total intensity of a powder spectrum is calculated, and examples of simulations of such spectra are given. A method for the determination of total intensities from the area under an “absorption” peak in a first derivative powder spectrum is also given.
Chloroplasts were submitted to a sequence of saturating short flashes and then rapidly mixed with dichlorophenyldimethylurea (DCMU). The amount of singly reduced secondary acceptor (B−) present was estimated from the DCMU-induced increase in fluorescence in the dark caused by the reaction: QB− Q−B. By varying the time interval between the preillumination and the mixing, the time course of B− reoxidation by externally added benzoquinone was investigated. It was found that benzoquinone oxidizes B− in a bimolecular reaction, and does not interact directly with Q−. When a sufficient delay after the preillumination was allowed in order to let benzoquinone reoxidize B− before the injection of DCMU, the fluorescence increase caused by one subsequent flash fired in the presence of DCMU was followed by a fast decay phase (). The amplitude of this phase was proportional to the amount of B− produced by the preillumination. This fast decay was observed only after the first flash in the presence of DCMU. These results are interpreted by assuming a binding of the singly reduced benzoquinone to Photosystem II where it acts as an efficient, DCMU-insensitive, secondary (exogenous) acceptor.
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.
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.
Polycrystalline samples of iron‐containing ferrichrome A, a cyclic hexapeptide obtained from the fungus Ustillago sphaerogena, have been investigated by paramagnetic resonance. Spectra were obtained at several temperatures between 300° and 1°K; a prominent line of 400‐Oe width located at g=4.3 was observed at all temperatures, while at 1°K additional resonances at g values of 9.6, 1.3, and 1.0 were observed. The spectra are interpreted by assuming a spin Hamiltonian containing crystal‐field terms large compared with the Zeeman splittings; the crystal‐field situation is intermediate between the case of axial symmetry, with H = D[Sz2−(1/3)S(S+1)]+gβS⋅H and a model proposed by Castner, Newell, Holton, and Slichter to explain certain iron resonances occurring at g=4.3, with H = E(Sx2−Sy2)+gβS⋅H. We have computed g values, energy eigenvalues, and eigenfunctions to be expected for the region between these two extremes, and the results should be useful in interpreting similar spectra due to iron situated in strong crystal fields of low symmetry.
The syntheses and physical properties of a series of Fe(salen)X and Fe(saloph)X complexes where X is phenolate or catecholate are reported. Magnetic susceptibility measurements as well as electronic, infrared, and NMR spectra indicate that the catecholate in Fe(salen)catH behaves very much like a phenolate and is concluded to be monodentate in its coordination to the iron. The abstraction of a proton from Fe(salen)catH results in an anionic complex, [Fe(salen)cat]-, with markedly different properties; the catecholate in this complex is chelated. Both monodentate and chelated catecholate complexes are high-spin ferric, demonstrating that catecholate coordination to a bis(phenolato)iron(III) complex does not result in the reduction of the ferric center. This is in agreement with observations made on dioxygenase-substrate complexes. In addition, studies on a series of Fe(salen)X complexes where X is phenolate, thiophenolate, benzoate, and catecholate show that the dominant salen-to-Fe(III) charge-transfer interaction is modulated by the coordination of these ligands. Comparisons with corresponding dioxygenase complexes show that the tyrosinate-to-iron(III) charge-transfer interactions are similarly affected, thus indicating that the salen ligand provides a reasonable approximation of the iron environment in the dioxygenases.
The phenolate-to-iron(III) charge-transfer transition in a series of iron(III) phenolate complexes has been investigated. NMR contact shifts for the phenolate protons are well correlated with the visible absorption maxima of the complexes and the FeIII/FeII redox potentials. The results indicate that the energy of the phenolate-to-iron(III) charge-transfer band is sensitive to the crystal field strength of the other ligands coordinated to the ferric center. The stronger the other ligands are, the higher the energy of the phenolate charge-transfer band. The blue shift of the charge-transfer band is also reflected in smaller contact shifts for the phenolate protons and a more negative FeIII/FeII redox potential. On the basis of these studies, the probable identities of ligating species in transient dioxygenase intermediates are deduced. These studies also demonstrate that a square-pyramidal complex with an apical and a basal phenolate can give rise to phenolate charge-transfer bands of quite different energies. A dioxygenase active site approaching such a structure is proposed. Lastly, the axial ligand in Fe(salen)OC6H4-4-CH3 is shown to be an excellent model for tyrosine in resonance Raman studies of iron-tyrosinate proteins. Isotopic substitution studies on the model complex show that the ca. 570-cm-1 feature found in these proteins cannot be assigned solely to an Fe-O stretching vibration.
