Temperature dependence of the oxidation kinetics of TyrZ and TyrD in oxygen-evolving photosystem II complexes throughout the range from 320K to 5K.

The photo-induced oxidation of TyrZ and TyrD by P680(•+), that involves both electron and proton transfer (PCET), has been studied in oxygen-evolving photosystem II from Thermosynechococcus elongatus. We used time-resolved absorption spectroscopy to measure the kinetics of P680(•+) reduction by tyrosine after the first flash given to dark-adapted PS II as a function of temperature and pH. The half-life of TyrZ oxidation by P680(•+) increases from 20ns at 300K to about 4µs at 150K. Analyzing the temperature dependence of the rate, one obtains a reorganization energy of about 770meV. Between 260K and 150K, the reduction of P680(•+) by TyrZ is increasingly replaced by charge recombination between P680(•+) and QA(•-). We propose that the driving force for TyrZ oxidation by P680(•+) decreases upon lowering the temperature. TyrZ oxidation cannot be excluded in a minority of PS II complexes at cryogenic temperatures. TyrD oxidation by P680(•+) with a half-life of about 30ns was observed at high pH. The pH dependence of the yield of TyrD oxidation can be described by a single protonable group with a pK of approximately 8.4. The rate of TyrD oxidation by P680(•+) is virtually identical upon substitution of solvent exchangeable protons with deuterons indicating that the rate is limited by electron transfer. The rate is independent of temperature between 5K and 250K. It is concluded that TyrD donates the electron to P680(•+) via PD2.

Spectroscopic properties of photosystem II core complexes from Thermosynechococcus elongatus revealed by single-molecule experiments.

In this study we use a combination of absorption, fluorescence and low temperature single-molecule spectroscopy to elucidate the spectral properties, heterogeneities and dynamics of the chlorophyll a (Chla) molecules responsible for the fluorescence emission of photosystem II core complexes (PS II cc) from the cyanobacterium Thermosynechococcus elongatus. At the ensemble level, the absorption and fluorescence spectra show a temperature dependence similar to plant PS II. We report emission spectra of single PS II cc for the first time; the spectra are dominated by zero-phonon lines (ZPLs) in the range between 680 and 705nm. The single-molecule experiments show unambiguously that different emitters and not only the lowest energy trap contribute to the low temperature emission spectrum. The average emission spectrum obtained from more than hundred single complexes shows three main contributions that are in good agreement with the reported bands F685, F689 and F695. The intensity of F695 is found to be lower than in conventional ensemble spectroscopy. The reason for the deviation might be due to the accumulation of triplet states on the red-most chlorophylls (e.g. Chl29 in CP47) or on carotenoids close to these long-wavelength traps by the high excitation power used in the single-molecule experiments. The red-most emitter will not contribute to the fluorescence spectrum as long as it is in the triplet state. In addition, quenching of fluorescence by the triplet state may lead to a decrease of long-wavelength emission.

Long-wavelength limit of photochemical energy conversion in Photosystem I.

In Photosystem I (PS I) long-wavelength chlorophylls (LWC) of the core antenna are known to extend the spectral region up to 750 nm for absorbance of light that drives photochemistry. Here we present clear evidence that even far-red light with wavelengths beyond 800 nm, clearly outside the LWC absorption bands, can still induce photochemical charge separation in PS I throughout the full temperature range from 295 to 5 K. At room temperature, the photoaccumulation of P700(+•) was followed by the absorbance increase at 826 nm. At low temperatures (T < 100 K), the formation of P700(+•)FA/B(-•) was monitored by the characteristic EPR signals of P700(+•) and FA/B(-•) and by the characteristic light-minus-dark absorbance difference spectrum in the QY region. P700 oxidation was observed upon selective excitation at 754, 785, and 808 nm, using monomeric and trimeric PS I core complexes of Thermosynechococcus elongatus and Arthrospira platensis, which differ in the amount of LWC. The results show that the LWC cannot be responsible for the long-wavelength excitation-induced charge separation at low temperatures, where thermal uphill energy transfer is frozen out. Direct energy conversion of the excitation energy from the LWC to the primary radical pair, e.g., via a superexchange mechanism, is excluded, because no dependence on the content of LWC was observed. Therefore, it is concluded that electron transfer through PS I is induced by direct excitation of a proposed charge transfer (CT) state in the reaction center. A direct signature of this CT state is seen in absorbance spectra of concentrated PS I samples, which reveal a weak and featureless absorbance band extending beyond 800 nm, in addition to the well-known bands of LWC (C708, C719 and C740) in the range between 700 and 750 nm. The present findings suggest that nature can exploit CT states for extending the long wavelength limit in PSI even beyond that of LWC. Similar mechanisms may work in other photosynthetic systems and in chemical systems capable of photoinduced electron transfer processes in general.

