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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.

Photosystem II (PSII) mutants are useful experimental tools to trap potential intermediates involved in the assembly of the oxygen-evolving PSII complex. Here, we focus on the subunit composition of the RC47 assembly complex that accumulates in a psbC null mutant of the cyanobacterium Synechocystis sp. PCC 6803 unable to make the CP43 apopolypeptide. By using native gel electrophoresis, we showed that RC47 is heterogeneous and mainly found as a monomer of 220 kDa. RC47 complexes co-purify with small Cab-like proteins (ScpC and/or ScpD) and with Psb28 and its homologue Psb28-2. Analysis of isolated His-tagged RC47 indicated the presence of D1, D2, the CP47 apopolypeptide, plus nine of the 13 low-molecular-mass (LMM) subunits found in the PSII holoenzyme, including PsbL, PsbM and PsbT, which lie at the interface between the two momomers in the dimeric holoenzyme. Not detected were the LMM subunits (PsbK, PsbZ, Psb30 and PsbJ) located in the vicinity of CP43 in the holoenzyme. The photochemical activity of isolated RC47-His complexes, including the rate of reduction of P680 + , was similar to that of PSII complexes lacking the Mn 4 CaO 5 cluster. The implications of our results for the assembly and repair of PSII in vivo are discussed.

Single-molecule fluorescence spectroscopy at 1.4 K 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 760 nm. These bands are similar to those found in bulk fluorescence spectra measured at cryogenic temperatures. The broad fluorescence bands at ≅ 726 and ≅ 760 nm belong to individual emitters that are broadened by strong electron–phonon coupling giving rise to a large Stokes-shift of about 20 nm 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 708 nm could be detected by its zero-phonon lines. The center of the zero-phonon line distribution is found at ≅ 714 nm. 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.

Circular dichroism (CD) spectra of photosystem I (PSI) complexes of the cyanobacteria Thermo� synechococcus elongatus, Arthrospira platensis and Synechocystis sp. PCC 6803 were studied. CD spectra of darkadapted PSI trimers and monomers, measured at 77 K, show common bands at 669�670(+), 673(+), 680(-), 683-685(-), 696-697(-), 702(-) and 711(-) nm. The intensities of these bands are species specific. In addition, bands at 683-685(-) and 673(+) nm differ in intensity for trimeric and monomeric PSI com� plexes. CD difference spectra (P700 + -P700) of PSI complexes at 283 K exhibit conservative bands at 701(-) and 691(+) nm due to changes in resonance interaction of chlorophylls in the reaction center upon oxidation of P700. Additional bands are observed at 671(-), 678(+), 685(-), 693(-) nm and in the region 720-725 nm those intensities correlate with intensities of analogous bands of antenna chlorophylls in darkadapted CD spectra. It is suggested that the variability of CD difference spectra of PSI complexes is determined by changes in resonance interaction of reaction center chlorophylls with closely located antenna chlorophylls.

We have studied the resonance Raman spectra of β-carotene in solution and in protein-complexes using laser excitation at various wavelengths in the visible region. Main focus was laid on the v 1 -band, which shows an anomalous behaviour upon variation of the excitation wavelength resulting in a frequency shift of the peak maximum. This shift is due to different excitation profiles of two closely spaced modes as shown previously for β-carotene in solution. In this work, we have extended the studies to the antenna pigment in photosystems I and II (PS I and PS II).

In the present work we have studied β-carotene dissolved in dichloromethane by means of resonance Raman spectroscopy. To obtain the Raman excitation profiles Raman spectra of /3-carotene have been acquired at various wavelengths throughout the visible region. It was found that the position of the prominent peak at ca. 1524 cm -1 varies with the excitation wavelength indicating the involvement of two different modes. Possible origins of the different enhancement pattern of the two modes are discussed.

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.

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).

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 (QA). 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, ChlD1. 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.

The photo-induced oxidation of Tyr Z and Tyr D 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 Tyr Z oxidation by P680 •+ increases from 20 ns at 300 K to about 4 μs at 150 K. Analyzing the temperature dependence of the rate, one obtains a reorganization energy of about 770 meV. Between 260 K and 150 K, the reduction of P680 •+ by Tyr Z is increasingly replaced by charge recombination between P680 •+ and Q A •− . We propose that the driving force for Tyr Z oxidation by P680 •+ decreases upon lowering the temperature. Tyr Z oxidation cannot be excluded in a minority of PS II complexes at cryogenic temperatures. Tyr D oxidation by P680 •+ with a half-life of about 30 ns was observed at high pH. The pH dependence of the yield of Tyr D oxidation can be described by a single protonable group with a pK of approximately 8.4. The rate of Tyr D 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 5 K and 250 K. It is concluded that Tyr D donates the electron to P680 •+ via P D2 .