This chapter discusses the photosynthetic oxygen-evolving process. The oxygen-evolving system in photosynthesis uses light energy to promote electrons from a plentiful substrate, water, to higher energy where they are ultimately used to reduce CO2 to organic products. The protons liberated in water oxidation are released vectorially, and contribute to the membrane free energy gradient that drives adenosine triphosphate (ATP) synthesis. With the progress made recently in understanding the interplay between the peripheral polypeptides and the required small ions, chloride, calcium and manganese, the effects of PS II oxidizing side inhibition have become both clearer and more complicated. A good deal of effort is currently being spent defining the loci of inhibitory treatments, particularly when manganese is not released by the inhibition. The situation when manganese is released is clearer and is dealt with first. The electron transfer cofactors have been identified to a large extent and their reactions characterized in some detail. Their protein binding sites and geometric arrangement is currently the subject of intense scrutiny. The mechanism of water oxidation and the organization of the active-site manganese atoms have lost a good deal of their nebulousness, and models based on solid metallobiochemical and inorganic chemical principles are being postulated. The uniqueness of the photosynthetic reaction whereby water is split is likely to be paralleled by uniqueness in the catalytic structures.
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.
The kinetics and concentration dependence of the binding of dichlorophenyldimethylurea (DCMU) to Photosystem II (PS II) were monitored through fluorescence measurements. According to whether the acceptor system is in the ‘odd’ state (QB− ag Q−B) or ‘even’ state (QB), very different results are obtained. The binding to centers in the even state is rapid ( at [DCMU] = 10−5 M and [chlorophyll] = 10 μg/ml), with a pH-independent rate. The concentration curve of the bound inhibitor (at equilibrium) corresponds to an association constant of about 3.3·107 M−1·1. The binding of the inhibitor to centers in the odd state is slow ( at pH 7, same DCMU and chlorophyll concentrations as above), and depends on pH. In the pH range 6–8, the lower the pH, the slower the kinetics. The association constant is also diminished by a factor of approx. 20 (at pH 7) compared to the even state centers. It is shown that these effects are in good agreement with predictions from Velthuys' hypothesis (Velthuys, B.R. (1981) FEBS Lett. 126, 277–281) that the mode of action of DCMU is a competition with plastoquinone for the binding to the secondary acceptor site. A large part of PS II photochemical quenching corresponds to acceptors which seem to possess a secondary acceptor distinct from B. They were called ‘non-B-type acceptors’ (Lavergne, J. (1982) Photobiochem. Photobiophys. 3, 257–285) and may be identified with Joliot's ‘Q2’ (Joliot P. and Joliot, A. (1977) Biochim. Biophys. Acta 462, 559–574). However, the rate at which the inhibition affects these non-B-type acceptors is similar to the rate of DCMU binding on the B site (i.e., slow in the odd state, fast in the even state).
The ferrous ion associated with the electron acceptors in Photosystem II can be oxidized by the unstable semiquinone form of certain high-potential quinones (phenyl-p-benzoquinone, dimethylbenzoquinone and benzoquinone) which are used as electron acceptors. In a flash sequence, alternating oxidation of the iron by the photoreduced semiquinone on odd-numbered flashes is followed by photoreduction of the iron on even-numbered flashes. These reactions are detected by monitoring EPR signals arising from Fe3+. The oxidation of the iron can also occur in the frozen state (−30°C) indicating that the high-potential quinone can occupy the QB site. The reaction also takes place when the exogenous quinone is added in the dark to samples in which QB is already in the semiquinone form. The inhibitors of electron transfer between QA− and QB, DCMU and sodium formate, block the photoreductant-induced iron oxidation. It is suggested that the iron oxidation takes place through the QB site. This unexpected photochemistry occurs under experimental conditions routinely used in studies of Photosystem II. Some previously reported phenomena can be reinterpreted on the basis of these new data.
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.