Investigating the photoprotective role of cytochrome b-559 in photosystem II in a mutant with altered ligation of the haem.

Despite many years of study, the physiological role of cytochrome b-559 (Cyt b-559) within the photosystem II (PSII) complex still remains unclear. Here we describe the analysis of a mutant of the green alga Chlamydomonas reinhardtii in which the His ligand to the haem, provided by the alpha subunit, has been replaced by a Cys residue. The mutant is unable to grow photoautotrophically but can assemble oxygen-evolving PSII supercomplexes to 15-20% of the levels found in the wild-type control. Haem is still detected in the isolated PSII supercomplexes but at sub-stoichiometric levels consistent with weaker binding to the mutated cytochrome. Analysis of PSII activity in cells indicates slowed electron transfer in the mutant between plastoquinones QA and QB. We show that PSII activity in the mutant is more sensitive to chronic photoinhibition than the WT control because of two effects: a faster rate of damage and an impaired PSII repair cycle at the level of synthesis and/or incorporation of D1 into PSII. We also demonstrate that Cyt b-559 plays a role during the critical stage of assembling the Mn4CaO5 cluster. Overall we conclude that Cyt b-559 optimises electron transfer on the acceptor side of PSII and plays physiologically important roles in the assembly, repair and maintenance of the complex.

Red antenna states of Photosystem I trimers from Arthrospira platensis revealed by single-molecule spectroscopy.

Single-molecule fluorescence spectroscopy at 1.4K was used to investigate the spectral properties of red (long-wavelength) chlorophylls in trimeric Photosystem I (PSI) complexes from the cyanobacterium Arthrospira platensis. Three distinct red antenna states could be identified in the fluorescence spectra of single PSI trimers from A. platensis in the presence of oxidized P700. Two of them are responsible for broad emission bands centered at 726 and 760nm. These bands are similar to those found in bulk fluorescence spectra measured at cryogenic temperatures. The broad fluorescence bands at ≅726 and ≅760nm belong to individual emitters that are broadened by strong electron-phonon coupling giving rise to a large Stokes-shift of about 20nm and rapid spectral diffusion. An almost perpendicular orientation of the transition dipole moments of F726 and F760 has to be assumed because direct excitation energy transfer does not occur between F726 and F760. For the first time a third red state assigned to the pool absorbing around 708nm could be detected by its zero-phonon lines. The center of the zero-phonon line distribution is found at ≅714nm. The spectral properties of the three red antenna states show a high similarity to the red antenna states found in trimeric PSI of Thermosynechoccocus elongatus. Based on these findings a similar organization of the red antenna states in PSI of these two cyanobacteria is discussed.

Fluorescence of the various red antenna states in photosystem I complexes from cyanobacteria is affected differently by the redox state of P700.

Photosystem I of cyanobacteria contains different spectral pools of chlorophylls called red or long-wavelength chlorophylls that absorb at longer wavelengths than the primary electron donor P700. We measured the fluorescence spectra at the ensemble and the single-molecule level at low temperatures in the presence of oxidized and reduced P700. In accordance with the literature, it was observed that the fluorescence is quenched by P700(+). However, the efficiency of the fluorescence quenching by oxidized P700(+) was found to be extremely different for the various red states in PS I from different cyanobacteria. The emission of the longest-wavelength absorbing antenna state in PS I trimers from Thermosynechococcus elongatus (absorption maximum at 5K: ≈ 719nm; emission maximum at 5K: ≈ 740nm) was found to be strongly quenched by P700(+) similar to the reddest state in PS I trimers from Arthrospira platensis emitting at 760nm at 5K. The fluorescence of these red states is diminished by more than a factor of 10 in the presence of oxidized P700. For the first time, the emission of the reddest states in A. platensis and T. elongatus has been monitored using single-molecule fluorescence techniques.

