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ABSTRACT: The semiquinone-iron complex of photosystem II was studied using electron spin resonance (ESR) spectroscopy and density functional theory calculations. Two forms of the signal were investigated: 1), the native g approximately 1.9 form; and 2), the g approximately 1.84 form, which is well known in purple bacterial reaction centers and occurs in photosystem II when treated with formate. The g approximately 1.9 form shows low- and high-field edges at g approximately 3.5 and g < 0.8, respectively, and resembles the g approximately 1.84 form in terms of shape and width. Both types of ESR signal were simulated using the theoretical approach used previously for the BRC complex, a spin Hamiltonian formalism in which the semiquinone radical magnetically interacts (J approximately 1 cm(-1)) with the nearby high-spin Fe(2+). The two forms of ESR signal differ mainly by an axis rotation of the exchange coupling tensor (J) relative to the zero-field tensor (D) and a small increase in the zero-field parameter D ( approximately 6 cm(-1)). Density functional theory calculations were conducted on model semiquinone-iron systems to identify the physical nature of these changes. The replacement of formate (or glutamate in the bacterial reaction centers) by bicarbonate did not result in changes in the coupling environment. However, when carbonate (CO(3)(2-)) was used instead of bicarbonate, the exchange and zero-field tensors did show changes that matched those obtained from the spectral simulations. This indicates that 1), the doubly charged carbonate ion is responsible for the g approximately 1.9 form of the semiquinone-iron signal; and 2), carbonate, rather than bicarbonate, is the ligand to the iron.
Biophysical Journal 10/2009; 97(7):2024-33. · 3.65 Impact Factor
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ABSTRACT: Low-temperature absorption and CD spectra, measured simultaneously, are reported from Photosystem II (PS II) reduced with sodium dithionite. Spectra were obtained using PS II core complexes before and after photoaccumulation of Pheo(D1)(-), the anion of the primary acceptor. For plant PS II, Pheo(D1)(-) was generated under conditions in which the primary plastoquinone was present as an anion (Q(A)(-)) and as a modified species taken to be the neutral doubly reduced hydroquinone (Q(A)H(2)). The bleaches observed upon Pheo(D1)(-) formation in the presence of Q(A)(-) are shifted to the blue compared those in the presence of Q(A)H(2). This is attributed to the influence of the charge on Q(A)(-), and this effect mirrors the well-known electrochromic effect of Q(A)(-) on the neutral pigments. The absorption bleaches induced by Pheo(D1) reduction are species dependent. Structured changes of the CD in the 680-690 nm spectral region are seen upon photoaccumulation of Pheo(D1)(-) in PS II from plant, Synechocystis and Thermosynechococcus vulcanus. These CD changes are shown to be consistent with the overall electronic assignments of Raszewski et al. [Raszewski et al. Biophys. J. 2008, 95, 105], which place the dominant Pheo(D1) excitation near 672 nm. CD changes associated with Pheo(D1) reduction are modeled to arise from the shift and intensity changes of two CD features: one predominately of Chl(D1) character, the other predominately Pheo(D2) in character. The assignments are also shown to account for the Q(Y) absorption changes in samples where the quinone is its charged (Q(A)(-)) and neutral (Q(A)H(2)) states.
The Journal of Physical Chemistry B 09/2009; 113(36):12364-74. · 3.70 Impact Factor
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Nicholas Cox,
Felix M Ho,
Naray Pewnim,
Ronald Steffen, Paul J Smith,
Kajsa G V Havelius,
Joseph L Hughes,
Lesley Debono,
Stenbjörn Styring,
Elmars Krausz,
Ron J Pace
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ABSTRACT: Detailed optical and EPR analyses of states induced in dark-adapted PS II membranes by cryogenic illumination permit characterization and quantification of all pigment derived donors and acceptors, as well as optically silent (in the visible, near infrared) species which are EPR active. Near complete turnover formation of Q(A)((-)) is seen in all centers, but with variable efficiency, depending on the donor species. In minimally detergent-exposed PS II membranes, negligible (<5%) oxidation of chlorophyll or carotenoid centers occurs for illumination temperatures 5-20 K. An optically silent electron donor to P680(+) is observed with the same decay kinetics as the S(1) split signal. Cryogenic donors to P680(+) seen are: (i) transient (t(1/2) approximately 150 s) tyrosine related species, including 'split signals' ( approximately 15% total centers), (ii) reduced cytochrome b(559) ( approximately 30-50% centers), and (iii) an organic donor, possibly an amino acid side chain, ( approximately 30% centers).