Photosystem II (PSII) performs one of the key reactions on our planet: the light-driven oxidation of water. This fundamental but very complex process requires PSII to act in a highly coordinated fashion. Despite detailed structural information on the fully assembled PSII complex, the dynamic aspects of formation, processing, turnover, and degradation of PSII with at least 19 subunits and various cofactors are still not fully understood. Transient complexes are especially difficult to characterize due to low abundance, potential heterogeneity, and instability. Here, we show that Psb27 is involved in the assembly of the water-splitting site of PSII and in the turnover of the complex. Psb27 is a bacterial lipoprotein with a specific lipid modification as shown by matrix-assisted laser-desorption ionization time of flight mass spectrometry. The combination of HPLC purification of four different PSII subcomplexes and (15)N pulse label experiments revealed that lipoprotein Psb27 is part of a preassembled PSII subcomplex that represents a distinct intermediate in the repair cycle of PSII.

In this work, we present the two-photon excitation spectrum and two-photon-sensitized fluorescence spectra of photosystem I (PS I) of Thermosynechococcus elongatus. The two-photon excitation spectr...

We applied optical spectroscopy, magnetic resonance techniques, and redox titrations to investigate the properties of the primary electron donor P700 in photosystem I (PS I) core complexes from cyanobacteria (Thermosynechococcus elongatus, Spirulina platensis, and Synechocystis sp. PCC 6803), algae (Chlamydomonas reinhardtii CC2696), and higher plants (Spinacia oleracea). Remarkable species-specific differences of the optical properties of P700 were revealed monitoring the (3P700–P700) and (P700+·–P700) absorbance and CD difference spectra. The main bleaching band in the Qy region differs in peak position and line width for the various species. In cyanobacteria the absorbance of P700 extends more to the red compared with algae and higher plants which is favorable for energy transfer from red core antenna chlorophylls to P700 in cyanobacteria. The amino acids in the environment of P700 are highly conserved with two distinct deviations. In C. reinhardtii a Tyr is found at position PsaB659 instead of a Trp present in all other organisms, whereas in Synechocystis a Phe is found instead of a Trp at the homologous position PsaA679. We constructed several mutants in C. reinhardtii CC2696. Strikingly, no PS I could be detected in the mutant YW B659 indicating steric constraints unique to this organism. In the mutants WA A679 and YA B659 significant changes of the spectral features in the (3P700–P700), the (P700+·–P700) absorbance difference and in the (P700+·–P700) CD difference spectra are induced. The results indicate structural differences among PS I from higher plants, algae, and cyanobacteria and give further insight into specific protein-cofactor interactions contributing to the optical spectra.

The structure of photosystem I from the thermophilic cyanobacterium Synechococcus elongatus has been recently resolved by x-ray crystallography to 2.5-A resolution. Besides the reaction center, photosystem I consists also of a core antenna containing 90 chlorophyll and 22 carotenoid molecules. It is their function to harvest solar energy and to transfer this energy to the reaction center (RC) where the excitation energy is converted into a charge separated state. Methods of steady-state optical spectroscopy such as absorption, linear, and circular dichroism have been applied to obtain information on the spectral properties of the complex, whereas transient absorption and fluorescence studies reported in the literature provide information on the dynamics of the excitation energy transfer. On the basis of the structure, the spectral properties and the energy transfer kinetics are simultaneously modeled by application of excitonic coupling theory to reveal relationships between structure and function. A spectral assignment of the 96 chlorophylls is suggested that allows us to reproduce both optical spectra and transfer and emission spectra and lifetimes of the photosystem I complex from S. elongatus. The model calculation allowed to study the influence of the following parameters on the excited state dynamics: the orientation factor, the heterogeneous site energies, the modifications arising from excitonic coupling (redistribution of oscillator strength, energetic splitting, reorientation of transition dipoles), and presence or absence of the linker cluster chlorophylls between antenna and reaction center. For the Förster radius and the intrinsic primary charge separation rate, the following values have been obtained: R(0) = 7.8 nm and k(CS) = 0.9 ps(-1). Variations of these parameters indicate that the excited state dynamics is neither pure trap limited, nor pure transfer (to-the-trap) limited but seems to be rather balanced.