The protolytic reactions of PSII membrane fragments were analyzed by measurements of absorption changes of the water soluble indicator dye bromocresol purple induced by a train of 10 s flashes in dark-adapted samples. It was found that: a) in the first flash a rapid H+-release takes place followed by a slower H+-uptake. The deprotonation is insensitive to DCMU but is completely eliminated by linolenic acid treatment of the samples; b) the extent of the H+-uptake in the first flash depends on the redox potential of the suspension. In this time domain no H+-uptake is observed in the subsequent flashes; c) the extent of the H+-release as a function of the flash number in the sequence exhibits a characteristic oscillation pattern. Multiphasic release kinetics are observed. The oscillation pattern can be satisfactorily described by a 1, 0, 1, 2 stoichiometry for the redox transitions Si Si+1 (i=0, 1, 2, 3) in the water oxidizing enzyme system Y. The H+-uptake after the first flash is assumed to be a consequence of the very fast reduction of oxidized Q400(Fe3+) formed due to dark incubation with K3[Fe(CN)6]. The possible participation of component Z in the deprotonation reactions at the PSII donor side is discussed.
Reaction centers from wild-type Rhodobacter sphaeroides (formerly called Rhodopseudomonas sphaeroides) were separated into two components: the LM complex and H subunit. LM was isolated after brief treatment of reaction centers with SDS by affinity chromatography with cytochrome c as ligand. A stable H preparation was obtained after dissociation of reaction centers with lithium perchlorate. LM was depleted of the transition metal, Mn, which interacts with QA and QB in native reaction centers. It retained only 30% of primary photochemistry which could be restored to 50–80% by addition of Q6, Q10 or other quinones. A stable semiquinone radical Q−A could be flash-induced in LM. Its absorption properties are similar to those of Q−A in native reaction centers. The quantum yield of photochemistry in an LM unit reconstituted with Q6 is the same as in intact reaction center and in LM in the presence of H. This result was confirmed by the rapid electron-transfer rate between I− and QA in LM + H (τ ≈ 0.45 ns). Ubiquinone in LM incubated with H becomes tightly bound at the QA site. Flash production of a Q2− species was not detected in LM and LM + H. We conclude that the depletion of the reaction center both of the H subunit and of the metal does not necessarily lower the quantum yield of the primary reaction or greatly modify the rate of electron transfer from I− to QA. These results contrast with observations of others that seemed to demonstrate that the metal is essential for high-rate electron transfer between I− and QA (Debus, R.J., Feher, G. and Okamura, M.Y. (1986) Biochemistry 25, 2276–2287). In our experiments, secondary electron transfer to QB was not restored in LM + H, unlike in reconstitution experiments reported with R26 Rb. sphaeroides reaction centers (Debus, R.J., Feher, G. and Okamura, M.Y. (1985) Biochemistry 24, 2488–2500). Apparently, interactions between H and LM were too weak for restoring QB activity.
Four representative inhibitors of Photosystem II (PS II) Q−A to QB electron transfer were shown to bind, at high concentrations, to PS II reaction centers having the acceptor-side non-heme iron in the Fe(III) state. Three of the inhibitors studied, DCMU, o-phenanthroline and dinoseb, modified the EPR spectrum of the Fe(III) relative to that obtained by ferricyanide oxidation in the absence of inhibitor. o-Phenanthroline gave particularly axial symmetry, while DCMU and dinoseb gave more rhombic configurations. The herbicide inhibitor, atrazine and its analogue, terbutryn, had no effect. The dissociation constants for inhibitor binding to reaction centers in the Fe(III) state were measured directly and also estimated from shifts in the midpoint potential of the Fe(III)/Fe(II) couple and were shown to increase by factors of approx. 100, approx. 10 and 10–15 for DCMU (pH 7.5), atrazine (pH 7.0) and o-phenanthroline (pH 7.0), respectively, upon oxidation of the iron. Atrazine and o-phenanthroline, which induce the smallest changes in the midpoint potential of the Fe(III)/Fe(II) couple, were shown to inhibit light-induced oxidation of the Fe(II) by phenyl-p-BQ, described in the preceding paper (Petrouleas, V. and Diner, B.A. (1987) Biochim. Biophys. Acta 893, 126–137). The extent of inhibition was much greater than would be predicted from a simple shift in the midpoint potential for Fe(III)/Fe(II) and we conclude that phenyl-p-BQ and the other quinones, which show light-induced oxidation, act through the QB binding site. It is also argued that reduction and oxidation of the iron by ferro- and ferricyanide, respectively, occur through this site. The effects of these inhibitors and of various quinones on the Fe(III) environment are discussed with reference to the known contact points between the protein and o-phenanthroline and terbutryn in the QB binding pocket of Rhodopseudomonas viridis reaction centers (Michel, H., Epp, O. and Deisenhofer, J. (1986) EMBO J. 5, 2445–2451). The Fe(III) EPR spectrum is thus a new and sensitive probe of the contact points at which molecules bind to the QB binding site.