Biochimica et Biophysica Acta 04/2009; 1787(7):882-9. · 4.66 Impact Factor
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ABSTRACT: The stoichiometry of photosystem II to photosystem I reaction centres in spinach leaf segments was determined by two methods,
each capable of monitoring both photosystems in a given sample. One method, based on the fast electrochromic (EC) signal,
was applied to leaf segments, thereby avoiding potential artefacts associated with the isolation of thylakoids. Two variations
of the EC method were used, either suppression of PSII activity by prior photoinactivation or suppression PSI by photo- oxidation
of P700, gave the separate contribution of each photosystem to the fast EC signal. The PSII/PSI stoichiometries obtained by
the EC methods ranged from 1.5 to 1.8 for spinach, and 1.5 to 1.9 for two other plant species. A second method, based on electron
paramagnetic resonance (EPR), gave comparable values of 1.7–2.1 for spinach. A third method consisting of separate determination
of the contents of functional PSII by oxygen yield per single turnover flash and of P700 gave a PSII/PSI stiochiometry consistent
with above values. We conclude that the content of functional PSII is greater than that of PSI, and PSII/PSI reaction centre
ratios considerably higher than unity in typical higher plants.
12/2007: pages 7-10;
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ABSTRACT: Optical spectra of chemically reduced PSII core complexes isolated from spinach are presented. In these samples, QA is pre-reduced in darkness, allowing the photo-accumulation of its electron transfer pathway precursor, PheoD1 —. We report low-temperature (2200 K) spectral changes in circular dichroism (CD) and absorption spectra associated with
PheoD1 photo-reduction. The area of the narrow (2 nm FWHM) bleach at 683.8 nm is fully commensurate with that of an isolated Pheoa, indicating weak coupling to its neighboring pigment, the accessory chlorophyll ChlD1. Also, a highly structured, second-derivative-like pattern is seen in the change in the CD at 683.8 nm upon photoreduction.
This can be interpreted as indicative of a weak PheoD1-ChlD1interaction.
12/2007: pages 43-46;
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ABSTRACT: The stoichiometry of Photosystem II (PSII) to Photosystem I (PSI) reaction centres in spinach leaf segments was determined by two methods, each capable of being applied to monitor the presence of both photosystems in a given sample. One method was based on a fast electrochromic (EC) signal, which in the millisecond time scale represents a change in the delocalized electric potential difference across the thylakoid membrane resulting from charge separation in both photosystems. This method was applied to leaf segments, thus avoiding any potential artefacts associated with the isolation of thylakoid membranes. Two variations of this method, suppressing PSII activity by prior photoinactivation (in spinach and poplar leaf segments) or suppressing PSI by photo-oxidation of P700 (the chlorophyll dimer in PSI) with background far-red light (in spinach, poplar and cucumber leaf segments), each gave the separate contribution of each photosystem to the fast EC signal; the PSII/PSI stoichiometry obtained by this method was in the range 1.5-1.9 for the three plant species, and 1.5-1.8 for spinach in particular. A second method, based on electron paramagnetic resonance (EPR), gave values in a comparable range of 1.7-2.1 for spinach. A third method, which consisted of separately determining the content of functional PSII in leaf segments by the oxygen yield per single turnover-flash and that of PSI by photo-oxidation of P700 in thylakoids isolated from the corresponding leaves, gave a PSII/PSI stoichiometry (1.5-1.7) that was consistent with the above values. It is concluded that the ratio of PSII to PSI reaction centres is considerably higher than unity in typical higher plants, in contrast to a surprisingly low PSII/PSI ratio of 0.88, determined by EPR, that was reported for spinach grown in a cabinet under far-red-deficient light in Sweden [Danielsson et al. (2004) Biochim. Biophys. Acta 1608: 53-61]. We suggest that the low PSII/PSI ratio in the Swedish spinach, grown in far-red-deficient light with a lower PSII content, is not due to greater accuracy of the EPR method of measurement, as suggested by the authors, but is rather due to the growth conditions.