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.

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.

Photosystem I (PS-I) contains a small fraction of chlorophylls (Chls) that absorb at wavelengths longer than the primary electron donor P700. The total number of these long wavelength Chls and their spectral distribution are strongly species dependent. In this contribution we present room temperature time-resolved fluorescence data of five PS-I core complexes that contain different amounts of these long wavelength Chls, i.e., monomeric and trimeric photosystem I particles of the cyanobacteria Synechocystis sp. PCC 6803, Synechococcus elongatus, and Spirulina platensis, which were obtained using a synchroscan streak camera. Global analysis of the data reveals considerable differences between the equilibration components (3.4 -15 ps) and trapping components (23-50 ps) of the various PS-I complexes. We show that a relatively simple compartmental model can be used to reproduce all of the observed kinetics and demonstrate that the large kinetic differences are purely the result of differences in the long wavelength Chl content. This procedure not only offers rate constants of energy transfer between and of trapping from the compartments, but also well-defined room temperature emission spectra of the individual Chl pools. A pool of red shifted Chls absorbing around 702 nm and emitting around 712 nm was found to be a common feature of all studied PS-I particles. These red shifted Chls were found to be located neither very close to P700 nor very remote from P700. In Synechococcus trimeric and Spirulina monomeric PS-I cores, a second pool of red Chls was present which absorbs around 708 nm, and emits around 721 nm. In Spirulina trimeric PS-I cores an even more red shifted second pool of red Chls was found, absorbing around 715 nm and emitting at 730 nm.

The dynamics of triplet excited states in light harvesting complexes from a range of different purple bacteria have been investigated by time-resolved EPR and optical absorption spectroscopy. The spin-polarized EPR spectra show for the LH2 complexes from Rhodobacter sphaeroides strains 2.4.1 and G1C at early times after light excitation a superposition of bacteriochlorophyll and carotenoid triplet states. In LH1 complexes from Rhodospirillum rubrum and LH2 complexes from Chromatium purpuratum and Rhodopseudomonas acidophila, only a carotenoid triplet spectrum is observed. In all complexes from every species, a time-dependent change in the polarization pattern of the carotenoid triplet is seen. In those complexes that show a superposition of bacteriochlorophyll and carotenoid triplets, the decay of the bacteriochlorophyll triplet is accompanied by an apparently simultaneous rise of the carotenoid triplet. Transient optical absorption spectroscopy demonstrates identical kinetics for the decay and rise of ba...

Two de noo synthesized proteins that form water-soluble four-helix bundle structures were constructed based on the modular synthesis strategy of template-assembled synthetic proteins. In the hydrophobic interior of these modular proteins one bis-histidine binding site was introduced to incorporate a single heme group or other metalloporphyrin cofactors. The exchange of two tryptophan against two arginine residues near the heme-binding site leads to variations in the stability of the modular proteins. The structural characterization of the de noo synthesized heme proteins by EPR spectroscopy revealed a heterogeneous binding situation of the cofactor in both systems. The heme group is coordinated by two axial histidine ligands, whose planes are oriented either parallel or twisted relative to each other. In one of the two modular proteins this heterogeneity was reduced by addition of guanidinium hydrochloride that aided in the proper folding of the heme-binding site. By using different central metals for the protoporphyrin IX cofactor (Fe3+, Co3+, Zn2+) different functional properties were introduced into this modular protein. While the Fe3+ porphyrin is easily reduced, the reduction of Co3+ porphyrin is very difficult and finally leads to the release of this metalloporphyrin cofactor into the aqueous surrounding. By incorporating a porphyrin with a Zn2+ central metal a modular protein was obtained that was capable of acting as electron donor in light induced triplet electron transfer.