Structure and function of intact photosystem 1 monomers from the cyanobacterium Thermosynechococcus elongatus.

Until now, the functional and structural characterization of monomeric photosystem 1 (PS1) complexes from Thermosynechococcus elongatus has been hampered by the lack of a fully intact PS1 preparation; for this reason, the three-dimensional crystal structure at 2.5 A resolution was determined with the trimeric PS1 complex [Jordan, P., et al. (2001) Nature 411 (6840), 909-917]. Here we show the possibility of isolating from this cyanobacterium the intact monomeric PS1 complex which preserves all subunits and the photochemical activity of the isolated trimeric complex. Moreover, the equilibrium between these complexes in the thylakoid membrane can be shifted by a high-salt treatment in favor of monomeric PS1 which can be quantitatively extracted below the phase transition temperature. Both monomers and trimers exhibit identical posttranslational modifications of their subunits and the same reaction centers but differ in the long-wavelength antenna chlorophylls. Their chlorophyll/P700 ratio (108 for the monomer and 112 for the trimer) is slightly higher than in the crystal structure, confirming mild preparation conditions. Interaction of antenna chlorophylls of the monomers within the trimer leads to a larger amount of long-wavelength chlorophylls, resulting in a higher photochemical activity of the trimers under red or far-red illumination. The dynamic equilibrium between monomers and trimers in the thylakoid membrane may indicate a transient monomer population in the course of biogenesis and could also be the basis for short-term adaptation of the cell to changing environmental conditions.

Primary photophysical processes in photosystem II: bridging the gap between crystal structure and optical spectra.

This Minireview summarizes our current knowledge of the optical properties of photosystem II (PS-II) and how these properties are related to the photosynthetic function, that is, excitation energy transfer from the antenna complexes to the reaction center (RC) and the subsequent transmembrane charge separation in the latter. Interpretation of the optical spectra of PS-II is much more difficult than for the RC of purple bacteria, due to the "spectral congestion" problem, namely, the strong spectral overlap of optical bands in PS-II. Recent developments in deciphering the optical properties of the pigments in PS-II, the identification of functional states, and the kinetic details of the primary excitation energy and charge-transfer reactions are summarized. The spectroscopic term P(680) that is generally used in the literature no longer indicates the same entity in its cationic and singlet excited form but different subsets of the six innermost pigments of the RC. The accessory chlorophyll Chl(D1) forms a sink for singlet excitation and triplet energy and most likely represents the primary electron donor in PS-II. In this respect, a special chlorophyll monomer in PS-II plays the role of the special pair in purple bacteria. Evidence that exciton transfer between the core antenna complexes CP43 and CP47 and the RC is the bottleneck for the overall photochemical trapping of excitation energy in PS-II is discussed. A short summary is provided of PS-II of Acaryochloris marina, which mainly contains chlorophyll d instead of the usual chlorophyll a. This system does not suffer from the spectral congestion problem and, therefore, represents an interesting model system. The final part of this Minireview provides a discussion of challenging problems to be solved in the future.

Spectroscopic studies of the chlorophyll d containing photosystem I from the cyanobacterium, Acaryochloris marina.

Absorbance difference spectroscopy and redox titrations have been applied to investigate the properties of photosystem I from the chlorophyll d containing cyanobacterium Acaryochloris marina. At room temperature, the (P740(+)-P740) and (F(A/B)(-)-F(A/B)) absorbance difference spectra were recorded in the range between 300 and 1000 nm while at cryogenic temperatures, (P740(+)A(1)(-)-P740A(1)) and ((3)P740-P740) absorbance difference spectra have been measured. Spectroscopic and kinetic evidence is presented that the cofactors involved in the electron transfer from the reduced secondary electron acceptor, phylloquinone (A(1)(-)), to the terminal electron acceptor and their structural arrangement are virtually identical to those of chlorophyll a containing photosystem I. The oxidation potential of the primary electron donor P740 of photosystem I has been reinvestigated. We find a midpoint potential of 450+/-10 mV in photosystem I-enriched membrane fractions as well as in thylakoids which is very similar to that found for P700 in chlorophyll a dominated organisms. In addition, the extinction difference coefficient for the oxidation of the primary donor has been determined and a value of 45,000+/-4000 M(-1) cm(-1) at 740 nm was obtained. Based on this value the ratio of P740 to chlorophyll is calculated to be 1 : to approximately 200 chlorophyll d in thylakoid membranes. The consequences of our findings for the energetics in photosystem I of A. marina are discussed as well as the pigment stoichiometry and spectral characteristics of P740.