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).
The mechanism of the bicarbonate effect was investigated by monitoring flash-induced pH changes. In control chloroplasts the proton yields exhibit a binary oscillation with a period of four. In CO2-depleted chloroplasts the binary oscillation disappears and only the period four pattern remains, which can be described by proton liberation in the water-oxidizing system. It is concluded that bicarbonate is involved in the protonation of Q2−B. The affinity of bicarbonate to its binding site is much lower in the presence of dithionite. It is suggested that bicarbonate exerts its influence through being a ligand for the non-haem iron between QA and QB.
Electron paramagnetic resonance (EPR) spectroscopy of the iron-semiquinone complex in photosynthetic bacterial cells and chromatophores of Rhodopseudomonas viridis is reported. Magnetic fields are used to orient the prolate ellipsoidal-shaped cells which possess a highly ordered internal structure, consisting of concentric, nearly cylindrical membranes. The field-oriented suspension of cells exhibits a highly dichroic EPR signal for the iron-semiquinone complex, showing that the iron possesses a low-symmetry ligand field and exists in a preferred orientation within the native reaction-center membrane complex. The EPR spectrum is analyzed utilizing a spin hamiltonian formalism to extract physical information describing the electronic structure of the iron and the nature of its interaction with the semiquinones. Exact numerical solutions and analytical expressions for the transition frequencies and intensities derived from a perturbation theory expansion are presented, and a computer-simulated spectrum is given. It has been found that, for a model which assumes no preferred orientation within the plane of the membranes, the orientation of the Fe2+ ligand axis of largest zero-field splitting (Z, the principal magnetic axis) is titled 64±6° from the membrane normal. The ligand field for Fe2+ has low symmetry, with zero-field splitting parameters of |D1|=7.0±1.3 cm−1 and |E1|=1.7±0.5 cm−1 and for the redox state Q1−Fe2+Q2−. The rhombic character of the ligand field is increased in the redox state Q1Fe2+Q−2, where . This indicates that the redox state of the quinones can influence the ligand field symmetry and splitting of the Fe2+. There exists an electron-spin exchange interaction between Fe2+ and Q−1 and Q−2, having magnitudes |J1|=0.12±0.03 cm−1 and , respectively. Such weak interactions indicate that a proper electronic picture of the complex is as a pair of immobilized semiquinone radicals having very little orbital overlap (probably fostered by superexchange) with the Fe2+ orbitals. The exchange interaction is analyzed by comparison with model systems of paramagnetic metals and free radicals to indicate an absence of direct coordination between Fe2+ and Q−1 and Q−2. Selective line-broadening of some of the EPR transitions, involving Q− coupling to the magnetic sublevels of the Fe2+ ground state, is interpreted as arising from an electron-electron dipolar interaction. Analysis of this line-broadening indicates a distance of 6.2–7.8 Ȧ between Fe2+ and Q−1, thus placing Q1 outside the immediate coordination shell of Fe2+.
The decay of fluorescence yield following each of a series of saturating laser flashes has been used to monitor the kinetics of reoxidation of the primary acceptor of Photosystem II under conditions of varied redox potential. 1.1. In dark-adapted chloroplasts, a damped binary oscillation as a function of flash number was observed in the kinetics of the decay of the fluorescence yield. The decay was faster on odd than on even-numbered flashes.2.2. In the presence of low concentrations of 1,4-benzoquinone, the oscillation was more marked, and over the range approx 200–350 mV, independent of redox potential. The decay following flash 1 under these conditions had a half-time of approx. 200–400 μs. The decay following flash 2 was decelerated; the initial rate was up to 10-fold slower than after flash 1.3.3. We suggest that the kinetics following a single flash reflect the rate of the reaction Q−B → QB−, and following the second flash, Q−B− → QB2−. Benzoquinone at low concentrations oxidises a residual fraction of B− which is usually reduced in the dark before the flash sequence.4.4. A faster component in the decay () following the first flash titrated in over the range Eh > 350 mV. The binary oscillation was still apparent but delayed by one flash.5.5. We discuss the relative redox potentials of the couples and , and the role of the component which titrates in at Eh > 350 mV.