Two histidines provide the axial ligands of the two chlorophyll a (Chl a) molecules which form the primary electron donor (P700) of photosystem I (PSI). Histidine 676 in the protein subunit PsaA, His(A676), and histidine 656 in subunit PsaB, His(B656), were replaced in the green algae Chlamydomnas reinhardtii by site-directed mutagenesis with nonpolar, uncharged polar, acidic, and basic amino acid residues. Only the substitutions with uncharged polar residues led to a significant accumulation of PSI in the thylakoid membranes. These PSI complexes were isolated and the physical properties of the primary donor characterized. The midpoint potential of P700(+)(*)/P700 was increased in all mutants (up to 140 mV) and showed a dependence on size and polarizability of the residues when His(B656) was substituted. In the light-minus-dark absorbance spectra, all mutations in PsaB exhibited an additional bleaching band at 665 nm at room temperature comparable with the published spectrum for the replacement of His(B656) with asparagine [Webber, A. N., Su Hui, Bingham, S. E., Käss, H., Krabben, L., Kuhn, M., Jordan, R., Schlodder, E., and Lubitz, W. (1996) Biochemistry 35, 12857-12863]. Substitutions of His(A676) showed an additional shoulder around 680 nm. In the low-temperature absorbance difference spectra of P700(+)(*)/P700, a blue shift of the main bleaching band by 2 nm and some changes in the spectral features around 660 nm were observed for mutations of His(B656) in PsaB. The analogous substitution in PsaA showed only a shift of the main bleaching band. Similar effects of the mutations were found in the (3)P700/P700 absorbance difference spectra at low temperatures (T = 2 K). The zero-field splitting parameters of (3)P700 were not significantly changed in the mutated PSI complexes. The electron spin density distribution of P700(+)(*), determined by ENDOR spectroscopy, was only changed when His(B656) was replaced. In all measurements, two general observations were made. (i) The replacement of His(B656) had a much stronger impact on the physical properties of P700 than the mutation of His(A676). (ii) The exchange of His(B656) with glutamine induces the smallest changes in the spectra or the midpoint potential, whereas the other replacements exhibited a stronger but very similar influence on the spectroscopic features of P700. The data provide convincing evidence that the unpaired electron in the cation radical and the triplet state of P700 are mainly localized on the Chl a of the dimer which is axially coordinated by His(B656).

The photochemically trapped bacteriopheophytin (BPh) b radical anion in the active branch (Φ A − ) of reaction centers (RCs) from Blastochloris (formerly called Rhodopseudomonas ) viridis is characterized by 1 H-ENDOR as well as optical absorption spectroscopy. The two site-directed mutants YF(M208) and YL(M208), in which tyrosine at position M208 is replaced by phenylalanine and leucine, respectively, are investigated and compared with the wild type. The residue at M208 is in close proximity to the primary electron donor, P, the monomeric bacteriochlorophyll (BChl), B A , and the BPh, Φ A , that are involved in the transmembrane electron transfer to the quinone, Q A , in the RC. The analysis of the ENDOR spectra of Φ A − at 160 K indicates that two distinct states of Φ A − are present in the wild type and the mutant YF(M208). Based on a comparison with Φ A − in RCs of Rhodobacter sphaeroides the two states are interpreted as torsional isomers of the 3-acetyl group of Φ A . Only one Φ A − state occurs in the mutant YL(M208). This effect of the leucine residue at position M208 is explained by steric hindrance that locks the acetyl group in one specific position. On the basis of these results, an interpretation of the optical absorption difference spectrum of the state Φ A − Q A − is attempted. This state can be accumulated at 100 K and undergoes an irreversible change between 100 and 200 K [Tiede et al., Biochim. Biophys. Acta 892 (1987) 294–302]. The corresponding absorbance changes in the BChl Q x and Q y regions observed in the wild type also occur in the YF(M208) mutant but not in YL(M208). The observed changes in the wild type and YF(M208) are assigned to RCs in which the 3-acetyl group of Φ A changes its orientation. It is concluded that this distinct structural relaxation of Φ A can significantly affect the optical properties of B A and contribute to the light-induced absorption difference spectra.

