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Oxygen-evolving photosystem II preparation from wild type and photosystem II mutants of Synechocystis sp. PCC 6803
Abstract
We present here a simple and rapid method which allows relatively large quantities of oxygen-evolving photosystem II- (PS-II-) enriched particles to be obtained from wild-type and mutants of the cyanobacterium Synechocystis 6803. This method is based on that of Burnap et al. [Burnap, R., Koike, H., Sotiropoulou, G., Sherman, L. A., & Inoue, Y. (1989) Photosynth. Res. 22, 123-130] but is modified so that the whole preparation, from cells to PS-II particles, is achieved in 10 h and involves only one purification step. The purified preparation exhibits a 5-6-fold increase of O2-evolution activity on a chlorophyll basis over the thylakoids. The ratio of PS-I to PS-II is about 0.14:1 in the preparation. The secondary quinone electron acceptor, QB, is present in this preparation as demonstrated by thermoluminescence studies. These PS-II particles are well-suited to spectroscopic studies as demonstrated by the range of EPR signals arising from components of PS-II that are easily detectable. Among the EPR signals presented are those from a formal S3-state, attributed to an oxidized amino acid interacting magnetically with the Mn complex in Ca(2+)-deficient PS-II particles, and from S2 modified by the replacement of Ca2+ by Sr2+. Neither of these signals has been previously reported in cyanobacteria. Their detection under these conditions indicates a similar lesion caused by Ca2+ depletion in both plants and cyanobacteria. The protocol has also been applied to mutants which have site-specific changes in PS-II. Data are presented on mutants having changes on the electron donor (Y160F) and electron acceptor (G215W) side of the D2 polypeptide.
... The cells were harvested, washed in washing buffer (see Materials and methods), and resuspended in cell disruption buffer. After this, a 60-min incubation at 4°C in a high sucrose buffer was performed, which increased lysis yield without any adverse effects on the oxygen evolution, similar to findings in other cyanobacterial preparations (Astier et al. 1986, Burnap and Trench 1989, Kirilovsky et al. 1992, Nilsson et al. 1992, Nyhus et al. 1992. After incubation, cells were pneumatically lysed by three passages through a Parr cell disruption bomb at 150 bar. ...
... The supernatant was discarded and the pellet resuspended in thylakoid washing buffer using gentle homogenization. This final ultracentrifugation step serves primarily to remove soluble proteins and phycobiliproteins (Kirilovsky et al. 1992, Nyhus et al. 1992. We tested pelleting the thylakoids using lower centrifugation force (48 000 g) but this lead to loss of light membrane fragments, and an overall lower yield of the final membrane preparation. ...
... This thylakoid preparation protocol was adapted from earlier methods developed for preparation of thylakoid membranes from Synechocystis 6803 (Astier et al. 1986, Burnap and Trench 1989, Kirilovsky et al. 1992 and Anabaena variabilis (Nyhus et al. 1992). In these protocols, a combination of osmotic shock and mechanical shearing was used to disrupt cells and extract the thylakoid membranes. ...
Nostoc punctiforme strain Pasteur Culture Collection (PCC) 73102, a sequenced filamentous cyanobacterium capable of nitrogen fixation, is used as a model organism for characterization of bioenergetic processes during nitrogen fixation in Nostoc. A protocol for isolating thylakoid membranes was developed to examine the biochemical and biophysical aspects of photosynthetic electron transfer. Thylakoids were isolated from filaments of N. punctiforme by pneumatic pressure-drop lysis. The activity of photosynthetic enzymes in the isolated thylakoids was analysed by measuring oxygen evolution activity, fluorescence spectroscopy and electron paramagnetic resonance spectroscopy. Electron transfer was found functional in both PSII and PSI. Electron transfer measurements in PSII, using diphenylcarbazide as electron donor and 2,6-dichlorophenolindophenol as electron acceptor, showed that 80% of the PSII centres were active in water oxidation in the final membrane preparation. Analysis of the membrane protein complexes was made by 2D gel electrophoresis, and identification of representative proteins was made by mass spectrometry. The ATP synthase, several oligomers of PSI, PSII and the NAD(P)H dehydrogenase (NDH)-1L and NDH-1M complexes, were all found in the gels. Some differences were noted compared with previous results from Synechocystis sp. PCC 6803. Two oligomers of PSII were found, monomeric and dimeric forms, but no CP43-less complexes. Both dimeric and monomeric forms of Cyt b(6)/f could be observed. In all, 28 different proteins were identified, of which 25 are transmembrane proteins or membrane associated ones.
... the triplet signal. This unidentified EPR signal is often detected in the His-tag PSII in the presence of a high amount of dithionite as is the case here [44] but was also detected in PSII from Synechocystis 6803 which had no His 6 -tag [45]. Fig. 6B and C show the external resonances with an expanded magnetic field scale. ...
... Although the triplet EPR signal is not expected to be very sensitive to the environment of the 3 Chl species, e.g. [20,[45][46][47][48], the spectral changes detected in Fig. 6 show that the modifications in the environment of the 3 Chl D1 in the T179H-PSII mutant became detectable with the total width of the signal smaller bỹ 10 gauss. This narrowing could reflect a change from water to His as the Chl D1 ligand in the D1/T179H mutant. ...
The monomeric chlorophyll, ChlD1, which is located between the PD1PD2 chlorophyll pair and the pheophytin, PheoD1 is the longest wavelength chlorophyll in the heart of Photosystem II and is thought to be the primary electron donor. Its central Mg²⁺ is liganded to a water molecule that is H-bonded to D1/T179. Here, two site-directed mutants, D1/T179H and D1/T179V, were made in the thermophilic cyanobacterium, Thermosynechococcus elongatus, and characterized by a range of biophysical techniques. The Mn4CaO5 cluster in the water-splitting site is fully active in both mutants. Changes in thermoluminescence indicate that i) radiative recombination occurs via the repopulation of *ChlD1 itself; ii) non-radiative charge recombination reactions appeared to be faster in the T179H-PSII; and iii) the properties of PD1PD2 were unaffected by this mutation, and consequently iv) the immediate precursor state of the radiative excited state is the ChlD1⁺PheoD1⁻ radical pair. Chlorophyll bleaching due to high intensity illumination correlated with the amount of ¹O2 generated. Comparison of the bleaching spectra with the electrochromic shifts attributed to ChlD1 upon QA⁻ formation, indicates that in the T179H-PSII and in the WT*3-PSII, the ChlD1 itself is the chlorophyll that is first damaged by ¹O2, whereas in the T179V-PSII a more red chlorophyll is damaged, the identity of which is discussed. Thus, ChlD1 appears to be one of the primary damage site in recombination-mediated photoinhibition. Finally, changes in the absorption of ChlD1 very likely contribute to the well-known electrochromic shifts observed at ~430 nm during the S-state cycle.
... However, detailed functional and biochemical characterization of PSll mutants generally is complex because the PSlllPSl reaction center ratio is unfavorable in this cyanobacterium (Fujita and Murakami, 1988). Even though severa1 useful PSll preparation procedures are available for wild-type Synechocystis 6803 (Burnap et al., 1989; Noren et al., 1991; Kirilovsky et al., 1992; Nilsson et al., 1992 ), preparation of oxygen-evolving PSll particles from a number of mutants has been unsuccessful, possibly due to a destabilized oxygenevolving complex in these mutants. Apart from PSI, the presente of phycobilisome components may also complicate the To whom correspondence should be addressed. ...
To design an in vivo system allowing detailed analysis of photosystem II (PSII) complexes without significant interference from other pigment complexes, part of the psaAB operon coding for the core proteins of photosystem I (PSI) and part of the apcE gene coding for the anchor protein linking the phycobilisome to the thylakoid membrane were deleted from the genome of the cyanobacterium Synechocystis sp strain PCC 6803. Upon transformation and segregation at low light intensity (5 microE m-2 sec-1), a PSI deletion strain was obtained that is light tolerant and grows reasonably well under photoheterotrophic conditions at 5 microE m-2 sec-1 (doubling time approximately 28 hr). Subsequent inactivation of apcE by an erythromycin resistance marker led to reduction of the phycobilin-to-chlorophyll ratio and to a further decrease in light sensitivity. The resulting PSI-less/apcE- strain grew photoheterotrophically at normal light intensity (50 microE m-2 sec-1) with a doubling time of 18 hr. Deletion of apcE in the wild type resulted in slow photoautotrophic growth. The remaining phycobilins in apcE- strains were inactive in transferring light energy to PSII. Cells of both the PSI-less and PSI-less/apcE- strains had an approximately sixfold enrichment of PSII on a chlorophyll basis and were as active in oxygen evolution (on a per PSII basis) as the wild type at saturating light intensity. Both PSI-less strains described here are highly appropriate both for detailed PSII studies and as background strains to analyze site- and region-directed PSII mutants in vivo.
... A width of <90 G is observed in PS II preparations from Synechocystis 6803 which have been washed with a Ca-free medium containing EDTA. 12 Similar signals have also been observed in a number of differently inhibited preparations. The exact width of the signal differs depending on the type of treatment used; the largest widths (~230 G) are found in acetate-treated PS II, 13 while smaller splittings (<160 G) are found in F − -treated, 14 NH 3 -treated, 15,16 and Cl − -depleted samples. ...
The structural consequences of calcium depletion of Photosystem II (PS II) by treatment at pH 3.0 in the presence of citrate has been determined by Mn K-edge X-ray absorption spectroscopy. X-ray absorption edge spectroscopy of Ca-depleted samples in the S1‘, S2‘, and S3‘ oxidation states reveals that there is Mn oxidation on the S1‘−S2‘ transition, although no evidence of Mn oxidation was found for the S2‘−S3‘ transition. This result is in keeping with the results from EPR studies where it has been found that the species oxidized to give the S3‘ broad radical signal found in Ca-depleted PS II is tyrosine Yz. The S2‘ state can be prepared by two methods: illumination followed by dark adaptation and illumination in the presence of DCMU to limit to one turnover. Illumination followed by dark adaptation was found to yield a lower Mn K-edge inflection-point energy than illumination with DCMU, indicating vulnerability to reduction of the Mn complex, even over the relatively short times used for dark adaptation (15 min). EXAFS measurements of Ca-depleted samples in the three modified S states (referred to here as S‘ states) reveals that the Fourier peak due to scatterers at 3.3 Å from Mn is strongly diminished, consistent with our previous assignment of a Ca-scattering contribution at this distance. Even after Ca depletion, there is still significant amplitude in the third peak, further supporting our conclusions from earlier studies that the third peak in native samples is comprised of both Mn and Ca scattering. The Mn−Mn contributions making up the second Fourier peak at 2.7 Å are largely undisturbed by Ca-depletion, but there is some evidence that S1‘-state samples contain significant amounts of reduced Mn(II), which is then photooxidized in the preparation of higher S‘ states.
... Other authors have invoked structural changes [27,46]. The here observed suppression, say at pH 7.5 is, however, neither attributable to Mn-depletion, which occurs only at pH around 9-10 [47][48][49][50][51], nor to the release of the extrinsic proteins occurring at pH values greater than 8 [52][53][54] because these effects are irreversible, whereas the here observed inhibition was reversible. ...
In cyanobacteria, algae and plants Photosystem II produces the oxygen we breathe. Driven and clocked by light quanta, the catalytic Mn(4)Ca-tyrosine centre accumulates four oxidising equivalents before it abstracts four electrons from water, liberating dioxygen and protons. Aiming at intermediates of the terminal four-electron cascade, we previously have suppressed this reaction by elevating the oxygen pressure, thereby stabilising one redox intermediate. Here, we established a similar suppression by increasing the proton concentration. Data were analysed in terms of only one (peroxy) redox intermediate between the fourfold oxidised Mn(4)Ca-tyrosine centre and oxygen release. The surprising result was that the release into the bulk of one proton per dioxygen is linked to the first and rate-limiting electron transfer in the cascade rather than to the second which produces free oxygen. The penultimate intermediate might thus be conceived as a fully deprotonated peroxy-moiety.
... The S 2 -state multiline signal from the CaMn 4 O x -cluster in PSII is centered around g = 2 and exhibits at least 18 well resolved lines spaced by 80-90 G, spread over roughly 1800 G (Fig. 5, spectra a and b, Table 2) [87][88][89]. Our multiline signal obtained from Arabidopsis is similar to multiline signals reported from spinach [87,90,91], green algae [44] and cyanobacterial preparations [60,92,93]. In addition to the multiline signal, illumination at 200 K also produced the g = 4.1 signal (Fig. 5, spectrum b). ...
Arabidopsis thaliana is widely used as a model organism in plant biology as its genome has been sequenced and transformation is known to be efficient. A large number of mutant lines and genomic resources are available for Arabidopsis. All this makes Arabidopsis a useful tool for studies of photosynthetic reactions in higher plants. In this study, photosystem II (PSII) enriched membranes were successfully isolated from thylakoids of Arabidopsis plants and for the first time the electron transfer cofactors in PSII were systematically studied using electron paramagnetic resonance (EPR) spectroscopy. EPR signals from both of the donor and acceptor sides of PSII, as well as from auxiliary electron donors were recorded. From the acceptor side of PSII, EPR signals from Q(A)- Fe²(+) and Phe- Q(A)- Fe²(+) as well as from the free Phe- radical were observed. The multiline EPR signals from the S₀- and S₂-states of CaMn₄O(x)-cluster in the water oxidation complex were characterized. Moreover, split EPR signals, the interaction signals from Y(Z) and CaMn₄O(x)-cluster in the S₀-, S₁-, S₂-, and the S₃-state were induced by illumination of the PSII membranes at 5K and characterized. In addition, EPR signals from auxiliary donors Y(D), Chl(+) and cytochrome b₅₅₉ were observed. In total, we were able to detect about 20 different EPR signals covering all electron transfer components in PSII. Use of this spectroscopic platform opens a possibility to study PSII reactions in the library of mutants available in Arabidopsis.