Introduction to optical methods in photosynthesis.

Optical spectroscopy is widely used to study structure and function of photosynthetic systems. Due to the large variety of different methods, these studies have contributed a lot to the identification of the cofactors involved in the primary reactions of photosynthesis and to the elucidation of the kinetics of the light-induced energy and electron transfer reactions. Within other aspects of photosynthesis research as e.g. photoinhibition, these techniques play an important role as well. In this brief introduction, I will focus on the basic principles of the different methods and the information obtained by applying these various techniques. In the reviews that follow, under the section "Optical Methods", these methods are discussed in detail.

Resonance Raman spectra of beta-carotene in solution and in photosystems revisited: an experimental and theoretical study.

The resonance Raman (RR) spectra of beta-carotene have been studied in solution and in the protein complexes of photosystems I and II (PS I, PS II). The experimental studies are complemented by density functional theory (DFT) calculations that allow for a consistent assignment of most of the experimental RR bands in the region between 900 and 1650 cm(-1). Thus, it was shown that the prominent peak at ca. 1525 cm(-1) is composed of two closely spaced modes, both of which dominated by C=C stretching coordinates of the polyene chain. These two modes exhibit different excitation profiles in the region of the allowed electronic S(0) --> S(2) transition with maxima that are separated by ca. 1300 cm(-1). Calculated RR spectra and excitation profiles obtained by time-dependent DFT in conjunction with the transform method indicate that the enhancement pattern of these modes cannot be rationalised within the Condon approximation (A-term scattering). Furthermore, symmetry considerations rule out enhancement via vibronic coupling. Instead, we suggest that the different excitation profiles of the two modes result from force constant changes and mode mixing upon electronic transition (Dushinsky rotation). RR spectra and excitation profiles are very similar in solution and in the protein complexes. Thus, the excitation-dependent frequency variation of the 1525-cm(-1) peak in PS I and PS II is an intrinsic molecular property of beta-carotene and does not reflect different pools of the pigments in specific molecular environments.

Spectroscopic properties of reaction center pigments in photosystem II core complexes: revision of the multimer model.

Absorbance difference spectra associated with the light-induced formation of functional states in photosystem II core complexes from Thermosynechococcus elongatus and Synechocystis sp. PCC 6803 (e.g., P(+)Pheo(-),P(+)Q(A)(-),(3)P) are described quantitatively in the framework of exciton theory. In addition, effects are analyzed of site-directed mutations of D1-His(198), the axial ligand of the special-pair chlorophyll P(D1), and D1-Thr(179), an amino-acid residue nearest to the accessory chlorophyll Chl(D1), on the spectral properties of the reaction center pigments. Using pigment transition energies (site energies) determined previously from independent experiments on D1-D2-cytb559 complexes, good agreement between calculated and experimental spectra is obtained. The only difference in site energies of the reaction center pigments in D1-D2-cytb559 and photosystem II core complexes concerns Chl(D1). Compared to isolated reaction centers, the site energy of Chl(D1) is red-shifted by 4 nm and less inhomogeneously distributed in core complexes. The site energies cause primary electron transfer at cryogenic temperatures to be initiated by an excited state that is strongly localized on Chl(D1) rather than from a delocalized state as assumed in the previously described multimer model. This result is consistent with earlier experimental data on special-pair mutants and with our previous calculations on D1-D2-cytb559 complexes. The calculations show that at 5 K the lowest excited state of the reaction center is lower by approximately 10 nm than the low-energy exciton state of the two special-pair chlorophylls P(D1) and P(D2) which form an excitonic dimer. The experimental temperature dependence of the wild-type difference spectra can only be understood in this model if temperature-dependent site energies are assumed for Chl(D1) and P(D1), reducing the above energy gap from 10 to 6 nm upon increasing the temperature from 5 to 300 K. At physiological temperature, there are considerable contributions from all pigments to the equilibrated excited state P*. The contribution of Chl(D1) is twice that of P(D1) at ambient temperature, making it likely that the primary charge separation will be initiated by Chl(D1) under these conditions. The calculations of absorbance difference spectra provide independent evidence that after primary electron transfer the hole stabilizes at P(D1), and that the physiologically dangerous charge recombination triplets, which may form under light stress, equilibrate between Chl(D1) and P(D1).