The catalytic center (CC) of water oxidation in photosystem II passes through four stepwise increased oxidized states (S0–S4) before O2 evolution takes place from 2H2O in the S4 → S0 transition. The pattern of the release of the four protons from the CC cannot be followed directly in the medium, because proton release from unknown amino acid residues also takes place. However, pH-independent net charge oscillations of 0:0:1:1 in S0:S1:S2:S3 have been considered as an intrinsic indicator for the H+ release from the CC. The net charges have been proposed to be created as the charge difference between electron abstraction and H+release from the CC. Then the H+ release from the CC is 1:0:1:2 for the S0 → S1 → S2 → S3 → S0 transition. Strong support for this conclusion is given in this work with the analysis of the pH-dependent pattern of H+ release in the medium measured directly by a glass electrode between pH 5.5 and 7.2. Improved and crystallizable photosystem II core complexes from the cyanobacterium Synechococcus elongatus were used as material. The pattern can be explained by protons released from the CC with a stoichiometry of 1:0:1:2 and protons from an amino acid group (pK ≈ 5.7) that is deprotonated and reprotonated through electrostatic interaction with the oscillating net charges 0:0:1:1 in S0:S1:S2:S3. Possible water derivatives that circulate through the S states have been named.

Electron-transfer reactions following the formation of P700(+)A1- have been studied in isolated Photosystem I complexes from Synechococcus elongatus between 300 and 5 K by flash absorption spectroscopy. (1) In the range from 300 to 200 K, A1- is reoxidized by electron transfer to the iron-sulfur cluster FX. The rate slows down with decreasing temperature, corresponding to an activation energy of 220 +/- 20 meV in this temperature range. Analyzing the temperature dependence of the rate in terms of nonadiabatic electron-transfer theory, one obtains a reorganization energy of about 1 eV and an edge-to-edge distance between A1 and FX of about 8 A assuming the same distance dependence of the electron-transfer rate as in purple bacterial reaction centers. (2) At temperatures below 150 K, different fractions of PS I complexes attributed to frozen conformational substates can be distinguished. A detailed analysis at 77 K gave the following results: (a) In about 45%, flash-induced electron transfer is limited to the formation and decay of the secondary pair P700(+)A1-. The charge recombination occurs with a t1/2 of about 170 micros. (b) In about 20%, the state P700(+)FX- is formed and recombines with complex kinetics (t1/2 = 5-100 ms). (c) In about 35%, irreversible formation of P700(+)FA- or P700(+)FB- is possible. (3) The transition from efficient forward electron transfer at higher temperatures to heterogeneous photochemistry at low temperatures has been investigated in different glass-forming solvents. The yield of forward electron transfer to the iron-sulfur clusters decreases in a narrow temperature interval. The temperature of the half-maximal effect varies between different solvents and appears to be correlated with their liquid to glass transition. It is proposed that reorganization processes in the surroundings of the reactants which are required for the stabilization of the charge-separated state become arrested near the glass transition. This freezing of protein motions and/or solvent reorganization may affect electron-transfer reactions through changes in the free-energy gap and the reorganization energy. (4) The rate of charge recombination between P700(+) and A1- increases slightly (about 1.5-fold) when the temperature is decreased from 300 to 5 K. This charge recombination characterized by a large driving force is much less influenced by the solvent properties than the forward electron-transfer steps from A1- to FX and FA/B.

The effects of detergents on the electronic structure of the oxidized primary donor P+ and the time constant τAP of the P+Q A − charge recombination at ambient temperatures have been investigated in native and mutant reaction centers (RCs) from Rhodobacter sphaeroides. It is shown that N-lauryl-N,N-dimethyl-3-ammonio-1-propane sulfonate (SB12) induces a transition to a second distinct conformation of the RC. In the case of the wild type and the mutant FY(M197), in which a hydrogen bond is introduced to the 2-acetyl group of the dimer half of P that is associated with the M-subunit of the RC, the conformational change causes a more asymmetric spin density distribution between the two bacteriochlorophyll moieties of P+ in favor of the L-half. For both types of RCs the time constant τAP depends on the SB12/RC ratio as does the position of the long-wavelength band of P, λmax. The increase of τAP by ∼30 ms and the shift of λmax from ∼866 nm to ∼851 nm are indicative for the conformational change. In addition, a smaller linear increase of τAP with increasing SB12/RC ratio is superimposed on the variation of τAP due to the conformational change. Similar effects of SB12 on the optical spectra as well as on τAP are also observed for the two heterodimer mutants HL(L173) and HL(M202), in which one of the bacteriochlorophylls of P is replaced by a bacteriopheophytin. There are no clear indications for a correlation of τAP with the localization of the positive charge in P+. Furthermore, it is concluded from the dependence of τAP on the SB12/RC ratio that the single-site mutations do not affect the standard free energy difference of the two conformations to a measurable extent.