Quinone and inhibitor binding to Rhodopseudomonas sphaeroides (R-26 and GA) reaction centers were studied using spectroscopic methods and by direct adsorption of reaction centers onto anion exchange filters in the presence of 14C-labelled quinone or inhibitor. These measurements show that as secondary acceptor, QB, ubiquinone (UQ) is tightly bound in the semiquinone form and loosely bound in the quinone and quinol forms. The quinol is probably more loosely bound than the quinone. o-Phenanthroline and terbutryn, a triazine inhibitor, compete with UQ and with each other for binding to the reaction center. Inhibition by o-phenanthroline of electron transfer from the primary to the secondary quinone acceptor (QA to QB) occurs via displacement of UQ from the QB binding site. Displacement of UQ by terbutryn is apparently accessory to the inhibition of electron transfer. Terbutryn binding is lowered by reduction of QB to Q−B but is practically unaffected by reduction of QA to Q−A in the absence of QB. UQ-9 and UQ-10 have a 5- to 6-fold higher binding affinity to the QB site than does UQ-1, indicating that the long isoprenoid chain facilitates the binding to the QB site.
We have recently shown by optical, EPR and Mössbauer spectroscopy that the high spin Fe(II) of the quinone-iron acceptor complex of Photosystem II can be oxidized by ferricyanide to high-spin Fe(III). The midpoint potential of the Fe(III)/Fe(II) couple is 370 mV at pH 7.5 and shows an approximate pH-dependence of −60 mV/pH unit. The iron was identified as being responsible for the high potential Photosystem II acceptor known as Q400, discovered by Ikegami and Katoh ((1975) Plant Cell Physiol. 14, 829–836) but until now not identified chemically. We establish here that QA and the oxidized Fe(III) are linked in series, with QA the first to be reduced in the primary charge separation of Photosystem II. At pH 7.5, an electron is then transferred from Q−A to Fe(III) with a of 25 μs, reforming QA Fe(II). The Fe(II) can also be oxidized to Fe(III) in oxygen-evolving thylakoid membranes through a photoreduction-induced oxidation in the presence of exogenous quinones, where . Single turnover illumination of the Photosystem II reaction center at 200 K, followed by warming to 0°C, results in photoreduction of these quinones to the semiquinone form which in turn oxidizes the Fe(II) to Fe(III). A second turnover of the reaction center reduces Fe(III) back to Fe(II). These reactions, similar to those reported by Zimmermann and Rutherford (Zimmermann, J.L. and Rutherford, A.W. (1986) Biochim. Biophys. Acta 851, 416–423) at room temperature, in work largely done in parallel, are summarized below: where QA and Qex are the primary quinone acceptor of Photosystem II and exogenous quinone, respectively. Detection of Fe(III) at g = 8 by EPR spectroscopy shows this signal to oscillate with period two upon successive turnovers of the Photosystem II reaction center. Different exogenous quinones give different EPR spectra for Fe(III), indicating that these bind close to the Fe binding site and modify the symmetry of the Fe(III) environment. A study of the pH-dependence of the light-induced oxidation of the Fe(III) by phenyl-p-BQ shows a pH-optimum at 6–7. The decline at higher pH is consistent with a pH-dependence of −60 mV/pH unit and −120 mV/pH unit, respectively, for redox couples Fe(III)/Fe(II) and Q−/QH2. The decline at lower pH was not foreseen and appears associated with a transformation of the quinone-iron environment from that showing a Q−AFe(II) EPR resonance of g = 1.9 at high pH to one at g = 1.84 below pH 6.5. The latter form appears not to support light-induced oxidation of the Fe(II) by exogenous quinones.
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.
A wild-type strain of Rhodopseudomonas sphaeroides was found to have manganese, instead of the expected iron, associated with the quinone acceptor complex in a high proportion of its photosynthetic reaction centres. Almost homogeneous reaction centre preparations containing either of these metals were obtained by appropriate depletion or enrichment of the metals in the growth media. An EPR signal attributed to reaction centre bound manganese was present in the dark in manganese-containing centres. This signal was absent under conditions where either QA or QB were in the semiquinone form. New EPR signals attributed to QA− Mn and QB−Mn are reported.PhotosynthesisManganese-semiquinoneESRIron-semiquinone
Absorbance changes and fluorescence yield changes induced by irradiating spinach chloroplasts with red light at −196° were measured as a function of the redox potential of the chloroplast suspension. Absorbance changes at 546 nm indicate the photoreduction of C-550 and changes at 556 nm indicate the photooxidation of cytochrome b 559. The changes of fluorescence yield indicate the photoreduction of Q, the fluorescence quencher of chlorophylla a in Photosystem II. The titration curves for all three changes were essentially the same and showed the same midpoint potential. In other experiments as well, it was found that when C-550 is in the reduced state the fluorescence yield of the chloroplasts is high and the low-temperature photooxidation of cytochrome b 559 is blocked. These data indicate that C-550 may be equivalent to Q and that cytochrome b 559 serves as the electron donor for the photoreduction of C-550 at low temperature.