... This might be due to different stability of structure of OEC and interaction among helices because of different species and amount of lipids in PSII complexes between the mesophilic and the thermophilic cyanobacteria. Actually, isolated PSII complexes from T. elongatus contain at least 2 or 3 functional plastoquinone Q B as probed by thermoluminescence measurements, for example [17,26,29,[33][34][35], whereas PSII isolated from mesophilic cells such as Synechocystis PCC 6803 and Chlamydomonas showed no or a decreased thermoluminescence band derived from the recombination of S 2 and Q B without addition of DCMU [36,37]. ...
A deletion mutant that lacks the Psb30 protein, one of the small subunits of Photosystem II, was constructed in a Thermosynechococcus elongatus strain in which the D1 protein is expressed from the psbA(3) gene (WT*). The DeltaPsb30 mutant appears more susceptible to photodamage, has a cytochrome b(559) that is converted into the low potential form, and probably also lacks the PsbY subunit. In the presence of an inhibitor of protein synthesis, the Psb30 lost more rapidly the water oxidation function than the WT* under the high light conditions. These results suggest that Psb30 contributes to structurally and functionally stabilise the Photosystem II complex in preventing the conversion of cytochrome b(559) into the low potential form. Structural reasons for such effects are discussed.
... Despite being designed to study specifically the features of TyrZ, the TyrD-less mutant provides an excellent opportunity to investigate the puzzling functions of TyrD and the possible influence on the early electron transfer steps. The point mutation was introduced in the D2 protein of Chlamydomonas [18], Synechocystis [20], and finally T. elongatus [21]. In view of the fact that the structural data are available for the cyanobacterial system, it is very suitable to study the latter system. ...
Redox-active tyrosine (Tyr) D is indirectly involved in controlling the primary electron transfer in PSII. The presence of the oxidized TyrD renders P680+ more oxidizing by localizing the charge more on PD1 and thus facilitates trapping of the excitation energy in PSII. We also conclude that the mechanism of the primary charge separation and stabilization is altered upon QA reduction.
... However, detailed functional and biochemical characterization of PSll mutants generally is complex because the PSlllPSl reaction center ratio is unfavorable in this cyanobacterium (Fujita and Murakami, 1988). Even though severa1 useful PSll preparation procedures are available for wild-type Synechocystis 6803 (Burnap et al., 1989;Noren et al., 1991;Kirilovsky et al., 1992;Nilsson et al., 1992), preparation of oxygen-evolving PSll particles from a number of mutants has been unsuccessful, possibly due to a destabilized oxygenevolving complex in these mutants. Apart from PSI, the presente of phycobilisome components may also complicate the To whom correspondence should be addressed. ...
To design an in vivo system allowing detailed analysis of photosystem II (PSII) complexes without significant interference from other pigment complexes, part of the psaAB operon coding for the core proteins of photosystem I (PSI) and part of the apcE gene coding for the anchor protein linking the phycobilisome to the thylakoid membrane were deleted from the genome of the cyanobacterium Synechocystis sp strain PCC 6803. Upon transformation and segregation at low light intensity (5 microE m-2 sec-1), a PSI deletion strain was obtained that is light tolerant and grows reasonably well under photoheterotrophic conditions at 5 microE m-2 sec-1 (doubling time approximately 28 hr). Subsequent inactivation of apcE by an erythromycin resistance marker led to reduction of the phycobilin-to-chlorophyll ratio and to a further decrease in light sensitivity. The resulting PSI-less/apcE- strain grew photoheterotrophically at normal light intensity (50 microE m-2 sec-1) with a doubling time of 18 hr. Deletion of apcE in the wild type resulted in slow photoautotrophic growth. The remaining phycobilins in apcE- strains were inactive in transferring light energy to PSII. Cells of both the PSI-less and PSI-less/apcE- strains had an approximately sixfold enrichment of PSII on a chlorophyll basis and were as active in oxygen evolution (on a per PSII basis) as the wild type at saturating light intensity. Both PSI-less strains described here are highly appropriate both for detailed PSII studies and as background strains to analyze site- and region-directed PSII mutants in vivo.
... PCC 6803 has been widely used because it is naturally transformable and it can grow heterotrophically at the expense of glucose (Williams 1988, Vermaas 1993, Pakrasi 1995. Although there are several reports describing the isolation of an active O 2evolving PSII complex from this organism (Burnap et al. 1989, Kirilovsky et al. 1992, Tang and Diner 1994, the isolation of the active PSII complex from the PSII mutants is not yet fully established. Instead, mutant cells were examined to monitor the effects of directed mutations on the O 2 -evolving properties (Debus 1992). ...
PSII-X is a small hydrophobic protein, which is universally present in photosystem II (PSII) core complex among cyanobacteria and plants. The role of PSII-X was studied by directed mutagenesis and biochemical analysis in the thermophilic cyanobacterium Synechococcus elongatus. The psbX-disrupted mutant could grow photoautotrophically indicative of non-essential function, while it showed growth defect under low CO(2) conditions. An active O(2)-evolving PSII complex was successfully isolated from the mutant and wild type. Protein composition of the isolated PSII complex was the same as wild type except for the absence of PSII-X. O(2) evolution supported by artificial quinones was affected in the psbX-disrupted mutant. At high concentration of 2,6-dichlorobenzoquinone or 2,6-dimethylbenzoquinone, the mutant showed much lower activity than wild type, while not much difference was found at low concentration. These results imply that binding or turnover of quinones at the Q(B) site depends, at least in part, on PSII-X protein in the PSII complex. Gel filtration chromatography of the PSII complex revealed that the dimeric structure of the complex was not greatly affected in the psbX-disrupted mutant.
... In the second, cosolvent/water mixtures have been used to study PSII kinetics. Sucrose and/or glycerol have been used in many PSII purification procedures as cryoprotectants and to improve PSII stability (for examples, see Berthold et al., 1981;Bricker et al., 1998;Kirilovsky et al., 1992;MacDonald and Barry, 1992;Noren et al., 1991). For example, glycerol has been shown to stabilize oxygen evolution and maintain binding of a 9-kDa PSII subunit in the cyanobacterium, Phormidium laminosum (Stewart et al., 1985). ...
Photosystem II catalyzes the oxidation of water and the reduction of plastoquinone. The active site cycles among five oxidation states, which are called the S(n) states. PSII purification procedures include the use of the cosolvents, sucrose and/or glycerol, to stabilize water splitting activity and for cryoprotection. In this study, the effects of sucrose and glycerol on PSII were investigated. Sucrose addition was observed to stimulate the steady-state rate of oxygen evolution in the range from 0 to 1.35 M. Glycerol addition was observed to stimulate oxygen evolution in the range from 0 to 30%. Both cosolvents were observed to be inhibitory at higher concentrations. Sucrose addition was shown to have no effect on the rate of Q(A)(-) oxidation or on the K(M) for exogenous acceptor. PSII was then treated to remove extrinsic proteins. In these samples, sucrose addition stimulated activity, but glycerol addition was inhibitory at concentrations higher than approximately 0.5 M. This inhibitory effect of glycerol at relatively low concentrations is attributed to glycerol binding to the active site, when extrinsic subunits are not present. Reaction induced FTIR spectra, associated with the S(1) to S(2) transition of the water-oxidizing complex, exhibited significant differences throughout the 1,800-1,200 cm(-1) region, when glycerol- and sucrose-containing samples were compared. These measurements suggest a cosolvent-induced shift in the pK(A) of an aspartic or glutamic acid side chain, as well as structural changes at the active site. These structural alterations are attributed to a change in preferential hydration of the oxygen-evolving complex.
... This signal is spread over roughly 1800 G and is made up of at least 18 lines, each separated by approximately 80 G, and arises from a magnetic tetramer910111213. The S 2 -state can be quantitatively formed either by flash illumination in non-frozen samples [8] or by continuous illumination at 200 K. Similar S 2 -multiline signals have been detected in PSII isolated from plants [8,14151617181920, from the cyanobacteria, Thermosynechococcus elongatus212223 and Synechocystis PCC 6803242526 and from the green alga, Scenedesmus obliquus [27,28]. Under some experimental conditions (in the presence of sucrose in the buffer) the S 2 -state gives rise to a g=4.1 signal that is stable at temperatures above 200 K, rather than a multiline signal [29,30]. ...
The X-band EPR spectra of the IR sensitive untreated PSII and of MeOH- and NH(3)-treated PSII from spinach in the S(2)-state are simulated with collinear and rhombic g- and Mn-hyperfine tensors. The obtained principal values indicate a 1Mn(III)3Mn(IV) composition for the Mn(4) cluster. The four isotropic components of the Mn-hyperfine tensors are found in good agreement with the previously published values determined from EPR and (55)Mn-ENDOR data. Assuming intrinsic isotropic components of the Mn-hyperfine interactions identical to those of the Mn-catalase, spin density values are calculated. A Y-shape 4J-coupling scheme is explored to reproduce the spin densities for the untreated PSII. All the required criteria such as a S=1/2 ground state with a low lying excited spin state (30 cm(-1)) and an easy conversion to a S=5/2 system responsible for the g=4.1 EPR signal are shown to be satisfied with four antiferromagnetic interactions lying between -290 and -130 cm(-1).
... To date, however, the g = 4.1 form of the S 2 state of the Mn 4 cluster has not been characterized in cyanobacterial PS II (McDermott et al. 1988;Haddy et al. 1992;Kirilovsky et al. 1992;Boussac et al. 1998a), although EPR studies of cyanobacterial PS II have revealed formation of the g = 2 multiline and the high-spin g = 4.1, 5.5 and 8.5 EPR signals similar to those observed in spinach PS II (Campbell et al. 1998a;Boussac et al. 1998a. Cyanobacterial PS II lacks the 23 kDa and 17 kDa extrinsic polypeptides that are present in spinach PS II (Boussac et al. 1998a). ...
The Mn4 cluster of PS II advances through a series of oxidation states (S states) that catalyze the breakdown of water to dioxygen in the oxygen-evolving complex. The present study describes the engineering and purification of highly active PS II complexes from mesophilic His-tagged Synechocystis PCC 6803 and purification of PS II core complexes from thermophilic wild-type Synechococcus lividus with high levels of the extrinsic polypeptide, cytochrome c
550. The g = 4.1 S2 state EPR signal, previously not characterized in untreated cyanobacterial PS II, is detected in high yields in these PS II preparations. We present a complete characterization of the g = 4.1 state in cyanobacterial His-tagged Synechocystis PCC 6803 PS II and S. lividus PS II. Also presented are a determination of the stoichiometry of cytochrome c
550 bound to His-tagged Synechocystis PCC 6803 PS II and analytical ultracentrifugation results which indicate that cytochrome c
550 is a monomer in solution. The temperature-dependent multiline to g = 4.1 EPR signal conversion observed for the S2 state in cyanobacterial PS II with high cytochrome c
550 content is very similar to that previously found for spinach PS II. In spinach PS II, the formation of the S2 state g = 4.1 EPR signal has been found to correlate with the binding of the extrinsic 17 and 23 kDa polypeptides. The finding of a similar correlation in cyanobacterial PS II with the binding of cytochrome c
550 suggests a functional homology between cytochrome c
550 and the 17 and 23 kDa extrinsic proteins of spinach PS II.
Photosynthetic oxygen evolution is powered by Photosystem II (PSII) through the oxidized P680⁺ and catalyzed by the oxygen-evolving complex (OEC=Mn4X-entity) and a tyrosine residue (YZ). We investigated oxygen-evolving and Mn-depleted core preparations from the wildtype of Synechocystis sp. PCC 6803 and a point-mutant D1-D61N. Studies with whole cells have revealed the following properties of D61N (1): oxygen release under continuous illumination is decreased to 17% of WT, PSII contains photooxidizable Mn, at least one S-state transition is slowed drastically, the rate of reduction of P680⁺ in hydroxylamine-treated cells is the same as in WT, and the midpoint potential of S2/S1 is slightly increased. Our previous studies with cells and oxygen-evolving core particles of D61N (2) have revealed that in comparison to WT, the transitions S1⇒S2 and S2⇒S3 were slowed about twofold and the flash-induced oxygen-liberation about tenfold. By measurements of the flash-induced absorption transients at 360nm we now present evidence that this drastic retardation of oxygen release (t1/2.≈13ms) was paralleled by a tenfold increased lifetime of YZOX during S3⇒S0, because no fast oxidation of the OEC by YZOX was observed.
The cyanobacterium Synechocystis PCC 6803 is especially useful in site-directed mutagenesis studies of photosystem II (PSII). Unfortunately, methods which rely on ion-exchange chromatography for recovery of the mutant PSII (1–4) are lengthy and can even be inadequate for the generation of material in sufficient yield and purity for biophysical study when the level of PSII expression in the mutant is low. Here we present the use of an engineered hexahistidine tag fused to the carboxy-terminus of the CP47 subunit for the rapid purification of PSII core complexes from Synechocystis PCC 6803 by Ni²⁺-affinity chromatography. A recent paper also reported purification of PSII from Chlamydomonas using a His-tagged D2 subunit (5).
Old and very recent experiments on the extent and the rate of proton release during the four reaction steps of photosynthetic water oxidation are reviewed. Proton release is discussed in terms of three main sources, namely the chemical production upon electron abstraction from water, protolytic reactions of Mn-ligands (e.g. oxo-bridges), and electrostatic response of neighboring amino acids. The extent of proton release differs between the four oxidation steps and greatly varies as a function of pH both, but differently, in thylakoids and PS II-membranes. Contrastingly, it is about constant in PS II-core particles. In any preparation, and on most if not all reaction steps, a large portion of proton transfer can occur very rapidly (<20 μs) and before the oxidation of the Mn-cluster by Yz (+) is completed. By these electrostatically driven reactions the catalytic center accumulates bases. An additional slow phase is observed during the oxygen evolving step, S3⇒S4→S0. Depending on pH, this phase consists of a release or an uptake of protons which accounts for the balance between the number of preformed bases and the four chemically produced protons. These data are compatible with the hypothesis of concerted electron/proton-transfer to overcome the kinetic and energetic constraints of water oxidation.