Biochimica et Biophysica Acta 09/2007; 1767(8):1064-72. · 4.66 Impact Factor
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ABSTRACT: We present the wavelength dependence of homogeneous holewidths of persistent spectral holes burnt in O2-evolving Photosystem II core complexes isolated from spinach, in the temperature range 2.5-8 K. The data supports the assignment that those chlorophylls which undergo persistent spectral hole-burning are specific CP43 and CP47-trap states that transfer their excitation energy to the reaction center. The lifetime-limited holewidths show that when PS II is in the S1(QA -) (closed) state, the CP43/CP47-trap states have excited-state lifetimes in the range from 70 to 270 ps. These lifetimes correspond to excitation transfer rates to the reaction center, and are far slower than required for models in which the PS II reaction center (P680) acts as a 'shallow-trap' for excitations. For wavelengths at which both traps absorb, the hole shape is clearly a composite of two Lorentzians, corresponding to hole-burning in both states simultaneously. The temperature dependence of the homogeneous holewidth does not follow the usual T1.3 dependence found in many chlorophyll-protein systems. Our data indicates T 2 temperature dependence, typically found in crystalline systems where the chromophore is coupled to localized phonon modes.
Photosynthesis Research 07/2005; 84(1-3):93-8. · 3.24 Impact Factor
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ABSTRACT: Low-temperature absorption and fluorescence spectra of fully active cores and membrane-bound PS II preparations are compared. Detailed temperature dependence of fluorescence spectra between 5 and 70 K are presented as well as 1.7-K fluorescence line-narrowed (FLN) spectra of cores, confirming that PS II emission is composite. Spectra are compared to those reported for LHCII, CP43, CP47 and D1/D2/cytit b559 subunits of PS II. A combination of subunit spectra cannot account for emission of active PS II. The complex temperature dependence of PS II fluorescence is interpretable by noting that excitation transfer from CP43 and CP47 to the reaction centre is slow, and strongly dependent on the precise energy at which a 'slow-transfer' pigment in CP43 or CP47 is located within its inhomogeneous distribution. PS II fluorescence arises from CP43 and CP47 'slow-transfer' states, convolved by this dependence. At higher temperatures, thermally activated excitation transfer to the PS II charge-separating system bypasses such bottlenecks. As the charge-separating state of active PS II absorbs at >700 nm, PS II emission in the 680-700 nm region is unlikely to arise from reaction centre pigments. PS II emission at physiological temperatures is discussed in terms of these results.
Photosynthesis Research 07/2005; 84(1-3):193-9. · 3.24 Impact Factor
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ABSTRACT: Active Photosystem II (PS II) cores were prepared from spinach, pea, Synechocystis PCC 6803, and Thermosynechococcus vulcanus, the latter of which has been structurally determined [Kamiya and Shen (2003) Proc Natl Acad Sci USA 100: 98-103]. Electrochromic shifts resulting from QA reduction by 1.7-K illumination were recorded, and the Qx and Qy absorption bands of the redox-active pheophytin a thus identified in the different organisms. The Qx transition is approximately 3 nm (100 cm-1) to higher energy in cyanobacteria than in the plants. The predominant Qy shift appears in the range 683-686 nm depending on species, and does not appear to have a systematic shift. Low-temperature absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of the chlorophyll Qy region are very similar in spinach and pea, but vary in cyanobacteria. We assigned CP43 and CP47 trap-chlorophyll absorption features in all species, as well as a P680 transition. Each absorption identified has an area of one chlorophyll a. The MCD deficit, introduced previously for spinach as an indicator of P680 activity, occurs in the same spectral region and has the same area in all species, pointing to a robustness of this as a signature for P680. MCD and CD characteristics point towards a significant variance in P680 structure between cyanobacteria, thermophilic cyanobacteria, and higher plants.