Photosystem I is a member of the iron-sulfur center or type I reaction centers. The primary electron donor in photosystem I is a chlorophyll a dimer termed P700. The biophysical properties of P700 are well understood, but the protein environment that gives it such unique properties is unknown. We have characterized site-directed mutants of the photosystem I reaction center protein PsaB and identified an amino acid, His-656, that interacts closely with one of the P700 chlorophylls. Mutation of His-656 to Asn or Ser increases the oxidation midpoint potential of P700/P700+. by 40 mV. The P700/P700+. optical difference spectra show the appearance of a new bleaching band at 667 nm. Electron nuclear double resonance spectroscopy indicates a significant increase in the hyperfine coupling corresponding to methyl protons at position 12 of the spin carrying chlorophyll a of P700+. The implication of these results to current structural models of the photosystem I reaction center is discussed.

Recombination reactions of the primary radical pair in photosystem II (PS II) have been studied in the nanosecond to millisecond time scales by flash absorption spectroscopy. Samples in which the first quinone acceptor (QA) was in the semiquinone form (QA-) or in the doubly reduced state (presumably QAH2) were used. The redox state of QA and the long-lived triplet state of the primary electron donor chlorophyll (3P680) were monitored by EPR. The following results were obtained at cryogenic temperatures (around 20 K). (1) the primary radical pair, P680+Pheo-, is formed with a high yield irrespective of the redox state of QA. (2) The decay of the primary pair is faster with QA- than with QAH2 and could be described biexponentially with t1/2 approximately 20 ns (approximately 65%)/150 ns (approximately 35%) and t1/2 approximately 60 ns (approximately 35%)/250 ns (approximately 65%), respectively. The different kinetics may be due to electrostatic and/or magnetic effects of QA- on charge recombination or due to conformational changes caused by the double reduction treatment. (3) The yield of the triplet state 3P680 was high both with QA- and QAH2. (4) The triplet decay was much faster with QA- [t1/2 approximately 2 microseconds (approximately 50%)/20 microseconds (approximately 50%)] than with QAH2 [t1/2 approximately 1 ms (approximately 65%)/3 ms (approximately 35%)]. The short lifetime of the triplet with QA- explains why it was not detected earlier. The mechanism of triplet quenching in the presence of QA- is not understood; however it may represent a protective process in PS II. (5) Almost identical data were obtained for PS II-enriched membranes from spinach and PS II core preparations from Synechococcus. Room temperature optical studies were performed on the Synechococcus preparation. In samples containing sodium dithionite to form QA- in the dark, EPR controls showed that multiple excitation flashes given at room temperature led to a decrease of the QA-Fe2+ signal, indicating double reduction of QA. During the first few flashes, QA- was still present in the large majority of the centers. In this case, the yield of the primary pair at room temperature was around 50%, and its decay could be described monoexponentially with t1/2 approximately 8 ns (a slightly better fit was obtained with two exponentials: t1/2 approximately 4 ns (approximately 80%)/25 ns (approximately 20%).(ABSTRACT TRUNCATED AT 400 WORDS)

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 5 K: ≅ 719 nm; emission maximum at 5 K: ≅ 740 nm) was found to be strongly quenched by P700+ similar to the reddest state in PS I trimers from Arthrospira platensis emitting at 760 nm at 5 K. 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.

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.

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.

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.

The activities of photosynthetic water oxidation and of the primary stable charge separation have been investigated as a function of the concentration of various salts by measuring flash-induced oxygen yields and absorption changes at 320 nm due to Q A reduction. (1) At low salt concentrations, water oxidation is inhibited, whereas the charge separation is not affected. (2) The former can be reactivated by salts containing neithercalcium nor chloride (e.g., Na 2 SO 4 ), indicating that these ions are not essential cofactors of water oxidation. (3) At higher concentrations, certain salts (e.g. CaCl 2 ) reversibly inhibit water oxidation and in the range of molar concentration also the primary stable charge separation. (4) These activating and inhibiting effects are explained by equilibria between active and inactive conformational states of PS II. These equilibria depend on the concentrations and properties of the various salts. (5) Arguments are given that, also in higher plants, salt-dependent conformational states are responsible for the salt effects.

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.