Photosystem II (PS II) is the site of oxygen evolution. Activation of dark adapted samples by a train of saturating flashes produces oxygen with a yield per flash which oscillates with a periodicity of four. Damping of the oxygen oscillations is accounted for by misses and double hits. The mechanisms hidden behind these parameters are not yet fully understood. The components which participate in charge transfer and storage in PS II are believed to be anchored to the heterodimer formed by the D1 and D2 proteins. The secondary plastoquinone acceptor QB binds on D1 in a loop connecting the fourth and fifth helices (the QB pocket). Several D1 mutants, mutated in the QB binding region, have been studied over the past ten years.In the present report, our results on nine D1 mutants of Synechocystis PCC 6714 and 6803 are analyzed. When oxygen evolution is modified, it can be due to a change in the electron transfer kinetics at the level of the acceptor side of PS II and also in some specific mutants to a long ranging effect on the donor side of PS II. The different properties of the mutants enable us to propose a classification in three categories. Our results can fit in a model in which misses are substantially determined by the fraction of centers which have QA (-) before each flash due to the reversibility of the electron transfer reactions. This idea is not new but was more thoroughly studied in a recent paper by Shinkarev and Wraight (1993). However, we will show in the discussion that some doubts remain as to the true origin of misses and double hits.
Cytochrome b559 (Cyt b559) is an intrinsic and essential component of the photosystem II (PSII) reaction center, but its physiological function remains yet undefined. In this study, a partial redox characterization of Cyt b559 in photosynthetic membranes from the transformable unicellular cyanobacterium Synecborystis sp. PCC 6803 (hereafter called Synecborystis 6803) has been performed. In thylakoid membranes extracted by a very mild cell-breaking procedure, as developed in this work, only one redox form of Cyt b559 was found, with a redox potential proper to a low-potential (LP) form. The midpoint redox potential value of this LP form has been shown to be + 170 mV at pH 7.5 in PSII-enriched membranes. In spite of a great variety of cell-breaking treatments used to prepare thylakoid membranes, it has not been possible to detect any high-potential (HP) form of Cyt b559. The absence of this HP form is discussed in terms of its higher lability in Synecborystis 6803 as compared with other cyanobacteria and higher plants. Alternatively, the possibility is considered that a HP form of Cyt b559 does not occur in Synecborystis 6803 because it can not be properly stabilized in the membrane environment.
The Mn-4-cluster of photosystem II (PSII) from Synechococcus elongatus was studied by electron paramagnetic resonance (EPR) spectroscopy after a series of saturating laser flashes given in the presence of either methanol or ethanol. Results were compared to those obtained in similar experiments done on PSII isolated from plants. The flash-dependent changes in amplitude of the EPR multiline signals were virtually identical in all samples. In agreement with earlier work [Messinger, J., Nugent, J, H. A., and Evans, M. C. W. (1997) Biochemistry, 36, 11055-11060; Ahrling, K. A., Peterson, S., and Styring, S, (1997) Biochemistry 36, 13148-13152], detection of an EPR multiline signal from the So state in PSII from plants was only possible with methanol present. In PSII from S. elongatus, it is shown that the S-0 state exhibits an EPR multiline signal in the absence of methanol (however, ethanol was present as a solvent for the artificial electron acceptor). The hyperfine lines are better resolved when methanol is present. The S-0 multiline signals detected in plant PSII and in S, elongatus were similar but not identical. Unlike the situation seen in plant PSII, the S-2 state in S. elongatus is not affected by the addition of methanol in that (i) the S-2 multiline EPR signal is not modified by methanol and (ii) the spin state of the S-2 state is affected by infrared light when methanol is present. It is also shown that the magnetic relaxation properties of an oxidized low-spin heme, attributed to cytochrome c(550), vary with the S states. This heme then is in the magnetic environment of the Mn-4 cluster.
We report the high-frequency (139.5 GHz) electron paramagnetic resonance (EPR) spectrum of the tyrosyl radical of photosystem II. A rhombic powder pattern with principal g values g1 = 2.007 82, g2 = 2.004 50, and g3 = 2.002 32 is observed. The well-defined turning points and the value of the largest principal g value are indicative of ordered hydrogen bonding to the tyrosyl phenyl oxygen. Hyperfine structure is resolved on all three turning points. Proton hyperfine couplings obtained from the simulation of the 139.5 GHz EPR spectrum are in good agreement with X-band electron spin echo−electron nuclear double resonance studies. The high-frequency EPR spectrum was acquired under conditions of saturation in which the dispersion signal is detected. Proper replication of the high-frequency EPR spectral features is only achieved in simulations which account for the line shapes characteristic of saturated dispersion signals. Comparison of the spectrum with spectra of non-hydrogen bonded tyrosyl radicals indicates that the largest principal g value (g1), oriented along the C−O bond, is sensitive to hydrogen bonding at the phenyl oxygen. Density functional calculations indicate that the decreased downfield shift in g1 from the free electron g value with increasing hydrogen bond strength arises from both a decreased spin density on the phenyl oxygen5−30% over a range of reasonable hydrogen bond distances (2.0−1.1 Å)and an increased splitting between ground state and excited state singly occupied molecular orbitals.
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.
Oxygenic photosynthesis occurs in plants, green algae, and procaryotic cyanobacteria.Two chlorophyll-containing photosystems
cooperate to transfer electrons from water to NADP+. Photosystem II is the membrane protein complex that carries out the light-catalyzed oxidation of water and reduction of
plastoquinone. The reaction center is composed ofboth intrinsic and extrinsic proteins; the prosthetic groups involved in
electron transfer include chlorophyll, pheophytin, quinone, tyrosine residues, and a manganese cluster.Cyanobacteria have
emerged as a convenient system with which to study the structure and function of Photosystem II for two reasons. Firstly,
isotopic labeling experiments are possible in this organism, facilitating many types of biophysical experiments. Secondly,
site-directed mutagenesis is easily performed. This chapter will review what is known about the structure and function of
Photosystem II with particular emphasis on the use of cyanobacteria in such studies. Areas in which there are significant
differences between plants and cyanobacteria will be highlighted.
A novel purification procedure was developed for the isolation of oxygen evolving photosystem 2 (PS2) from Mastigocladus laminosus. The isolation procedure involves dodecyl maltoside extraction followed by column chromatography using anion exchange resins. The isolated PS2 reaction center (RC) was analyzed for its biochemical and biophysical characteristics. Analysis by SDS polyacrylamide gel electrophoresis revealed that the complex contained five intrinsic membrane proteins (CP 47, CP 43, D1, D2, and cyt b
559) and at least three low molecular mass proteins. The complex exhibited high rates of oxygen evolution [333 mmol(O2) kg–1(Chl) s–1] in the presence of 2.5 mM 2,6-dimethylbenzoquinone (DMBQ) as an artificial electron acceptor. The red chlorophyll a absorption peak of this complex was observed at 673.50.2 nm. The isolated PS2 core complex was free of photosystem 1 as inferred from its SDS-PAGE and fluorescence spectrum. The electron transfer properties of the Mastigocladus cells and the purified PS2 core complex were further probed by measuring thermoluminescence signals, which indicated the presence of a primary quinone electron acceptor (QA) in the purified PS2 core complex.
Manganese K-edge X-ray spectra have been obtained for Photosystem II samples treated to inhibit oxygen evolution without displacement of manganese. Inhibition treatments included the use of ammonia, acetate and high concentrations of sodium chloride. These treatments affect the binding of calcium and chloride cofactors and result in either a block or slowing of OEC S-state cycling at the S3 → S0 transition. Following each inhibition treatment the S-states show characteristic K-edge energies and EPR spectra. The differences between each type of inhibitory treatment in the K-edge energy for each S-state may result from conformational and/or ligand changes to the manganese complex. However for each of the inhibitory treatments, the K-edge energies showed edge shifts between S-states consistent with manganese oxidation on both the S1 → S2 and S2 → S3 transitions.
Spirulina platensis is a cyanobacterium which usually lives under high-light conditions. Nonetheless, it is thought to contain the most red-shifted antenna pigment of all known Chl a-containing phototrophic organisms, as shown by its 77 K fluorescence peaking at 760 nm. To exclude preparation artifacts and to exclude the possibility that long wavelength-absorbing pigments form only when the temperature is lowered to 77 K, we carried out experiments with whole cells at room temperature. The combined analysis of stationary absorption and fluorescence spectra as well as fluorescence induction and time-resolved fluorescence decays shows that the pigment responsible for the 77 K fluorescence at 760 nm (i) has the oscillator strength of approximately one Chl a molecule, (ii) absorbs maximally at 738 nm (), (iii) is present only in the antenna system of PS I, (iv) participates in light collection, and (v) does not entail a low photochemical quantum yield. Other, more abundant but less red-shifted Chl a antenna pigments lead to a significantly larger absorption cross section of the photosynthetic unit of PS I above 700 nm compared to units that would not possess these long wavelength-absorbing pigments. These results support the hypothesis that the physiological role of long wavelength-absorbing pigments is to increase the absorption cross section at wavelengths of >700 nm when in densely populated mats the spectrally filtered light is relatively more intense at these wavelengths [Trissl, H.-W. (1993) Photosynth. Res. 35, 247-263].
Genome sequence of Arabidopsis thaliana (Arabidopsis) revealed two psbO genes (At5g66570 and At3g50820) which encode two distinct PsbO isoforms: PsbO1 and PsbO2, respectively. To get insights into the function of the PsbO1 and PsbO2 isoforms in Arabidopsis we have performed systematic and comprehensive investigations of the whole photosynthetic electron transfer chain in the T-DNA insertion mutant lines, psbo1 and psbo2. The absence of the PsbO1 isoform and presence of only the PsbO2 isoform in the psbo1 mutant results in (i) malfunction of both the donor and acceptor sides of Photosystem (PS) II and (ii) high sensitivity of PSII centers to photodamage, thus implying the importance of the PsbO1 isoform for proper structure and function of PSII. The presence of only the PsbO2 isoform in the PSII centers has consequences not only to the function of PSII but also to the PSI/PSII ratio in thylakoids. These results in modification of the whole electron transfer chain with higher rate of cyclic electron transfer around PSI, faster induction of NPQ and a larger size of the PQ-pool compared to WT, being in line with apparently increased chlororespiration in the psbo1 mutant plants. The presence of only the PsbO1 isoform in the psbo2 mutant did not induce any significant differences in the performance of PSII under standard growth conditions as compared to WT. Nevertheless, under high light illumination, it seems that the presence of also the PsbO2 isoform becomes favourable for efficient repair of the PSII complex.
A chlorophyll-protein complex has been isolated from the cyanobacterium Synechocystis sp. PCC 6803 that closely resembles higher plant photosystem II reaction centers in spectral properties. The Synechocystis complex has a pigment content of 5-7 chlorophyll a molecules:1 Cyt b559:2 pheophytins; an optical absorption redmost transition at approximately 675 nm; and a nonconservative circular dichroism red signal, with extrema at 682 (+) and 652 (-) nm. Upon illumination, the Synechocystis D1/D2/Cyt b559 complex accumulates reduced pheophytin. LDS-PAGE and/or immunoblotting showed the D1, D2, and Cyt b559 proteins, aggregated and degraded forms of D1 and possibly D2, and traces of ATP synthase and the CP47 photosystem II chlorophyll protein. The availability of such a Synechocystis preparation opens the way for employing site-directed mutagenesis in studying primary reactions of oxygenic photosynthesis.
Oxygen-evolving photosystem II complexes were isolated from the green alga Chlamydomonas reinhardtii by selective solubilization of thylakoid membranes with dodecyl maltoside followed by density gradient centrifugation and anion-exchange chromatography. In the presence of CaCl2 and K3[Fe(CN)6] the complexes evolved oxygen at rates exceeding 1000 mumol (mg of chl)-1 h-1. The particles contained 40 chlorophylls a and had properties very similar to those of PSII isolated from higher plants. Chlamydomonas reinhardtii is now the first organism which can be used for both site-directed mutagenesis and detailed biochemical and biophysical characterization of oxygen-evolving photosystem II. It seems therefore to be an ideal model organism for investigation of structure-function relationships in photosynthetic oxygen evolution.
Several site-directed photosystem II mutants with substitutions at Asp-170 of the D1 polypeptide were characterized by noninvasive methods in vivo. In several mutants, including some that evolve oxygen, a significant fraction of photosystem II reaction centers are shown to lack photooxidizable Mn ions. In this fraction of reaction centers, either the high-affinity site from which Mn ions rapidly reduce the oxidized secondary electron donor, YZ+, is devoid of Mn ions or the Mn ion(s) bound at this site are unable to reduce YZ+. It is concluded that the Mn clusters in these mutants are unstable or are assembled inefficiently in vivo. Mutants were constructed in the unicellular cyanobacterium Synechocystis sp. PCC 6803. The in vivo characterization procedures employed in this study involved measuring changes in the yield of variable chlorophyll a fluorescence following a saturating flash or brief illumination given in the presence of the electron transfer inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, or following each of a series of saturating flashes given in the absence of this inhibitor. These procedures are easily applied to mutants that evolve little or no oxygen, facilitate the characterization of mutants with labile oxygen-evolving complexes, permit photosystem II isolation efforts to be concentrated on mutants having the stablest Mn clusters, and guide systematic spectroscopic studies of isolated photosystem II particles to mutants of particular interest.