Photosynthesis Research 07/2005; 84(1-3):309-16. · 3.24 Impact Factor
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ABSTRACT: We report and compare highly resolved, simultaneously recorded absorption and CD spectra of active Photosystem II (PSII) samples in the range 440-750 nm. From an appropriately scaled comparison of spinach membrane fragment (BBY) and PSII core spectra, we show that key features of the core spectrum are quantitatively represented in the BBY data. PSII from the cyanobacterium Synechocystis 6803 display spectral features in the Qy region of comparable width (50-70 cm(-1) fwhm) to those seen in plant PSII but the energies of the resolved features are distinctly different. A comparison of spectra taken of PSII poised in the S1QA and S2QA(-) redox states reveals electrochromic shifts largely attributable to the influence of QA(-) on Pheo(D1). This allows accurate determinations of the Pheo(D1) Qy absorption positions to be at 685.0 nm for spinach cores, 685.8 nm for BBY particles, and 683.0 nm for Synechocystis. These are discussed in terms of earlier reports of the Pheo(D1) energies in PSII. The Qx transition of Pheo(D1) undergoes a blue shift upon Q(A) reduction, and we place a lower limit of 80 cm(-1) on this shift in plant material. By comparing the magnitude of the Stark shifts of the Qx and Qy bands of Pheo(D1), the directions of the transition-induced dipole moment changes, Deltamu(x) and Deltamu(y), for this functionally important pigment could be determined, assuming normal magnitudes of the Deltamu's. Consequently, Deltamu(x) and Deltamu(y) are determined to be approximately orthogonal to the directions expected for these transitions. Low-fluence illumination experiments at 1.7 K resulted in very efficient formation of QA(-). This was accompanied by cyt b(559) oxidation in BBYs and carotenoid oxidation in cores. No chlorophyll oxidation was observed. Our data allow us to estimate the quantum efficiency of PSII at this temperature to be of the order 0.1-1. No Stark shift associated with the S1-to-S2 transition of the Mn cluster is evident in our samples. The similarity of Stark data in plants and Synechocystis points to minimal interactions of Pheo(D1) with nearby chloropyll pigments in active PSII preparations. This appears to be at variance with interpretations of experiments performed with inactive solubilized reaction-center preparations.
Journal of the American Chemical Society 10/2003; 125(43):13063-74. · 9.91 Impact Factor
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ABSTRACT: Preparation of a minimum PSII core complex from spinach is described, containing four Mn per reaction center (RC) and exhibiting high O2 evolving activity [approximately 4000 micromol of O2 (mg of chl)(-1) x h(-1)]. The complex consists of the CP47 and CP43 chlorophyll binding proteins, the RC D1/D2 pair, the cytochrome b559 subunits, and the Mn-stabilizing psbO (33 kDa) protein, all present in the same stoichiometric amounts found in the parent PSII membranes. Several small subunits are also present. The cyt b559 content is 1.0 per RC in core complexes and PSII membranes. The total chlorophyll content is 32 chl a and <1 chl b per RC, the lowest yet reported for any active PSII preparation. The core complex exhibits the characteristic EPR signals seen in the S2 state of higher plant PSII. A procedure for preparing low-temperature samples of very high optical quality is developed, allowing detailed optical studies in the S1 and S2 states of the system to be made. Optical absorption, CD, and MCD spectra reveal unprecedented detail, including a prominent, well-resolved feature at 683.5 nm (14630 cm(-1)) with a weaker partner at 187 cm(-1) to higher energy. On the basis of band intensity, CD, and MCD arguments, these features are identified as the exciton split components of P680 in an intact, active reaction center special pair. Comparisons are made with solubilized D1/D2/cyt b559 material and cyanobacterial PSII.