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.
A new EPR signal is reported in Rhodospirillum rubrum chromatophores. The signal is attributed to Q−BFe2+, the semiquinone-iron complex of the secondary quinone electron acceptor, on the basis of the following observations. (1) It is induced by a single laser flash given a room temperature and is stable. (2) It is present after odd-numbered flashes and absent after even-numbered flashes when a series of flashes is given. (3) When it is already present, low-temperature illumination results in the disappearance of the signal due to formation of the Q−AFe2+Q−B state. (4) Its formation is inhibited by the presence of orthophenanthroline at normal values of pH. The Q−BFe2+ signal has two main features, one at g = 1.93 and the other at g = 1.82. The two features have different microwave power and temperature dependences, with the g = 1.82 signal being more difficult to saturate and requiring lower temperatures to be observable. Raising the pH leads to an increase in the g = 1.82 feature, while the g = 1.93 signal decreases in amplitude. It is suggested that the two parts of the signal may represent two EPR forms due to structural heterogeneity. The low-field feature of the Q−BFe2+ signal shifts to lower field as the pH is raised and a pK for this change seems to occur at pH 9.4. The Q−AFe2+ signal at g = 1.88 also shifts as the pH is increased; however, the shift is less marked than that seen for Q−BFe2+, the shift is to higher field and the range over which it occurs is wider and depends upon the temperature of Q−AFe2+ formation. This effect may be due to a pK on a protein group being shifted to higher pH by the presence of Q−A. ortho-Phenanthroline broadens and shifts the Q−AFe2+ signal. The inhibition of electron transfer between Q−A and QB by ortho-phenanthroline becomes less effective at high pH. The new Q−BFe2+ signal is unlike other semiquinone-iron signals reported in the literature in bacteria; however, it is remarkably similar to the Q−BFe2+ signal reported in Photosystem II.
EPR signals in the high-spin region were studied at 10 K in photosystem II (PS II) particles and in a purified oxygen-evolving PS II reaction center complex under oxidizing conditions. PS II particles showed EPR peaks at g = 8.0 and 5.6, confirming the recent report by Petrouleas and Diner [(1986) Biochim. Biophys. Acta 849, 264-275]. Addition of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) or o-phenanthroline shifted the peaks to be closer to g = 6.0 depending on the medium pH. On the other hand, the PS II reaction center complex showed peaks at g = 6.1 and 7.8, and at g = 6.1 and 6.4, in the absence and presence of o-phenanthroline, respectively. All these peaks were found to be decreased by the illumination at 10 K. These results suggest that the high-spin signals are due to Q400, Fe(III) atom interacting with the PS II primary electron acceptor quinone QA as reported and that the Fe atom also interacts with the secondary acceptor quinone QB. This interaction seems to induce the highly asymmetric ligand coordination of the Fe atom and to be affected by DCMU and o-phenanthroline in a somewhat different manner.
Delayed luminescence from saturating flashes given to isolated chloroplasts was measured in the time range of 65–800 μsec with the following results: 1.1. Three distinct components having decay half times of approx. 10, 35 and 200 μsec could be detected.2.2. The yields of both the 35- and 200-μsec delayed luminescence components oscillate with a period of four, in phase with oscillations of O2 yield; no large oscillations of fluorescence paralleling those of luminescence or O2 were observed.3.3. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) abolished the 10- and 200-μsec components and the oscillatory behavior of the 35-μsec component.4.4. The 35- and 200-μsec components are not directly influenced by System I.The DCMU isolated 35-μsec component showed the following properties: 1.1. The decay is first order and the emission spectrum is essentially identical to that of chloroplast fluorescence;2.2. The yield saturates with a total emission of about 10-4 quanta/trap.3.3. The temperature dependence indicates an activation energy of about 250 mV for the yield and 200 mV for the decay.4.4. Maximal emission was obtained when Q, the acceptor of System II, was oxidized prior to the flash.The results are discussed in terms of possible mechanisms concerning the production and behavior of the luminescence.