The reaction center of photosystem II of oxygenic photosynthesis contains two redox-active tyrosines called Z and D, each of which can act as an electron donor to the oxidized primary electron donor, P680+. These tyrosines are located in homologous positions on the third transmembrane alpha-helix of each of the two homologous polypeptides, D1 and D2, that comprise the reaction center. Tyrosine D of polypeptide D2 has been proposed, upon oxidation, to give up its phenolic proton to a nearby basic amino acid residue, forming a neutral radical. Modeling studies have pointed to His190 (spinach numbering) as a likely candidate for this basic residue. As a test of this hypothesis, we have constructed three site-directed mutations in the D2 polypeptide of the cyanobacterium Synechocystis sp. PCC6803. His189 (the Synechocystis homologue of His190 of spinach) has been replaced by glutamine, aspartate, or leucine. Instead of the normal D. EPR signal (g = 2.0046; line width 16-19 G), PSII core complexes isolated from these three mutants show an altered dark-stable EPR signal with a narrowed line width (11-13 G), and g values of 2.0046, 2.0043, and 2.0042 for the His189Gln, His189Asp, and His189Leu mutants, respectively. Despite the reduced line width, these EPR signals show g values and microwave-power saturation properties similar to the normal D. signal. Furthermore, specific deuteration in one of those mutants at the 3 and 5 positions of the phenol ring of the photosystem II reaction center tyrosines results in a loss of hyperfine structure of the EPR signal, proving that the signal indeed arises from tyrosine.2+ This observation provides support for a model in which an imidazole nitrogen of His189 accepts the phenolic proton of Tyr160 upon oxidation of D, forming a back hydrogen bond to the phenolic oxygen of the neutral tyrosyl radical.
The oxidizing side of photosystem II contains two redox-active tyrosyl side chains, TyrZ and TyrD, and a cluster of Mn atoms involved in water oxidation. The structural environment of these components is unknown, and with computer-assisted modeling we have created a three-dimensional model for the structures around TyrZ and TyrD [Svensson et al. (1990) EMBO J. 9, 2051-2059]. Both tyrosines are proposed to form hydrogen bonds to nearby histidine residues (for Synechocystis 6803, these are His190 on the D1 and His189 on the D2 proteins). We have tested this proposal by electron paramagnetic resonance (EPR) spectroscopy of TyrDox in mutants of the cyanobacterium Synechocystis 6803 carrying site-directed mutations in the D2 protein. In two mutants, where His189 of the D2 protein is changed to either Tyr or Leu, the normal EPR spectrum from TyrDox is replaced by narrow, structureless radical signals with g-values similar to that of TyrDox (g approximately 2.0050). The new radicals copurify with photosystem II, are dark-stable, destabilized by elevated pH, and light-inducible, and originate from radicals formed by oxidation. These properties are similar to those of normal TyrDox, and we assign the new spectra to TyrDox in an altered environment induced by the point mutation in His189. In a third mutant, where Gln164 of the D2 protein was mutated to Leu, we also observed a modified EPR spectrum from TyrDox. This is also consistent with the model in which this residue is found in the immediate vicinity of TyrDox. Thus the results provide experimental evidence supporting essential aspects of the structural model.
An S3 electron paramagnetic resonance (EPR) signal is observed in a variety of photosystem 2 (PS2) samples in which the oxygen-evolving complex (OEC) has been inhibited. These signals have been proposed to be due to an interaction, S2X+, between the manganese cluster in an oxidation state equivalent to S2 and an organic radical, either oxidized histidine [Boussac et al. (1990) Nature 347, 303-306] or the tyrosine radical Yz+ [Hallahan et al. (1992) Biochemistry 31, 4562-4573]. We report that treatment of PS2 with acetate at pH 5.5 leads to a slowing of the reduction of Yz+ and allows the trapping of an S3-type state on freezing to 77 K following illumination at 277 K. The S3 EPR signal in acetate-treated PS2 has a broader and more complex line shape but otherwise has similar properties to other S3 signals. The addition to acetate-treated samples in the S1 state of the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), which allows only a single turnover of the reaction center, causes a large reduction in the yield of the S3 signal. Various anion and cation treatments change the S3 signal line shape and are used to show that acetate probably acts by binding and displacing chloride. We propose that a variety of treatments which affect calcium and chloride cofactor binding cause a modification of the S2 state of the manganese cluster, slow the reduction of Yz+, and allow an S3 EPR signal to be observed following illumination.(ABSTRACT TRUNCATED AT 250 WORDS)
The protein environment can dramatically affect the EPR line shape of tyrosine radicals. The alterations can be caused by: (1) a change in methylene geometry caused by different protein steric constraints; (2) a change in spin density caused by a change in protein environment; or (3) covalent modification of the tyrosine. Any or all of these effects may also be important, in some cases, in control of oxidation potential and chemical reactivity. The new signal that has been observed in the YF161D1 PS II mutant has an approximate 1:3:3:1 lineshape. There is no precedent for a 1:3:3:1 EPR signal from a tyrosine in a powder sample. However, as described above, given the diversity of signals from tyrosine radicals, it is impossible to exclude the possibility that the signal arises from tyrosine on this basis.
We compare primary charge separation in a photosystem II reaction center preparation isolated from a wild-type (WT) control
strain of the cyanobacterium Synechocystis sp. PCC 6803 and from two site-directed mutants of Synechocystis in which residue 130 of the D1 polypeptide has been changed from a glutamine to either a glutamate (mutant D1-Gln130Glu),
as in higher plant sequences, or a leucine residue (mutant D1-Gln130Leu). The D1-130 residue is thought to be close to the
pheophytin electron acceptor. We show that, when P680 is photoselectively excited, the primary radical pair state P680Ph is formed with a time constant of 20-30 ps in the WT and both mutants; this time constant is very similar to that observed
in Pisum sativum (a higher plant). We also show that a change in the residue at position D1-130 causes a shift in the peak of the pheophytin
Q-band. Nanosecond and picosecond transient absorption measurements indicate that the quantum yield of radical pair formation
(), associated with the 20-30-ps component, is affected by the identity of the D1-130 residue. We find that, for the isolated
photosystem II reaction center particle, > > > . Furthermore, the spectroscopic and quantum yield differences we observe between the WT Synechocystis and higher plant photosystem II, seem to be reversed by mutating the D1-130 ligand so that it is the same as in higher plants.
This result is consistent with the previously observed natural regulation of quantum yield in Synechococcus PS II by particular changes in the D1 polypeptide amino acid sequence (Clark, A. K., Hurry, V. M., Gustafsson, P. and Oquist,
G.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11985-11989).
The reaction center of photosystem II (PSII) of the oxygenic photosynthetic electron transport chain contains two redox-active tyrosines, Tyr160 (YD) of the D2 polypeptide and Tyr161 (YZ) of the D1 polypeptide, each of which may be oxidized by the primary electron donor, P680+. Spectroscopic characterization of YZ. has been hampered by the simultaneous presence of the much more stable YD., the short lifetime of YZ., and the difficulty in trapping the YZ. radical at low temperature. We present here a method for obtaining an uncontaminated YZ. radical, trapped by freezing under illumination of PSII core complexes isolated from YD-less mutants of Synechocystis 6803. Specific labeling with deuterium of the beta-methylene-3,3- or of the ring 3,5-protons of the PSII reaction center tyrosines in the YD-less D2-Tyr160Phe mutant results in a change in the hyperfine structure of the YZ. EPR signal, further confirming that this signal indeed arises from tyrosine. The trapped YZ. radical is also stable for several months at liquid nitrogen temperature. Due to both the absence of contaminating paramagnetic species and the stability at low temperature of YZ., this mutant core complex constitutes an excellent experimental system for the spectroscopic analysis of YZ.. We have compared the environments of YZ. and YD. by EPR, 1H ENDOR, and TRIPLE spectroscopies using both mutant and wild-type core complexes, with the following observations: (1) the EPR spectra of YZ. and YD. differ in line shape and line width. (2) Both YZ. and YD. exhibit D2O-exchangeable 1H hyperfine coupling near 3 MHz, consistent with the presence of a hydrogen bond from a proton donor to the phenolic oxygen atom of a neutral tyrosyl radical. This hyperfine coupling is sharp in the case of YD., indicating the hydrogen bond to be well-defined. In the case of YZ. it is broad, suggestive of a distribution of hydrogen-bonding distances. (3) YD. possesses three additional weak couplings that disappear in D2O, arising from three or fewer protons (protein or solvent) located within a shell between 4.5 and 8.5 A. (4) All of the 1H couplings of YD. are sharp, which is indicative of a well-ordered protein environment. (5) All of the 1H couplings in the YZ. spectrum are broad. The environment surrounding YZ. appears to be more disordered and solvent-accessible.
Photosystem II, the photosynthetic water-oxidizing complex, can be isolated from both plants and cyanobacteria. A variety of methods have been developed for purification of this enzyme, which can be isolated in several functional and structural forms. Knowledge of the pigment content of photosystem II preparations is important for precise spectroscopic, biochemical, and functional analysis. We have determined pigment stoichiometries in oxygen-evolving photosystem II preparations from plants and cyanobacteria. We have employed a solvent system for the isocratic elution of a reverse phase HPLC column in which we have determined the extinction coefficients of the relevant pigments. Pigments were extracted from four photosystem II preparations. These preparations included spinach photosystem II membranes [Berthold, D. A., Babcock, G. T., & Yocum, C. F. (1981) FEBS Lett. 134, 231-234], spinach photosystem II reaction center complexes [Ghanotakis, D. F., & Yocum, C. F. (1986) FEBS Lett. 197, 244-248], spinach photosystem II complexes [MacDonald, G. M., & Barry, B. A. (1992) Biochemistry 31, 9848-9856], and photosystem II particles isolated from the cyanobacterium, Synechocystis sp. PCC 6803 [Noren, G. H., Boerner, R. J., & Barry, B. A. (1991) Biochemistry 30, 3943-3950]. Pigment stoichiometries were determined using two different methods of data analysis and were based on the assumption that there are two pheophytin a molecules per photosystem II reaction center. The pigment stoichiometries obtained were comparable for the two methods of data analysis and agreed with previous biophysical and biochemical characterizations of the preparations. The average pigment stoichiometries (chlorophyll:plastoquinone-9 per 2 pheophytin a) determined using the two data analysis methods were as follows: photosystem II membranes, 274:3.2; photosystem II reaction center complexes, 78:2.5; Synechocystis PS II particles, 55:2.4; photosystem II complexes, 121:2.0.
The Mn cluster of Photosystem II (PSII) from Synechococcus elongatus was studied using EPR. A signal with features between g = 5 and g = 9 is reported from the S2-state. The signal is attributed to the manganese cluster in a state with a spin 5/2 state. Spectral simulations of the signal indicate zero field splitting parameters where the |E/D| was 0.13. The new signal is formed by irradiating PSII samples which contain the spin = 1/2 S2-state using 813 nm light below 200 K. This effect is attributed to a spin-state change in the manganese cluster due to absorption of the IR light by the Mn-cluster itself. The signal is similar to that reported recently in PSII of plants [Boussac, A., Un, S., Horner, O., and Rutherford, A. W. (1998) Biochemistry 37, 4001-4007]. In plant PSII the comparable signal is formed at a lower temperature (optimally below 77 K), and gradual warming of the sample in the dark leads to the formation of the state responsible for the well-known g = 4.1 signal prior to formation of the spin 1/2 multiline signal. In the present work using cyanobacterial PSII, warming of the sample in the dark leads to the formation of the spin 1/2 multiline signal without formation of the g = 4 type signal as an intermediate. These observations provide a partial explanation for the long-standing "mystery of the missing g = 4 state" in cyanobacterial PSII. The observations are rationalized in terms of three possible states which can exist for S2: (i) the spin 1/2 multiline signal, (ii) the state responsible for the g = 4.1 signal, and (iii) the new spin 5/2 state. The relative stability of these states differs between plants and cyanobacteria.
Photosynthetic oxygen evolution is powered by photosystem II (PSII), in particular by the oxidized chl a-aggregate P680+, and catalyzed by the oxygen-evolving complex (Mn4X-entity) as well as a tyrosine residue (YZ). The role of particular amino acids as cofactors of electron and proton transfer or as modulators of the activity is still ill-defined. The effects of single-site mutations at the donor side of PSII on the partial reactions of water oxidation have been primarily studied in whole cells. Because of better signal-to-noise in oxygen-evolving core preparations more detailed information on the electronic, protonic, and electrostatic events is expected from studies with such material. We investigated cells and oxygen-evolving core preparations from the wildtype of Synechocystis sp. PCC 6803 and point-mutants of D1-D61. In cells, oxygen-release was slowed drastically in D61A (8-fold) and D61N (10-fold) compared to WT, whereas it remained unchanged in D61E within the time resolution of the measurements. In core preparations, the S1 --> S2 and S2 --> S3 transitions were slowed approximately 2-fold in D61N compared to WT. However, the nanosecond components of electron transfer from YZ to P680+ were unchanged in the same mutant. We conclude that substitution of a neutral residue for D1-D61 selectively affects electron-transfer events on the donor side of YZ.
Recent models for water oxidation in photosystem II postulate that the tyrosine Y(Z) radical, Y(Z)(*), abstracts both an electron and a proton from the Mn cluster during one or more steps in the catalytic cycle. This coupling of proton- and electron-transfer events is postulated to provide the necessary driving force for oxidizing the Mn cluster in its higher oxidation states. The formation of Y(Z)(*) requires the deprotonation of Y(Z) by His190 of the D1 polypeptide. For Y(Z)(*) to abstract both an electron and a proton from the Mn cluster, the proton abstracted from Y(Z) must be transferred rapidly from D1-His190 to the lumenal surface via one or more proton-transfer pathways. The proton acceptor for D1-His190 has been proposed to be either Glu189 of the D1 polypeptide or a group positioned by this residue. To further define the role of D1-Glu189, 17 D1-Glu189 mutations were constructed in the cyanobacterium Synechocystis sp. PCC 6803. Several of these mutants are of particular interest because they appear to assemble Mn clusters in 70-80% of reaction centers in vivo, but evolve no O(2). The EPR and electron-transfer properties of PSII particles isolated from the D1-E189Q, D1-E189L, D1-E189D, D1-E189N, D1-E189H, D1-E189G, and D1-E189S mutants were examined. Intact PSII particles isolated from mutants that evolved no O(2) also exhibited no S(1) or S(2) state multiline EPR signals and were unable to advance beyond an altered Y(Z)(*)S(2) state, as shown by the accumulation of narrow "split" EPR signals under multiple turnover conditions. In the D1-E189G and D1-E189S mutants, the quantum yield for oxidizing the S(1) state Mn cluster was very low, corresponding to a > or =1400-fold slowing of the rate of Mn oxidation by Y(Z)(*). In Mn-depleted D1-Glu189 mutant PSII particles, charge recombination between Q(A)(*)(-) and Y(Z)(*) in the mutants was accelerated, showing that the mutations alter the redox properties of Y(Z) in addition to those of the Mn cluster. These results are consistent with D1-Glu189 participating in a network of hydrogen bonds that modulates the properties of both Y(Z) and the Mn cluster and are consistent with proposals that D1-Glu189 positions a group that accepts a proton from D1-His190.