Steady-state and transient polarized absorption spectroscopy of photosystem I complexes from the cyanobacteria Arthrospira platensis and Thermosynechococcus elongatus.

Core antenna and reaction centre of photosystem I (PS I) complexes from the cyanobacteria Arthrospira platensis and Thermosynechococcus elongatus have been characterized by steady-state polarized absorption spectroscopy, including linear dichroism (LD) and circular dichroism (CD). CD spectra and the second derivatives of measured 77 K CD spectra reveal the spectral components found in the polarized absorption spectra indicating the excitonic origin of the spectral forms of chlorophyll in the PS I complexes. The CD bands at 669-670(+), 673(+), 680(-), 683-685(-), 696-697(-), and 711(-) nm are a common feature of used PSI complexes. The 77 K CD spectra of the trimeric PS I complexes exhibit also low amplitude components around 736 nm for A. platensis and 720 nm for T. elongatus attributed to red-most chlorophylls. The LD measurements indicate that the transition dipole moments of the red-most states are oriented parallel to the membrane plane. The formation of P700(+)A(1)(-) or (3)P700 was monitored by time-resolved difference absorbance and LD spectroscopy to elucidate the spectral properties of the PS I reaction centre. The difference spectra give strong evidence for the delocalization of the excited singlet states in the reaction centre. Therefore, P700 cannot be considered as a dimer but should be regarded as a multimer of the six nearly equally coupled reaction centre chlorophylls in accordance with structure-based calculations. On the basis of the results presented in this work and earlier work in the literature it is concluded that the triplet state is localized most likely on P(A), whereas the cation is localized most likely on P(B).

Both chlorophylls a and d are essential for the photochemistry in photosystem II of the cyanobacteria, Acaryochloris marina.

We have measured the flash-induced absorbance difference spectrum attributed to the formation of the secondary radical pair, P(+)Q(-), between 270 nm and 1000 nm at 77 K in photosystem II of the chlorophyll d containing cyanobacterium, Acaryochloris marina. Despite the high level of chlorophyll d present, the flash-induced absorption difference spectrum of an approximately 2 ms decay component shows a number of features which are typical of the difference spectrum seen in oxygenic photosynthetic organisms containing no chlorophyll d. The spectral shape in the near-UV indicates that a plastoquinone is the secondary acceptor molecule (Q(A)). The strong C-550 change at 543 nm confirms previous reports that pheophytin a is the primary electron acceptor. The bleach at 435 nm and increase in absorption at 820 nm indicates that the positive charge is stabilized on a chlorophyll a molecule. In addition a strong electrochromic band shift, centred at 723 nm, has been observed. It is assigned to a shift of the Qy band of the neighbouring accessory chlorophyll d, Chl(D1). It seems highly likely that it accepts excitation energy from the chlorophyll d containing antenna. We therefore propose that primary charge separation is initiated from this chlorophyll d molecule and functions as the primary electron donor. Despite its lower excited state energy (0.1 V less), as compared to chlorophyll a, this chlorophyll d molecule is capable of driving the plastoquinone oxidoreductase activity of photosystem II. However, chlorophyll a is used to stabilize the positive charge and ultimately to drive water oxidation.

P700+- and 3P700-induced quenching of the fluorescence at 760 nm in trimeric Photosystem I complexes from the cyanobacterium Arthrospira platensis.