Biochemistry 02/2002; 41(6):1981-9. · 3.42 Impact Factor
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ABSTRACT: The two forms of the g ≈ 4.1 signal recently identified in photosystem II (Smith, P. J.; Pace, R. J. Biochim. Biophys. Acta 1996, 1275, 213) have been simulated at several frequencies as near-axial spin 3/2 centers. In both cases, an explicit spin coupling model is assumed, involving two magnetically isolated Mn pairs, one for each signal type. For that pair assumed to give rise to the spin 1/2 multiline signal as the ground state, the modeling of the first-excited-state 4.1 signal gives estimates of the fine structure parameters for the individual Mn centers and the exchange coupling constant for the pair. The fine structure terms suggest that one Mn ion is a conventional MnIII ion in a highly axially distorted environment. The other Mn center, which is formally spin 3/2, is unlikely to be a conventional MnIV ion, but rather a MnIII−radical ligand pair, strongly antiferromagnetically coupled to give a net spin 3/2 state. The coupling between this Mn−radical center and the other MnIII is weak (J = −2.3 cm-1) in the absence of alcohol in the buffer medium, as determined earlier (Smith and Pace). The model is shown to be quantitatively consistent with the behavior of other signals proposed to arise from this coupled dimer. Comparison of our own data with those of others (Haddy, A.; et al. Biochim. Biophys. Acta 1992, 1099, 25−34) on one-dimensionally ordered photosystem II samples shows a generally consistent orientation of the molecular axis system for the dimer in the membrane plane. The second 4.1 signal, which exhibits ground-state behavior, may be simulated at X- and Q-band frequencies as an isolated system with D = +1.1 cm-1 and E/D = 0.037. The spin center is suggested to arise from a radical-bridged Mn homodimer, and the modeling parameters have been interpreted within this framework. The resulting proposal, involving two isolated dimers for the Mn organization within the oxygen evolving center, is critically examined in the light of recent work from other groups.
12/1998;
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ABSTRACT: We present the first report of highly efficient persistent spectral hole-burning in actiVe (oxygen-evolving) Photosystem II (PSII) preparations. Samples are poised in the S 1 state of the Kok cycle, with the primary quinone (Q A) either neutral or photoreduced to Q A -via a low-temperature pre-illumination. Remarkably efficient hole-burning is observed within the chlorophyll Q y (0,0) absorption envelope in the wavelength range of 676-695 nm. The hole-burning action spectrum of a sample poised in the S 1 (Q A -) state is dominated by a narrow feature (∼40 cm -1) at 684 nm, where hole depths of 30% are attainable. The photoproduct for spectral holes burnt in this region is distributed across the ∼50 cm -1 absorption feature centered at 683.5 nm, independent of the excitation wavelength within this band. Saturated hole-burning experiments indicate weak electron-phonon coupling near 684 nm but stronger coupling for holes burnt near 690 nm. Selective excitation near 690 nm of samples in the S 1 (Q A) state also results in efficient Q A -formation. Negligible hole-burning activity is observed at higher energies (<676 nm). Holewidths extrapolated to zero fluence and temperature are 2.0 (0.5 GHz near 685 nm for PSII samples in the S 1 (Q A -) state. Holewidths are twice as large and hole-burning quantum efficiencies are up to an order of magnitude greater (approaching 1%) for samples in the S 1 (Q A) state. We ascribe hole-burning near 684 nm to slow (40-210 ps) excitation transfer from a CP43 chlorophyll to the PSII reaction center, and we ascribe hole-burning at ∼690 nm to excitation transfer from a chlorophyll in CP47. The unusually high hole-burning efficiency that we observe is attributed to a mechanism that involves charge separation in the reaction center that follows excitation transfer from these "slow transfer" states in CP43 and CP47. A key result of this work is the observation that selective excitation in the range 685-695 nm leads to efficient charge separation, as indicated by Q A -formation. This indicates the presence of (a relatively weak) P680 absorption in a native PSII, extending to low energy and underlying the CP47 chlorophyll trap absorption.