The Mn(4)-cluster and the cytochrome c(550) in histidine-tagged photosystem II (PSII) from Synechococcus elongatus were studied using electron paramagnetic resonance (EPR) spectroscopy. The EPR signals associated with the S(0)-state (spin = 1/2) and the S(2)-state (spin = 1/2 and IR-induced spin = 5/2 state) were essentially identical to those detected in the non-His-tagged strain. The EPR signals from the S(3)-state, not previously reported in cyanobacteria, were detectable both using perpendicular (at g = 10) and parallel (at g = 14) polarization EPR, and these signals are similar to those found in plant PSII. In the S(3)-state, near-infrared illumination at 50 K induced a 176-G-wide split signal at g = 2 and signals at g = 5.20 and g = 1.51. These signals differ slightly from those reported in plant PSII [Ioannidis, N., and Petrouleas, V. (2000) Biochemistry 39, 5246-5254]. In accordance with the cited work, the split signal presumably reflects a radical interacting with the Mn(4)-cluster in a fraction of centers, while the g = 5.20 and g = 1.51 signals are tentatively attributed to a high-spin state of the Mn(4)-cluster with zero field splitting parameters different from those in plant PSII, reflecting minor changes in the environment of the Mn(4)-cluster. Biochemical modifications (Sr(2+)/Ca(2+) substitution, acetate and NH(3) treatments) were also investigated. In Sr(2+)-reconstituted PSII, in addition to the expected modified S(2) multiline signal, a signal at g = 5.2 was present instead of the g approximately 4 signal seen in plant PSII. In NH(3)-treated samples, in addition to the expected modified S(2)-multiline signal, a g approximately 4 signal was detected in a small proportion of the reaction centers. This is of note since g approximately 4 spectra arising from the Mn(4)-cluster in the S(2) state have not yet been published in cyanobacterial PSII. The detection of modified S(3)-signals in both perpendicular (at g = 7.5) and parallel (at g = 12) polarization EPR from NH(3)-treated PSII indicate that NH(3) is still bound in the S(3)-state. The acetate-treated PSII behaves essentially as in plant PSII. A study using oriented samples indicated that the heme plane of the oxidized low spin Cytc(550) was perpendicular to the plane of the membrane.
The tetranuclear manganese cluster in photosystem II is ligated by one or more histidine residues, as shown by an electron spin echo envelope modulation (ESEEM) study conducted with [(15)N]histidine-labeled photosystem II particles isolated from the cyanobacterium Synechocystis sp. strain PCC 6803 [Tang, X.-S., Diner, B. A., Larsen, B. S., Gilchrist, M. L., Jr., Lorigan, G. A., and Britt, R. D. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 704-708]. One of these residues may be His332 of the D1 polypeptide. Photosystem II particles isolated from the Synechocystis mutant D1-H332E exhibit an altered S(2) state multiline EPR signal that has more hyperfine lines and narrower splittings than the corresponding signal in wild-type PSII particles [Debus, R. J., Campbell, K. A., Peloquin, J. M., Pham, D. P., and Britt, R. D. (2000) Biochemistry 39, 470-478]. These D1-H332E PSII particles are also unable to advance beyond an altered S(2)Y(Z)(*) state, and the quantum yield for forming the S(2) state is very low, corresponding to an 8000-fold slowing of the rate of Mn oxidation by Y(Z)(*). These observations are consistent with His332 being close to the Mn cluster and modulating the redox properties of both the Mn cluster and tyrosine Y(Z). To determine if D1-His332 ligates the Mn cluster, we have conducted an ESEEM study of D1-H332E PSII particles. The histidyl nitrogen modulation observed near 5 MHz in ESEEM spectra of the S(2) state multiline EPR signal of wild-type PSII particles is substantially diminished in D1-H332E PSII particles. This result is consistent with ligation of the Mn cluster by D1-His332. However, alternate explanations are possible. These are presented and discussed.
Electron paramagnetic resonance (EPR) spectroscopy at 94 GHz is used to study the dark-stable tyrosine radical Y(D)(*) in single crystals of photosystem II core complexes (cc) isolated from the thermophilic cyanobacterium Synechococcus elongatus. These complexes contain at least 17 subunits, including the water-oxidizing complex (WOC), and 32 chlorophyll a molecules/PS II; they are active in light-induced electron transfer and water oxidation. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with four PS II dimers per unit cell. High-frequency EPR is used for enhancing the sensitivity of experiments performed on small single crystals as well as for increasing the spectral resolution of the g tensor components and of the different crystal sites. Magnitude and orientation of the g tensor of Y(D)(*) and related information on several proton hyperfine tensors are deduced from analysis of angular-dependent EPR spectra. The precise orientation of tyrosine Y(D)(*) in PS II is obtained as a first step in the EPR characterization of paramagnetic species in these single crystals.
The essential involvement of manganese in photosynthetic water oxidation was implicit in the observation by Pirson in 1937 that plants and algae deprived of Mn in their growth medium lost the ability to evolve Oâ. Addition of this essential element to the growth medium resulted in the restoration of water oxidation within 30 min. There is increased interest in the study of Mn in biological chemistry and dioxygen metabolism in the last two decades with the discovery of several Mn redox enzymes. The list of enzymes where Mn is required for redox activity includes a Mn superoxide dismutase, a binuclear Mn-containing catalase, a binuclear Mn-containing ribonucleotide reductase, a proposed binuclear Mn site in thiosulfate oxidase, a Mn peroxidase that is capable of oxidative degradation of lignin, and perhaps the most complex and important, the tetranuclear Mn-containing oxygen-evolving complex in photosystem II (Mn-OEC). Mn is well suited for the redox role with accessible oxidation states of II, III, and IV, and possibly V: oxidation states that have all been proposed to explain the mechanisms of the Mn redox enzymes.
The H(2)O oxidizing domain of the cyanobacterial photosystem II (PSII) complex contains a low potential, c-type cytochrome termed c(550) that is essential for the in vivo stability of the PSII complex. A mutant lacking cytochrome c(550) (DeltapsbV) in Synechocystis sp. PCC6803 has been further analyzed together with a construct in which the distal axial heme iron ligand, histidine 92, has been substituted with a methionine (C550-H92M). Heme staining of SDS-PAGE showed that the C550-H92M mutation did not disturb the accumulation and heme-binding properties of the cytochrome. In DeltapsbV cells, the number of charge separating PSII centers was estimated to be 56% of the wild type, but of the existing centers, 33% lacked photooxidizable Mn ions. C550-H92M did not discernibly affect the intrinsic PSII electron-transfer kinetics compared to the wild type nor did it exhibit a significant fraction of centers lacking photooxidizable Mn; however, the number of charge separating PSII centers in mutant cells was 69% of the wild type. C550-H92M lost photoautotrophic growth ability in the absence of Ca(2+), but its growth was not affected by depletion of Cl(-), which differs from DeltapsbV. Taken together, the results suggest that in the absence of cytochrome c(550) electron transfer on the donor side is retarded perhaps at the level of Y(z) to P680(+) transfer, the heme ligand. His92 is not absolutely required for assembly of functional PSII centers; however, replacement by methionine prevents normal accumulation of PSII centers in the thylakoid membranes and alters the Ca(2+) requirement of PSII. The results are discussed in terms of current understanding of the Ca(2+) site of PSII.
Two D2 mutants were created with a site-directed mutation near the presumable binding site of QA. In one of the mutants, in which Trp-253, the aromatic residue potentially involved in facilitating electron transport from pheophytin to QAand/or in binding of QA, had been replaced by Leu, PS II was undetectable in thylakoids. This mutant is an obligate photoheterotroph. In another mutant the Gly-215 residue, located next to the His residue that is proposed to bind QA and Fe2+, was mutated to Trp. This mutation leads to a rapid inactivation of oxygen evolution capacity in the light, and to a virtual elimination of the potential to grow photoautotrophically, but does not greatly affect the number of photosystem II reaction centers on a chlorophyll basis. We propose that proper binding of QA to the photosystem II reaction center complex is a prerequisite for stability of the photosystem II complex. Impairment of QA binding leads to rapid inactivation of photosystem II, which may be followed by a structural disintegration of the complex.
Publisher Summary This chapter presents detailed information on chlorophylls and carotenoids to give practical directions toward their quantitative isolation and determination in extracts from leaves, chloroplasts, thylakoid particles, and pigment proteins. The chapter focuses on the spectral characteristics and absorption coefficients of chlorophylls, pheophytins, and carotenoids, which are the basis for establishing equations to quantitatively determine them. Therefore, the specific absorption coefficients of the pigments are re-evaluated. This is achieved by using a two-beam spectrophotometer of the new generation, which allows programmed automatic recording and printing out of the proper wavelengths and absorbancy values. Several procedures have been developed for the separation of the photosynthetic pigments, including column (CC), paper (PC), and thin-layer chromatography (TLC) and high-pressure liquid chromatography (HPLC). All chloroplast carotenoids exhibit a typical absorption spectrum that is characterized by three absorption maxima (violaxanthin, neoxanthin) or two maxima with one shoulder (lutein and β-carotene) in the blue spectral region.
Highly photoactive Photosystem I (PS I) and Photosystem II (PS II) core complexes have been isolated from the cyanobacterium Synechocystis Pasteur Culture Collection (PCC) 6803 and a phycocyanin-deficient mutant, enriched in PS II. Cell breakage using glass beads was followed by sucrose density gradient centrifugation and two high-performance liquid chromatography steps involving anion-exchange and hydroxyapatite. The PS I core complex has an apparent molecular mass of 300 +/- 20 kDa (including a detergent shell of about 50 kDa) and contains subunits of approximately 60, approximately 60, 18.5, 18.5, 16, 15, 10.5, 9.5, and 6.5 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblots; its antenna size is 75 +/- 5 chlorophyll/P-700. The PS II core complex has an apparent molecular mass of 310 +/- 20 kDa (including the detergent shell); subunits of 43, 37, 33, 29, and 10-11 kDa were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The antenna size of the average PS II complex is 45 +/- 5 chlorophyll/primary quinone electron acceptor (QA). This preparation procedure also yields, as a byproduct, a highly purified cytochrome b6f complex. This complex contains four subunits of 38, 24, 19, and 15 kDa and b- and c-type cytochromes in a ratio of 2:1. Its apparent molecular mass of 180 +/- 20 kDa (including the detergent shell) is consistent with a monomeric complex.
Photosynthetic oxygen evolution takes place in the thylakoid protein complex known as photosystem II. The reaction center core of this photosystem, where photochemistry occurs, is a heterodimer of homologous polypeptides called D1 and D2. Besides chlorophyll and quinone, photosystem II contains other organic cofactors, including two known as Z and D. Z transfers electrons from the site of water oxidation to the oxidized reaction center primary donor, P+.680, while D+. gives rise to the dark-stable EPR spectrum known as signal II. D+. has recently been shown to be a tyrosine radical. Z is probably a second tyrosine located in a similar environment. Indirect evidence indicates that Z and D are associated with the D1 and D2 polypeptides, respectively. To identify the specific tyrosine residue corresponding to D, we have changed Tyr-160 of the D2 polypeptide to phenylalanine by site-directed mutagenesis of a psbD gene in the cyanobacterium Synechocystis 6803. The resulting mutant grows photosynthetically, but it lacks the EPR signal of D+.. We conclude that D is Tyr-160 of the D2 polypeptide. We suggest that the C2 symmetry in photosystem II extends beyond P680 to its immediate electron donor and conclude that Z is Tyr-161 of the D1 polypeptide.
Absorption spectra in the near infra-red have been obtained for transient species obtained from chlorophyll a, by flash absorption spectroscopy. The triplet state of Chl a presents an absorption band around 760 nm (ϵ = 7,500 M−1 cm−1) and a very broad band (ϵ = 1,700 M−1 cm−1) around 1100 nm. At low temperature, evidence was obtained for the formation of the triplet state of aggregated Chl a. Its t1/2 of decay is around 0.5 ms instead of 1 ms for the monomeric species. The radical-cation Chl a+ was formed by reaction of 3Chl a with a quinone; it has an absorption band at 840 nm (in cyclohexanol) with a shoulder around 740 nm and no absorption between 1000 and 1650 nm. The cation has also been formed in vivo, as the oxidized state of the photosynthetic primary electron donors, P680 and P700. The spectrum of P+680 resembles that of Chl a+; its absorption maximum is at 820 nm. The spectrum of P+680, maximum at 810 nm, is significantly broader. P+700 has no absorption between 1000 and 1650 nm. These spectra are in favor of recent suggestions on a monomeric nature of P680 and of a dimeric nature for P700.