The 5 K absorption spectrum of Photosystem I (PS I) trimers from Arthrospira platensis (old name: Spirulina platensis) exhibits long-wavelength antenna (exciton) states absorbing at 707 nm (called C707) and at 740 nm (called C740). The lowest energy state (C740) fluoresces around 760 nm (F760) at low temperature. The analysis of the spectral properties (peak position and line width) of the lowest energy transition (C740) as a function of temperature within the linear electron-phonon approximation indicates a large optical reorganization energy of approximately 110 cm(-1) and a broad inhomogeneous site distribution characterized by a line width of approximately 115 cm(-1). Linear dichroism (LD) measurements indicate that the transition dipole moment of the red-most state is virtually parallel to the membrane plane. The relative fluorescence yield at 760 nm of PS I with P700 oxidized increases only slightly when the temperature is lowered to 77 K, whereas in the presence of reduced P700 the fluorescence yield increases nearly 40-fold at 77 K as compared to that at room temperature (RT). A fluorescence induction effect could not be resolved at RT. At 77 K the fluorescence yield of PS I trimers frozen in the dark in the presence of sodium ascorbate decreases during illumination by about a factor of 5 due to the irreversible formation of (P700+)F(A/B-) in about 60% of the centers and the reversible accumulation of the longer-lived state (P700+)FX-. The quenching efficiency of different functionally relevant intermediate states of the photochemistry in PS I has been studied. The redox state of the acceptors beyond A(0) does not affect F760. Direct kinetic evidence is presented that the fluorescence at 760 nm is strongly quenched not only by P700+ but also by 3P700. Similar kinetics were observed for flash-induced absorbance changes attributed to the decay of 3P700 or P700+, respectively, and flash-induced fluorescence changes at 760 nm measured under identical conditions. A nonlinear relationship between the variable fluorescence around 760 nm and the [P700red]/[P700total] ratio was derived from titration curves of the absorbance change at 826 nm and the variable fluorescence at 760 nm as a function of the redox potential imposed on the sample solution at room temperature before freezing. The result indicates that the energy exchange between the antennae of different monomers within a PS I trimer stimulates quenching of F760 by P700+.

Electric field effects on red chlorophylls, beta-carotenes and P700 in cyanobacterial Photosystem I complexes.

We have probed the absorption changes due to an externally applied electric field (Stark effect) of Photosystem I (PSI) core complexes from the cyanobacteria Synechocystis sp. PCC 6803, Synechococcus elongatus and Spirulina platensis. The results reveal that the so-called C719 chlorophylls in S. elongatus and S. platensis are characterized by very large polarizability differences between the ground and electronically excited states (with Tr(Deltaalpha) values up to about 1000 A(3) f(-2)) and by moderately high change in permanent dipole moments (with average Deltamu values between 2 and 3 D f(-1)). The C740 chlorophylls in S. platensis and, in particular, the C708 chlorophylls in all three species give rise to smaller Stark shifts, which are, however, still significantly larger than those found before for monomeric chlorophyll. The results confirm the hypothesis that these states originate from strongly coupled chlorophyll a molecules. The absorption and Stark spectra of the beta-carotene molecules are almost identical in all complexes and suggest similar or slightly higher values for Tr(Deltaalpha) and Deltamu than for those of beta-carotene in solution. Oxidation of P700 did not significantly change the Stark response of the carotenes and the red antenna states C719 and C740, but revealed in all PSI complexes changes around 700-705 and 690-693 nm, which we attribute to the change in permanent dipole moments of reduced P700 and the chlorophylls responsible for the strong absorption band at 690 nm with oxidized P700, respectively.

Optical properties, excitation energy and primary charge transfer in photosystem II: theory meets experiment.

In this review we discuss structure-function relationships of the core complex of photosystem II, as uncovered from analysis of optical spectra of the complex and its subunits. Based on descriptions of optical difference spectra including site directed mutagenesis we propose a revision of the multimer model of the symmetrically arranged reaction center pigments, described by an asymmetric exciton Hamiltonian. Evidence is provided for the location of the triplet state, the identity of the primary electron donor, the localization of the cation and the secondary electron transfer pathway in the reaction center. We also discuss the stationary and time-dependent optical properties of the CP43 and CP47 subunits and the excitation energy transfer and trapping-by-charge-transfer kinetics in the core complex.

Red antenna states of photosystem I from Synechococcus sp. PCC 7002.

Absorption, fluorescence and single-molecule spectroscopy at low temperatures were used to elucidate spectral properties, heterogeneities and dynamics of the red-shifted chlorophyll a (Chla) molecules responsible for the fluorescence in photosystem I (PSI) from the cyanobacterium Synechoccocus sp. PCC 7002. The 77 K absorption spectrum indicates the presence of 2-3 red-shifted Chla's absorbing at about 708 nm. The fluorescence emission spectrum is dominated by a broad band at 714 nm. The emission spectra of single PSI complexes show zero-phonon lines (ZPLs) as well as a broad intensity distribution without ZPLs. The spectral region below 710 nm often shows ZPLs, they form a spectral band with a maximum at 698 nm (F698). The region above 710 nm is dominated by broad intensity distributions and the observation of ZPLs is less frequent. The broad distributions are due to the emission of the C708 Chla's and the emission from F698 stems from a Chla species absorbing at the blue side of P700. The properties of these two emissions show a close relation to those of the C708 and C719 pools observed in T. elongatus. Therefore an assignment of F698 and C708 to Chla-species with similarities to C708 and C719 in T. elongatus is proposed.

Site-directed mutations at D1-Thr179 of photosystem II in Synechocystis sp. PCC 6803 modify the spectroscopic properties of the accessory chlorophyll in the D1-branch of the reaction center.

D1-Thr179, which overlies the reaction center chlorophyll Chl D1 of Photosystem II was replaced with His and Glu through site-directed mutation in Synechocystis sp. PCC 6803. Spectroscopic characterization of the mutants indicates that, compared to wild type, the main bleaching in the triplet-minus-singlet absorbance difference spectrum and the electrochromic band shift in the (P680 (+)Q A (-)-P680Q A) absorbance difference spectrum are displaced to the red by approximately 2 nm in the D1-Thr179His mutant and to the blue by approximately 1 nm in the D1-Thr179Glu mutant. These difference spectra are compared with the absorbance difference spectra, measured on the same states in the D1-His198Gln mutant in which the axial ligand D1-His198 of the special pair chlorophyll, P D1, was replaced by glutamine. Together, these results give direct evidence that (a) the reaction center triplet state, produced upon charge recombination from (3)[P (+)Pheo (-)], is primarily localized on Chl D1; (b) the cation of the oxidized donor P (+) is predominantly localized on chlorophyll P D1 of the special pair; and (c) the Q Y band of the accessory chlorophyll Chl D1 is electrochromically shifted in response to charges on P (+) and Q A (-). Light-induced absorbance difference spectra (between 650 and 710 nm), associated with the oxidation of secondary donors and the reduction of Q A, exhibit a bleaching attributed to the oxidation of a Chl Z and strong electrochromic band shifts. On the basis of mutation-induced spectroscopic changes and of structure-based calculations, we conclude that the experimental spectra are best explained by a blue-shift of the Q Y band of the accessory chlorophyll Chl D1, arising from charges on Car D2 (+) and Chl ZD2 (+) and on reduced Q A.

Site-directed mutations at D1-His198 and D1-Thr179 of photosystem II in Synechocystis sp. PCC 6803: deciphering the spectral properties of the PSII reaction centre.

Site-directed mutations were constructed in photosystem II of Synechocystis sp. PCC6803 in which the axial ligand, D1-His198, of special pair chlorophyll PD1 was replaced with Gln and where D1-Thr179, which overlies monomeric chlorophyll ChlD1, was replaced with His. The D1-His198Gln mutation produces a 3nm displacement to the blue of the bleaching minimum in the Soret and in the Qy region of the (P+QA--PQA) absorbance difference spectrum. To a first approximation, the bleaching can be assigned to the low-energy exciton transition of the special pair chlorophylls PD1/PD2. The D1-Thr179His mutation produces a 2nm displacement to the red of the bleaching minimum in the Qy region of the (3P-1P) absorbance difference spectrum. Analysis of the flash-induced (P+QA--PQA) and (3P-1P) absorbance difference spectra of both mutants compared with wild-type at 80K indicate that the cation of the oxidized donor P+ is predominantly localized on the chlorophyll PD1 of the special pair and that the reaction centre triplet state, produced upon charge recombination from 3[P+Pheo-], when the primary quinone electron acceptor QA is doubly reduced, is primarily localized on ChlD1.