The oxidation‐reduction midpoint potential (E m) of the primary quinone (QA) of the acceptor quinone complex of bacterial photosynthetic reaction centers has been measured as a function of pH in the presence and absence of ubiquinone and o‐phenanthroline (o‐Phen). Reaction centers, isolated from Rhodopseudomonas sphaeroides, were incorporated into egg phosphatidylcholine vesicles. Contrary to earlier reports, the E m was found to exhibit a pH‐dependence very similar to that observed in chromatophores, with a slope of ‐ 60 mV/pH up to a p K for Q−A/Q−A(H+) at pH 9.5–10.0. In the presence of ubiquinone to reconstitute the secondary quinone (QB), the E m/pH curve of QA was shifted to lower potentials, indicating that the binding of Qn (actually QBH2) was suppressed by reduction of QA. o‐Phen, an inhibitor of electron transport from QA to QB, raised the pK of Q−A/Q−A(H+) and, at pH‐values below but not above this pK, reversed the effects of QB. In the absence of QB, o‐Phen lowered the E m of QA above the pK but had no effect below it. These results are discussed in terms of interactions between the binding sites for QA and QB (A‐ and B‐sites). It is suggested that ubiquinone and o‐Phen compete for the B‐site in a mutually exclusive fashion, and that their relative binding strengths are modulated by the redox and protonation state of QA. In preparations with low quinone content, o‐Phen inhibits photochemistry suggesting that it can also compete with ubiquinone at the A‐site. Competitive displacement of quinone from the B‐site by o‐Phen and other inhibitors is suggested as the primary mode of action of a broad class of herbicides active in Photosystem II of plants. The relative binding affinities of the various redox states of QB are also discussed and it is concluded that the order of binding strength is: Q−B → QB → QBH2. This accounts for the atypical stability of the semiquinone and the lower average E m for reduction to the quinol, compared to free ubiquinone in the quinone pool. It may also be significant in the functioning of quinones in communicating reducing equivalents from the reaction center to other electron transport complexes in the intact membrane.
Membranes and PS II particles retaining high rates of O2-evolving activity have been isolated from the transformable cyanobacterium, Synechocystis sp. PCC6803. Membranes from cells grown under red light exhibit rates of O2-evolution ranging from 500-700 μmole O2/mg chl/h. PS II particles are prepared by a simple procedure involving DEAE column chromatography of detergent extracts obtained by simultaneous treatment of membranes with octylglucoside and dodecylmaltoside. The isolated PS II fraction is enriched in polypeptides immunologically cross-reactive with polypeptides present in core reaction center preparations of spinach, exhibits 77 K fluorescence emission maxima at 685 and 696 nm, but not emission and absorption due to phycobilines and is capable of rates of O2-evolution exceeding 1000 μmole O2/mg chl/h.
Redox events that occur in photosynthetic O2-evolving centers in NaCl/EDTA-washed PS II membranes were investigated by means of low temperature EPR and thermoluminescence. The following results have been obtained: (i) In the washed membranes, O2 centers could maintain the S2 state more than 3 h in darkness at room temperature. This dark-stable S2 was modified as seen by a multiline EPR signal with reduced hyperfine line spacing and also by a thermoluminescence band with upshifted peak temperature. (ii) This modified S2 state had an abnormally long life of at 20°C, and its appearance required the presence of EDTA in the medium. On addition of exogenous Ca2+, the modified S2 was converted in darkness to normal S2, and then decayed rapidly to be undetectable. (iii) On illuminating this modified S2, an EPR signal centering at aroung g = 2 was newly induced at no expense of the dark-stable EPR multiline signal. This EPR signal was accompanied by a new thermoluminescence band peaking at around 5°C, suggesting the presence of a new redox component whose oxidized form is capable of providing a positive charge for thermoluminescence in place of Mn. (iv) This new component was efficiently oxidized by illumination at −5°C but much less at −60°C, showing a half-inhibition temperature at around −40°C. (v) Addition of various divalent cations in place of Ca2+ variously affected both thermoluminescence glow peaks arising from the dark-stable S2 or from the new redox component, suggesting a cation-species-dependent modulation of the redox properties of both components. (vi) Both of these two thermoluminescence bands showed no dependency on flash number, suggesting interruption of further oxidation beyond their respective abnormal states. On addition of Ca2+, all these abnormal properties were abolished and normal period-four flash pattern was restored. These abnormal properties of the redox events in NaCl/EDTA-washed PS II membranes were discussed in relation to the demand for exogenous Ca2+ in recovery of normal properties.
A single flash given at − 15°C to chloroplasts results in charge separation in Photosystem II to form a stable state which, upon warming, recombines giving rise to luminescence. This recombination occurs at 25°C in untreated chloroplasts but is shifted to 0°C in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea or weak concentrations of a reducing agent. The luminescence at 0°C is attributed to recombination of the S2Q−A state while that at 25°C is attributed to recombination of S2QAQ−B (and S3QAQ−B upon further flash illumination). The identification of the thermoluminescence at 25°C is based upon the following experimental evidence: (1) illumination of chloroplasts in the presence of methyl viologen with 710 nm light before and after flash illumination has no effect on the extent or temperature of the thermoluminescence. This is taken as evidence that the plastoquinone pool is not involved in the recombination reaction. (2) Calculations of the extent of thermoluminescence expected after a number of flashes, assuming that S2QAQ−B and S3QAQ−B are the thermoluminescent reactants, give a good fit to the experimental results. (3) The effect of continuous illumination at 77 K (i.e., donation from cytochrome b-559 to QA and thence to QB or Q−B) results in predictable changes in the extent of flash-induced thermoluminescence.
The effect of the extrinsic 33-kDa protein on the photosynthetic oxygen evolution was studied by comparing spinach Photosystem II particles depleted of the 33-kDa protein with those reconstituted with the protein. The light-intensity dependence of the oxygen-evolution activity under continuous illumination suggests that a dark step, but not a light step, in the oxygen-evolving reaction is accelerated by the 33-kDa protein. Consistently, the pattern of oxygen yield with a series of short saturating flashes, which showed a maximum on the third flash and a damped oscillation with a period of 4, was not much affected by the removal and rebinding of the 33-kDa protein, when the dark interval between the flashes was long enough, i.e., longer than 0.5 s. The millisecond kinetics of oxygen release after the third flash was retarded by the removal of the 33-kDa protein and stimulated by its rebinding, suggesting that the transition from S3 to S0 is accelerated by the 33-kDa protein. The stability of the S2 and S3 states in darkness was higher in the absence of the 33-kDa protein than its presence.
In Photosystem II preparations at low temperature we were able to generate and trap an intermediate state between the S1 and S2 states of the Kok scheme for photosynthetic oxygen evolution. Illumination of dark-adapted, oxygen-evolving Photosystem II preparations at 140 K produces a 320-G-wide EPR signal centered near g = 4.1 when observed at 10 K. This signal is superimposed on a 5-fold larger and somewhat narrower background signal; hence, it is best observed in difference spectra. Warming of illuminated samples to 190 K in the dark results in the disappearance of the light-induced g = 4.1 feature and the appearance of the multiline EPR signal associated with the S2 state. Low-temperature illumination of samples prepared in the S2 state does not produce the g = 4.1 signal. Inhibition of oxygen evolution by incubation of PS II preparations in 0.8 M NaCl buffer or by the addition of 400 μM NH2OH prevents the formation of the g = 4.1 signal. Samples in which oxygen evolution is inhibited by replacement of Cl− with F− exhibit the g = 4.1 signal when illuminated at 140 K, but subsequent warming to 190 K neither depletes the amplitude of this signal nor produces the multiline signal. The broad signal at g = 4.1 is typical for a spin system in a rhombic environment, suggesting the involvement of non-heme Fe in photosynthetic oxygen evolution.
The multiline EPR signals arising from manganese in the S2 state of the oxygen-evolving system of spinach and the cyanobacterium Anacystis nidulans have very similar properties and are affected identically by NH3, suggesting that the system is highly conserved. The temperature dependence of the signal amplitude follows Curie behavior down to sub-helium temperatures. This is in contrast to previous reports, which were taken as evidence for a tetrameric manganese cluster. Thus, it seems that it is not yet possible from EPR data alone to distinguish between this model and a dimeric structure.
Redox titrations of the photo-induced pheophytin EPR signal in Photosystem II show two transitions which reflect the redox state of Q. The high potential wave (Em ⋍ −50 mV) can be photo-induced at 5 K and 77 K. The low potential wave (Em ⋍ −275 mV) required illumination at 200 K. This indicates the presence of two kinds of PS-II reaction centres differing in terms of the competence of their donors at low temperature and the Em-values of their acceptors. Measurements of the semiquinone-iron acceptor also demonstrate functional heterogeneity at low temperature. This is the first observation of the semiquinone-iron acceptor in a non-mutant species.
A complete medium of defined composition has been developed for quantitative growth of wild-type and auxotrophic mutant strains of Anacystis nidulans. This medium has proved to be more satisfactory than other complex media (for example casein hydrolysate, yeast extract) for both the isolation and the growth of auxo-trophs. Rigorous control of the pH of complete and other supplemented media is essential for quantitative growth on agar. Four diagnostic media are described which each contain a different combination of the supplements used in the complete medium and facilitate the identification of the nutritional requirements of mutants. By using these media a number of auxotrophs have been isolated including five with novel phenotypes which require respectively (i) thiamine, (ii) p-aminobenzoic acid, (iii) a combination of pyruvate or acetate plus malate or succinate or fumarate, (iv) serine or glycine and (v) adenine.
We have investigated the effects of temperature on the formation and decay of the light-induced multiline EPR signal species associated with photosynthetic oxygen evolution (Dismukes, G.C. and Siderer, Y. (1980) FEBS Lett. 121, 78–80). (1) The decay rate following illumination is temperature dependent: at 295 K the half-time of decay is about 40 s, at 253 K the half-time is approx. 40 min. (2) A single intense flash of light becomes progressively less effective in generating the multiline signal below about 240 K. (3) Continuous illumination is capable of generating the signal down to almost 160 K. (4) Continuous illumination after a preilluminating flash generates less signal above 200 K than at lower temperatures. Our results support the conclusion of Dismukes and Siderer that the S2 state gives rise to this multiline signal; we find that the S1 state can be fully advanced to the S2 state at temperatures as low as 160 K. The S2 state is capable of further advancement at temperatures above about 210 K, but not below that temperature.
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.
— Using isolated chloroplasts and techniques as described by Joliot and Joliot[6] we studied the evolution of O2 in weak light and light flashes to analyze the interactions between light induced O2 precursors and their decay in darkness. The following observations and conclusions are reported: 1. Light flashes always produce the same number of oxidizing equivalents either as precursor or as O2. 2. The number of unstable precursor equivalents present during steady state photosynthesis is ∼ 1.2 per photochemical trapping center. 3. The cooperation of the four photochemically formed oxidizing equivalents occurs essentially in the individual reaction centers and the final O2 evolution step is a one quantum process. 4. The data are compatible with a linear four step mechanism in which a trapping center, or an associated catalyst, (S) successively accumulates four + charges. The S4+ state produces O2 and returns to the ground state S0. 5. Besides S0 also the first oxidized state S+ is stable in the dark, the two higher states, S2+ and S3+ are not. 6. The relaxation times of some of the photooxidation steps were estimated. The fastest reaction, presumably S*1←S2, has a (first) half time ≤ 200 μsec. The S*2 state and probably also the S*0 state are processed somewhat more slowly (˜ 300–400 μsec).
Various approaches have been used to investigate the polypeptides required for oxygen evolution in cyanobacteria, in particular the thermophile Phormidium laminosum. Antibodies against the extrinsic 33 kDa protein from spinach Photosystem II cross-reacted clearly in immunoblotting experiments with a corresponding polypeptide in isolated thylakoids and Photosystem II particles from P. laminosum and with whole-cell homogenates of three species of cyanobacteria (Phormidium laminosum, Synechococcus leopoliensis and Anabaena variabilis). In contrast, no cyanobacterial proteins reacted with antibodies against the 23 and 16 kDa proteins of spinach Photosystem II. The lack of cross-reactivity and the absence of these polypeptides from highly active Photosystem II particles of Phormidium laminosum strongly suggest that cyanobacteria do not contain polypeptides corresponding to these two chloroplast proteins. Treatment of P. laminosum Photosystem II particles with 0.8 M alkaline Tris, 1 M NaCl, CaCl2 or MgCl2 inhibited O2 evolution, and quantitatively removed a 9 kDa polypeptide from the particles. None of these treatments removed comparable amounts of the 33 kDa polypeptide, and only Tris treatment removed manganese. The release of the 9 kDa polypeptide upon NaCl treatment correlated well with the deactivation at the donor side of Photosystem II. A direct connection between the 33 kDa polypeptide and O2 evolution was established by the finding that trypsin treatment digested this polypeptide and inhibited O2 evolution in parallel.
Freezing of spinach or barley chloroplasts during continuous illumination results in the trapping of a paramagnetic state or a mixture of such states characterized by a multiline EPR spectrum. Added Photosystem II electron acceptor enhances the signal intensity considerably. Treatments which abolish the ability of the chloroplasts to evolve oxygen, by extraction of the bound manganese, prevent the formation of the paramagnetic species. Restoration of Photosystem II electron transport in inhibited chloroplasts with an artificial electron donor (1,5-diphenylcarbazide) does not restore the multiline EPR spectrum. The presence of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) results in a modified signal which may represent a second paramagnetic state. The paramagnetic forms appear to originate on the donor side in Photosystem II and are dependent on a functional oxygenevolving site and bound, intact manganese. It is suggested that magnetically interacting manganese ions in the oxygen-evolving site may be responsible for the EPR signals. This suggestion is supported by calculations.
Photosystem 2 preparations with very high rates of oxygen evolution from the thermophilic cyanobacterium Phormidium laminosum have been studied by EPR spectrometry. In the presence of DCMU the g = 1.82 signal of the iron—quinone electron acceptor (Q) can be observed. It is proposed that DCMU is necessary to disrupt a magnetic interaction, between the semiquinone forms of Q and the secondary acceptor B, which otherwise prevents detection of the Q−Fe signal. A doublet EPR signal arising from magnetic interaction between Q−Fe and the reduced intermediary electron acceptor pheophytin (I−), and a spin-polarized triplet signal assumed to arise from the back reaction between I− and P680+ can also be seen. Preliminary redox titrations of Q reduction have been carried out, indicating Em ⋍ 0 mV.
Binding of NH3 to the S2 state of the O2-evolving complex of photosystem II (PSII) causes a structural change in the Mn site that is detectable with low-temperature electron paramagnetic resonance (EPR) spectroscopy. Untreated spinach PSII membranes at pH 7.5 produce a S2 state multiline EPR spectrum when illuminated at either 210 K or at 0°C in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) having an average hyperfine line spacing of 87.5 G. The temperature dependence of the S2 state multiline EPR signal observed from untreated samples deviates from the Curie law above 5 K, with a maximum signal intensity at 6.9 K as has been previously observed. In contrast, 100 mM NH4Cl-treated PSII membranes at pH 7.5 exhibit a new S2 state EPR spectrum when illuminated at 0°C in the presence of DCMU. The novel S2 state EPR spectrum from NH4Cl-treated PSII membranes has an average hyperfine line spacing of 67.5 G and a temperature dependence obeying the Curie law except for small deviations at low temperature. We assign the new S2 state EPR signal from NH4Cl-treated PSII membranes to a form of the S2 state having one or more NH3 molecules directly coordinated to the Mn site. NH3 does not bind to Mn in the dark-stable S1 state present before illumination, since generation of the S2 state in NH4Cl-treated PSII membranes by illumination at 210 K does not yield the new S2 state EPR spectrum. Since inhibition of O2 evolution activity in the presence of NH4Cl probably occurs through binding of NH3 to the O2-evolving complex in competition with substrate H2O molecules, these results indicate that the EPR-detectable Mn site functions as the substrate-binding site of the O2-evolving complex.
Photosynthetic organisms are able to oxidize organic or inorganic compounds upon the absorption of light, and they use the extracted electron for the fixation of carbon dioxide. The most important oxidation product is oxygen due to the splitting of water. In eukaryotes these processes occur in photosystem II of chloroplasts. Among prokaryotes photosynthetic oxygen evolution is restricted to cyanobacteria and prochloron-type organisms. How water is split in the oxygen-evolving complex of photosystem II belongs to the most important question to be answered. The primary charge separation occurs in the reaction center of photosystem II. This reaction center is a complex consisting of peripheral and integral membrane proteins, several chlorophyll A molecules, two pheophytin A molecules, two and three plastoquinone molecules, and one non-heme iron atom. The location of the photosystem II reaction center is still a matter of debate. Nakatani et al. (l984) concluded from fluorescence measurements that a protein of apparent molecular weight 47,000 (CP47) is the apoprotein of the photosystem II reaction center. A different view emerged from work with the photosynthetic reaction centers from the purple bacteria. The amino acid sequence of the M subunit of the reaction center from Phodopseudomonas (Rps.) sphaeroides has sequence homologies with the D1 protein from spinach. A substantial amount of structural information can be obtained with the reaction center from Rhodopseudomonas viridis, which can be crystallized. Here the authors discuss the structure of the photosynthetic reaction center from the purple bacterium Rps. viridis and describe the role of those amino acids that are conserved between the bacterial and photosystem II reaction center.
A study of electron paramagnetic resonance (EPR) signals from components on the electron donor side of photosystem II has been performed. By measurement of EPR signal IIslow (D+) it is shown that, after three flashes, D+ decays slowly in the dark at room temperature in the fraction of the centers that was in the S0 state (t1/2 of 20 min in thylakoid membranes and 50 min in photosystem II enriched membranes). This reaction is accompanied by a conversion of S0 to S1. The concentration of S1 was estimated from the amplitude of the S2-state multiline EPR signal that could be generated by illumination at 200 K. These observations indicate that D+ accepts an electron from S0 in a dark reaction in which D and S1 are formed. In addition, the reactions by which D donates an electron to S2 or S3 have been directly measured by monitoring both signal IIslow and the multiline signal. The redox interactions between the D/D+ couple and the S states are explained in terms of a model in which D/D+ has a midpoint potential between those of S0/S1, and S1/S2. In addition, this model provides explanations for a number of previously unrelated phenomena, and the proposal is put forward that the reaction between D+ and Mn2+ is involved in the so-called photoactivation process.
Electron paramagnetic resonance (EPR) signals arising from components in photosystem II have been studied in membranes isolated from spinach chloroplasts. A broad EPR signal at g = 4.1 can be photoinduced by a single laser flash at room temperature. When a series of flashes is given, the amplitude of the g = 4.1 signal oscillates with a period of 4, showing maxima on the first and fifth flashes. Similar oscillations occur in the amplitude of a multiline signal centered at g ≃ 2. Such an oscillation pattern is characteristic of the S2 charge accumulation state in the oxygen-evolving complex. Accordingly, both EPR signals are attributed to the S2 state. Earlier data from which the g = 4.1 signal was attributed to a component different from the S2 state [Zimmermann, J.-L., & Rutherford, A. W. (1984) Biochim. Biophys. Acta 767, 160-167; Casey, J. L., & Sauer, K. (1984) Biochim. Biophys. Acta 767, 21-28] are explained by the effects of cryoprotectants and solvents, which are shown to inhibit the formation of the g = 4.1 signal under some conditions. The g = 4.1 signal is less stable than the multiline signal when both signals are generated together at low temperature. This indicates that the two signals arise from different populations of centers. The differences in structure responsible for the two different EPR signals are probably minor since both kinds of centers are functional in cyclic charge accumulation and seem to be interconvertible. The difference between the two EPR signals, which arise from the same redox state of the same component (a mixed-valence manganese cluster), is proposed to be due to a spin-state change, where the g = 4.1 signal reflects an S = 3/2 state and the multiline signal an S = 1/2 state within the framework of the model of de Paula and Brudvig [de Paula, J. C., & Brudvig, G. W. (1985) J. Am. Chem. Soc. 107, 2643-2648]. The spin-state change induced by cryoprotectants is compared to that seen in the iron protein of nitrogenase.
Mutants affected in their pigment content and in the structure of their phycobilosomes (PBS) were isolated in the cyanobacterium Synechocystis PCC 6803 by enriching a population with the inhibitor p-hydroxymercuribenzoate. Three of these mutants, PMB 2, PMB 10 and PMB 11, with original phenotypes, are described. Applying several criteria of analysis (77K absorption and fluorescence, protein electrophoretic patterns, electron microscopy), it was possible to assign the component polypeptides to each substructure of the phycobilisome. The model structure obtained fits with those described in other species PMB 10 and PMB 11, completely lacking PC, are the first source of pure PBS cores available, in which no contamination by residual PC can be feared, and are thus particularly interesting for further biochemical studies. The capacity of genetic transformation of Synechocystis PCC 6803 by chromosomal DNA makes this system very convenient for the analysis of the regulation of synthesis of the PBS constituents.
We have studied the conditions required to reactivate oxygen evolution in NaCl-washed Photosystem-II particles. Restoration of oxygen evolution by Ca2+ revealed an heterogeneity in these Photosystem-II particles: 30% possess a low affinity site for Ca2+ (1–2 mM), 70% a high affinity site for Ca2+ (50–100 μM), even in the absence of the 24 kDa protein. The sole effect of the 24 kDa protein added back to Photosystem-II particles shortly before illumination was to stabilize oxygen evolution. Added back more than half an hour before, to Photosystem II particles at a high chlorophyll concentration, it increased oxygen evolution from approx. 40% of the control to 60–70% of the control. After reconstitution, an appreciable fraction of the low-affinity site for Ca2+ was still present.
In this work we describe a new phenotype of herbicide-resistant mutants. We have selected and characterized several metribuzin resistant mutants from Synechocystis 6714. We found that an increase in metribuzin resistance involved a cross-resistance with other herbicides. Therefore, the mutants could be classified in three groups: (1) metribuzin resistant; (2) atrazine and metribuzin resistant; (3) DCMU, atrazine and metribuzin resistant. Mutants which did not present cross-resistance were up to 25-fold more resistant to metribuzin than the wild type. We have studied the electron transfer properties of Photosystem II in these mutants using several techniques (oxygen emission, fluorescence, and thermoluminescence measurements). They presented modifications in the electron transfer between QA and QB, as was generally observed in most herbicide-resistant mutants previously studied. However, unexpectedly, one of these mutants, M30, presented a modified oscillatory pattern of oxygen emission. After dark adaptation the maximum of the oscillation was shifted by one flash. The matrix analysis indicated that the shifted maximum of the oxygen sequence corresponded to an increased S0 concentration in the dark-adapted state. In whole cells S0 and S1 are in equilibrium. This equilibrium is shifted in favor of S0 in the M30 mutant. The mutation renders the S-states more accessible to cell reductants.
The purification and properties of a new oxygen-evolving Photosystem (PS) II particle from the thermophilic blue-green alga Phormidium laminosum are described. The activity of the lauryldimethylamine N-oxide PS II-enriched supernatant described previously (Stewart, A.C. and Bendall, D.S. (1979) FEBS Lett. 107, 308–312) was found to be stabilized for several days at 4°;C by the addition of a second detergent, dodecyl-β-d-maltoside (lauryl maltoside). The lauryl maltoside/lauryldimethylamine N-oxide extract could be fractionated by sucrose density gradient centrifugation. Very high rates of oxygen evolution, typically 1900–2400 μmol O2/mg chlorophyll a per h at pH 7 with dimethylbenzoquinone and ferricyanide as acceptors, were observed for the lowest green band from the gradient. This fraction contained cytochromes b-559 (high-potential) and c-549, but was completely devoid of P-700 and cytochromes b-563 and f. The purified oxygen-evolving particles comprised seven major polypeptides (Mr 58 900, 52 400, 43 200, 33 900, 30 000, 16 000 and 15 000) and approximately five minor polypeptides. The particles contained 3–4 Mn atoms per reaction centre and had a chlorophyll antenna of approx. 50 chlorophyll a. The fast phase of fluorescence induction curves in the presence of hydroxylamine and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) could be described by an exponential, suggesting that no energy transfer was occurring between the PS II units responsible for this phase. Comparison of the area above the fluorescence induction curves in the absence and presence of DCMU suggested an acceptor pool size of 2–3 equivalents per centre.
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.
A correlation is demonstrated between the loss of the QA−Fe2+ EPR signal and the ability to photoinduce the radical-pair-recombination triplet state in Photosystem II. The QA−Fe2+ signal is diminished by procedures which are thought to reduce the semiquinone by a further electron: (1) low quantum yield photoreduction in the presence of sodium dithionite at room temperature; (2) chemical reduction in the dark by sodium dithionite at pH 7.0. The chemical reduction proeess is extremely slow (t1/2≈ 5 h) but can be accelerated (t1/2≈1.5 h) by the presence of the redox mediator, benzyl viologen. In redox titrations at pH. 7.0 the QA−Fe2+ signal disappears with an irreversible transition at potentials lower than −350 mV. The ability to observe the triplet signal shows a corresponding potential dependence. The variations in the amplitude of the triplet EPR signal match variations in triplet yield measured by flash absorption spectroscopy at low temperature. From these observations the following conclusions are drawn: (1) The redox titration data that led to the suggestion that an extra component functions between pheophytin and QA−Fe2+ (Evans, M.C.W., Atkinson, Y. E. and Ford, R. C. (1985) Biochim. Biophys. Acta 806, 247–254) can probably be explained instead by the second reduction of QA−Fe2+. (2) The variable yield of triplet and of P680+ Ph−, and possibly the lifetime of the latter, which have been reported in the literature probably reflect, at least in part, different amounts of native QA−Fe2+ remaining in the various preparations used. From considerations of the literature, and increase in quantum yield of charge separation is thought to occur upon the second reduction of QA−Fe2+. The most likely explanation for this is the disappearance of an electrostatic interaction between QA−Fe2+ and P680+Ph− as QA−Fe2+ becomes further reduced. Other factors which may influence or be responsible for these phenomena and comparisons with the primary photochemistry in purple bacteria are discussed. In addition the relevance of these observations to the lesions involved in photoinhibition is pointed out.
Exposure of highly resolved Photosystem II preparations to 2 M NaCl produces an 80% inhibition of oxygen-evolution activity concomitant with extensive loss of two water-soluble polypeptides (23 and 17 kDa). Addition of Ca2+ to salt-washed PS II membranes causes an acceleration in the decay of Z⨥, the primary donor to P-680+, and we show here that this acceleration is due to reconstitution of oxygen-evolution activity by Ca2+. Other cations (Mg2+, Mn2+, Sr2+) are much less effective in restoring oxygen evolution. On the basis of these observations we propose that Ca2+, perhaps in concert with the 23 kDa polypeptide, is an essential cofactor for electron transfer from the ‘S’-states to Z on the oxidizing side of PS II.
The orientation properties of the reaction centre triplet in Photosystem II (PS II) were determined. A high triplet yield was generated in PS II membranes by double reduction of the primary quinone electron acceptor QA. It is deduced that the triplet state is localised on a chlorophyll, the tetrapyrrolic plane of which is tilted at 30° to the membrane. A similar orientation was found in D1 / D2 / cyt b-559 particles demonstrating that the triplet is confined to the reaction centre. Optical work in the literature has been interpreted as indicating that the triplet is localised on a monomeric chlorophyll and that, in the singlet state, P680 consists of this molecule weakly coupled to a second chlorophyll. The weakness of the coupling, compared to the coupling in the special pair of purple bacteria, allows P680 to be considered as a monomer. Taking the optical data into account, we propose that P680 is a chlorophyll molecule oriented at 30° to the membrane. This result is discussed in terms of the structural analogy between PS II and the reaction centre of purple bacteria. A model is favored in which P680 is a chlorophyll, structurally analogous to one of the monomeric bacteriochlorophylls of the bacterial reaction centre. In addition, the orientation data indicate that this chlorophyll is rotated by 45° in its ring plane compared to the monomeric bacteriochlorophylls in the reaction centre of Rhodopseudomonas viridis.
CO2 depletion leads to an approximately 10-fold increase in the light-induced EPR signal at g = 1.82, attributed to the QA− · Fe2+ complex, in Photosystem II-enriched thylakoid membrane fragments. Upon reconstitution with HCO3−the signal decreases to the size in control samples. The split pheophytin− signal is broader in control or reconstituted than in CO2-depleted samples. It is concluded that HCO2− strongly influences the localization and conformation of the QA− · Fe+ complex. The QA− · Fe2+ and split pheophytirr− EPR signals from triazine-resistant Brassica napus were virtually identical to those from triazine-susceptible samples, indicating that the change in the 32-kDa azidoatrazine-binding protein does not lead to a confonnational change of the Qa− · Fe2+ complex.
THE evolution of oxygen as a result of light-driven water oxidation
occurs in plants and is catalysed by photosystem-II (PS-II). A
manganese-cluster probably acts both as the active site and as a
charge-accumulating device (for a review, see ref. 1). The enzyme cycle
involves five redox states which are denoted as
S0-S4, depend-ing on the number of positive
equivalents stored2. Oxygen is released after formation of
the transient S4 state. Ca2+ is an obligatory
cofactor in this process and its depletion inhibits the enzyme cycle at
the step before water oxidation, that is, after formation of the
S3 state3. In chelator-treated,
Ca2+-depleted PS-II, a new electron paramagnetic resonance
(EPR) signal arising from a formal S3 state has been
reported4. It was suggested that the S3 EPR signal
could originate from the oxidation of an amino acid, interacting
magnetically with the manganese cluster4. There are only a
few examples of amino-acid oxidation in enzyme chemistry and these are
limited to tyrosine5 and tryptophan6. Here we
report evidence that the S2 to S3 transition
occurring in Ca2+-depleted PS-II corresponds to the oxidation
of histidine.
The transformable cyanobacterium Synechocystis 6803 has a photosynthetic apparatus that is similar to that of plants. Because of the ease with which this organism can be genetically manipulated and isotopically labeled, Synechocystis has been used extensively in recent studies of electron transfer in the water-splitting complex, photosystem II. Here, we present the first EPR characterization of a highly active oxygen-evolving preparation from this organism. This preparation shows oxygen-evolution activities in the range from 2400-2600 mumol of O2/(mg of chlorophyll.h). We show that this preparation is stable enough for room temperature EPR studies. We then use this assay to show that the lineshapes of the D+ and Z+ tyrosine radicals are identical in this preparation, as has been observed in photosystem II complexes from a wide variety of photosynthetic species. We also present the first multiline EPR spectrum that has been observed from the Synechocystis manganese cluster.
The effect of protonation events on the charge equilibrium between tyrosine-D and the water-oxidizing complex in photosystem II has been studied by time-resolved measurements of the EPR signal IIslow at room temperature. The flash-induced oxidation of YD by the water-oxidizing complex in the S2 state is a monophasic process above pH 6.5 and biphasic at lower pHs, showing a slow and a fast phase. The half-time of the slow phase increases from about 1 s at pH 8.0 to about 20 s at pH 5.0, whereas the half-time of the fast phase is pH independent (0.4-1 s). The dark reduction of YD+ was followed by measuring the decay of signal IIslow at room temperature. YD+ decays in a biphasic way on the tens of minutes to hours time scale. The minutes phase is due to the electron transfer to YD+ from the S0 state of the water-oxidizing complex. The half-time of this process increases from about 5 min at pH 8.0 to 40 min at pH 4.5. The hours phase of YD+ has a constant half-time of about 500 min between pH 4.7 and 7.2, which abruptly decreases above pH 7.2 and below pH 4.7. This phase reflects the reduction of YD+ either from the medium or by an unidentified redox component of PSII in those centers that are in the S1 state. The titration curve of the half-times for the oxidation of YD reveals a proton binding with a pK around 7.3-7.5 that retards the electron transfer from YD to the water-oxidizing complex. We propose that this monoprotic event reflects the protonation of an amino acid residue, probably histidine-190 on the D2 protein, to which YD is hydrogen bonded. The titration curves for the oxidation of YD and for the reduction of YD+ show a second proton binding with pK approximately 5.8-6.0 that accelerates the electron transfer from YD to the water-oxidizing complex and retards the process in the opposite direction. This protonation most probably affects the water-oxidizing complex. From the measured kinetic parameters, the lowest limits for the equilibrium constants between the S0YD+ and the S1YD as well as between the S1YD+ and S2YD states were estimated to be 5 and 750-1000, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
To probe the involvement of amino acid residues of the D2 protein in the water-splitting process in photosystem II, site-directed mutagenesis was applied to identify D2 residues that might contribute to binding the Mn cluster involved in oxygen evolution. Mutation of Glu-69 to Gln or Val in D2 of the cyanobacterium Synechocystis sp. PCC 6803 was found to lead to a loss of photoautotrophic growth. However, in cells of the Gln mutant (E69Q) a significant Hill reaction rate could be observed upon the start of illumination, but the oxygen evolution rate declined with a half-time of approximately 1 min. Addition of 1 mM Mn2+ stabilized oxygen evolution in E69Q thylakoids. Other divalent cations were ineffective in specific stabilization. When the water-splitting system was bypassed, the rate of electron transport remained stable during illumination, indicating that the inactivation of oxygen evolution is localized in the water-splitting complex. We interpret these observations to indicate that Glu-69 is a Mn ligand and that the loss of oxygen evolution in the E69Q mutant upon turnover of PS II is initiated by changes in the Mn cluster, possibly leading to Mn release from the water-splitting complex. The addition of exogenous Mn to E69Q thylakoids may help to keep the Mn cluster active for a longer time, perhaps by providing Mn to rebind in the cluster after release of one Mn and before the Mn cluster had disintegrated.(ABSTRACT TRUNCATED AT 250 WORDS)
NaCl/EGTA-washing of photosystem II (PS-II) results in the removal of Ca2+ and the inhibition of oxygen evolution. Two new EPR signals were observed in such samples: a stable and modified S2 multiline signal and an S3 signal [(1989) Biochemistry 28, 8984-8989]. Here, we report what factors are responsible for the modifications of the S2 signal and the observation of the S3 signal. The following results were obtained. (i) The stable, modified, S2 multiline signal can be induced by the addition of high concentrations of EGTA or citrate to PS-II membranes which are already inhibited by Ca(2+)-depletion. (ii) The carboxylic acids act in the S3-state, are much less effective in S2 and have no effect in the S1-state. (iii) The extrinsic polypeptides (17- and 23-kDa) are not required to observe either the modified S2 signal or the S3 signal. However, they do influence the splitting and the lifetime of the S3 signal, and they seem to have a slight influence on the hyperfine pattern of the S2 signal. (iv) The S3 signal can be observed in Ca(2+)-depleted PS-II which does not exhibit the modified multiline signal. Then, it is proposed that formation of histidine radical during the S2 to S3 transition in Ca(2+)-depleted PS-II [(1990) Nature 347, 303-306] also occurs in functional PS-II.
A microscopic interaction model for a fully hydrated lipid bilayer membrane containing cholesterol is used to calculate, as a function of temperature and composition, the membrane area, the membrane hydrophobic thickness, and the average acyl-chain orientational order parameter, S. The order parameter, S, is related to the first moment, M1, of the quadrupolar magnetic resonance spectrum which can be measured for lipids with perdeuterated chains. On the basis of these model calculations as well as recent experimental measurements of M1 using magnetic resonance and of membrane area using micromechanical measurements, a discussion of the possible relationships between membrane area, hydrophobic thickness, and moments of nuclear magnetic resonance spectra is presented. It is pointed out that S under certain circumstances may be useful for estimating the hydrophobic membrane thickness. This is particularly advantageous for multicomponent membranes where structural data are difficult to obtain by using diffraction techniques. The usefulness of the suggested relationships is demonstrated for cholesterol-containing bilayers.
In photosystem II, electrons are sequentially extracted from water at a site containing Mn atoms and transferred through an intermediate carrier (Z) to the photooxidized reaction-center chlorophyll (P680+). Two polypeptides, D1 and D2, coordinate the primary photoreactants of the reaction center. Recently Debus et al. [Debus, R.J., Barry, B.A., Babcock, G.T., & McIntosh, L. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 427-430], have suggested that Z is a tyrosine residue located at position 161 of the D1 protein. To test this proposal, we have engineered a strain of the cyanobacterium Synechocystis PCC 6803 to produce a D1 polypeptide in which Tyr-161 has been replaced by phenylalanine. Wild-type Synechocystis PCC 6803 contains three nonidentical copies of the psbA gene which encode the D1 polypeptide. In the mutant strain, two copies were deleted by replacement with antibiotic-resistance genes, and site-directed mutations were constructed in a cloned portion of the remaining gene (psbA-3), carrying a third antibiotic-resistance gene downstream. Transformants were selected for antibiotic resistance and then screened for a photoautotrophy-minus phenotype. The mutant genotype was verified by complementation tests and by amplification and sequencing of genomic DNA. Cells of the mutant cannot evolve oxygen and, unlike the wild type, are unable to stabilize, with high efficiency, the charge-separated state in the presence of hydroxylamine and DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea]. Analyses by optical and EPR spectroscopy of reaction centers purified from this mutant indicate that Z can no longer be photooxidized and, instead, a chlorophyll cation radical, Chl+, is produced in the light. In the wild type, charge recombination between Z+ and the reduced primary quinone electron acceptor QA- occurs with a t1/2 of 80 ms. In the mutant, charge recombination between Chl+ and QA- occurs with a t1/2 of 1 ms. From these observations, we conclude that Z is indeed Tyr-161 of the D1 polypeptide.
Photosystem II enriched membranes were depleted of Ca2+ and the 17- and 23-kDa polypeptides by treatment with NaCl and EGTA. The 17- and 23-kDa polypeptides were then reconstituted. This preparation was incapable of O2 evolution until Ca2+ was added. An EPR study revealed the presence of two new EPR signals. One of these is a modified S2 multiline signal with an isotropic g value of 1.96 with at least 26 hyperfine peaks (average spacing 55 G) distributed over approximately 1600 G. The other is a near-Gaussian signal with an isotropic g value of 2.004, which is attributed to a formal S3 state. Experiments involving the interconversion of these signals and the effect of Ca2+ and Sr2+ rebinding provide evidence for these assignments. From these results the following conclusions are drawn: (1) These results are consistent with our earlier demonstration that charge accumulation is blocked after formation of S3 when Ca2+ is deficient. (2) Binding of the 17- and 23-kDa polypeptides to photosystem II in the absence of Ca2+ results in the perturbation of the Mn cluster. This is taken as a further indication that the Ca2+-binding site is close to or even an integral part of the Mn cluster. (3) The S3 signal may arise from an organic free radical interacting magnetically with the Mn cluster. However, other possible origins for this signal, including the Mn cluster itself, must also be considered.
EPR studies have revealed that removal of calcium using citric acid from the site in spinach photosystem II which is coupled to the photosynthetic O2-evolving process produces a structural change in the manganese cluster responsible for water oxidation. If done in the dark, this yields a modified S1' oxidation state which can be photooxidized above 250 K to form a structurally altered S2' state, as seen by formation of a "modified" multiline EPR signal. Compared to the "normal" S2 state, this new S2'-state EPR signal has more lines (at least 25) and 25% narrower 55Mn hyperfine splittings, indicative of disruption of the ligands to manganese. The calcium-depleted S2' oxidation state is greatly stabilized compared to the native S2 oxidation state, as seen by a large increase in the lifetime of the S2' EPR signal. Calcium reconstitution results in the reduction of the oxidized tyrosine residue 161YD+ (Em approximately 0.7-0.8 V, NHE) within the reaction center D1 protein in both the S1' and S2' states, as monitored by its EPR signal intensity. We attribute this to reduction by Mn. Thus a possible structural role which calcium plays is to bring YD+ into redox equilibrium with the Mn cluster. Photooxidation of S2' above 250 K produces a higher S state (S3 or S4) having a new EPR signal at g = 2.004 +/- 0.003 and a symmetric line width of 163 +/- 3 G, suggestive of oxidation of an organic donor, possibly an amino acid, in magnetic contact with the Mn cluster. This EPR signal forms in a stoichiometry of 1-2 relative to YD+.(ABSTRACT TRUNCATED AT 250 WORDS)
The light-driven water-splitting/oxygen-evolving enzyme remains one of the great enigmas of plant biology. However, due to the recent expansion of research efforts on this enzyme, it is grudgingly giving up some of its secrets.
The Mn donor complex in the S1 and S2 states and the iron-quinone acceptor complex (Fe2+-Q) in O2-evolving photosystem II (PS II) preparations from a thermophilic cyanobacterium, Synechococcus sp., have been studied with X-ray absorption spectroscopy and electron paramagnetic resonance (EPR). Illumination of these preparations at 220-240 K results in formation of a multiline EPR signal very similar to that assigned to a Mn S2 species observed in spinach PS II, together with g = 1.8 and 1.9 EPR signals similar to the Fe2+-QA- acceptor signals seen in spinach PS II. Illumination at 110-160 K does not produce the g = 1.8 or 1.9 EPR signals, nor the multiline or g = 4.1 EPR signals associated with the S2 state of PS II in spinach; however, a signal which peaks at g = 1.6 appears. The most probable assignment of this signal is an altered configuration of the Fe2+-QA- complex. In addition, no donor signal was seen upon warming the 140 K illuminated sample to 215 K. Following continuous illumination at temperatures between 140 and 215 K, the average X-ray absorption Mn K-edge inflection energy changes from 6550 eV for a dark-adapted (S1) sample to 6551 eV for the illuminated (S2) sample. The shift in edge inflection energy indicates an oxidation of Mn, and the absolute edge inflection energies indicate an average Mn oxidation state higher than Mn(II). Upon illumination a significant change was observed in the shape of the features associated with 1s to 3d transitions. The S1 spectrum resembles those of Mn(III) complexes, and the S2 spectrum resembles those of Mn(IV) complexes. The extended X-ray absorption fine structure (EXAFS) spectrum of the Mn complex is similar in the S1 and S2 states. Simulations indicate O or N ligands at 1.75 +/- 0.05 A, transition metal neighbor(s) at 2.73 +/- 0.05 A, which are assumed to be Mn, and terminal ligands which are probably N and O at a range of distances around 2.2 A. The Mn-O bond length of 1.75 A and the transition metal at 2.7 A indicate the presence of a di-mu-oxo-bridged Mn structure. Simulations indicate that a symmetric tetranuclear cluster is unlikely to be present, while binuclear, trinuclear, or highly distorted tetranuclear structures are possible. The striking similarity of these results to those from spinach PS II suggests that the structure of the Mn complex is largely conserved across evolutionarily diverse O2-evolving photosynthetic species.