We present a theoretical analysis of the flash-induced absorbance difference spectrum assigned to the formation of the secondary radical pair P(+)QA(-) in photosystem II of the chlorophyll d-containing cyanobacterium Acaryochloris marina. An exciton Hamiltonian determined previously for chlorophyll a-containing photosystem II complexes is modified to take into account the occupancy of certain binding sites by chlorophyll d instead of chlorophyll a. Different assignments of the reaction center pigments to chlorophyll a or d from the literature are investigated in the calculation of the absorbance difference spectrum. A quantitative explanation of the experimental data requires one chlorophyll a molecule per reaction center, located at the site of P(D1). The remaining sites are occupied by chlorophyll d and pheophytin a. By far, the lowest site energy is found for the accessory chlorophyll of the D1 branch, Chl(D1), which represents the sink of excitation energy and therefore the primary electron donor. The cationic state is stabilized at the chlorophyll a, which drives the oxidation of water.

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.

The oxidation potential of the primary electron donor P740 of photosystem I (PS I) of the chlorophyll d dominated cyanobacterium, Acaryochloris marina, has been reinvestigated. We find a midpoint potential of about 450 mV in a photosystem I-enriched membrane fraction as well as in thylakoids, which is significantly higher than that originally reported by Hu et al. (1998). This value, which is, indeed, very similar to that found for P700 in chlorophyll a dominated organisms, is in agreement with results of Bailleul et al. (this Congress). In addition, the extinction difference coefficient for the oxidation of the primary donor has been determined and a value of 45 (±4) mM−1 cm−1 at 740 nm was obtained.

Transient absorbance difference spectroscopy has been used to study the reduction kinetics of P680+ after the first flash given to darkadapted oxygen-evolving PS II complexes from Thermosynechococcus elongatus as a function of temperature between 80 and 320 K. The half-life of P680+ reduction by TyrZ increases from 20 ns at 300 K to about 4 μs at 150 K corresponding to an activation energy of (122 ± 3) meV. Analyzing the temperature dependence of the rate in terms of nonadiabatic electron transfer theory, one obtains a reorganization energy of about 700 meV and an edge-to-edge distance of about 8.5 A which is in good agreement with the distance between PD1 and TyrZ in the recent structural model of PS II at 3.0 A resolution (Loll et al. 2005). In the range from 260 to 150 K, the re-reduction of P680+ by TyrZ is increasingly replaced by the charge recombination between P680+ and QA −. It is proposed that reorganization processes which are required for the stabilization of the state P680TyrZ ox become arrested around 200 K. Freezing out of the electron donation from TyrZ to P680+ is the result. The yield of TyrZ oxidation at low temperature is

At low temperatures (

Site-directed mutations were constructed in photosystem II of Synechocystis sp. PCC6803 in which the axial ligand, D1-His198, of special pair chlorophyll P D1 was replaced with Gln and where D1-Thr179, which overlies monomeric chlorophyll Chl D1 , was replaced with His. The D1-His198Gln mutation produces a 3 nm displacement to the blue of the bleaching minimum in the Soret and in the Qy region of the – absorbance difference spectrum. To a first approximation, the bleaching can be assigned to the low-energy exciton transition of the special pair chlorophylls P D1 /P D2 . The D1-Thr179His mutation produces a 2 nm displacement to the red of the bleaching minimum in the Qy region of the ( 3 P– 1 P) absorbance difference spectrum. Analysis of the flash-induced – and ( 3 P– 1 P) absorbance difference spectra of both mutants compared with wild-type at 80 K indicate that the cation of the oxidized donor P + is predominantly localized on the chlorophyll P D1 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 Q A is doubly reduced, is primarily localized on Chl D1 .

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.

Summary In cyanobacteria, long wavelength chlorophylls (LWC) are located in the Photosystem I (PS I) core complex, whereas in plants and algae they are distributed between the PS I core and the light-harvesting complexes (LHC I). LWC are most probably aggregates of (excitonically) coupled chlorophylls, mainly dimers or trimers. The total number of LWC is rather small (≤10% of the total chlorophylls). Depending on their location in the PS I antenna and their distance from P700, they can play a crucial role in the kinetics of energy transfer and in the trapping of the excitation energy by charge separation. Energy absorbed by LWC is transferred uphill to P700 with high efficiency at room temperature, thereby increasing the cross-section for the absorption of red light. LWC are involved also in the dissipation of excess energy, thus protecting the reaction center. Under physiological conditions, the excitations within the PS I antenna are nearly thermally equilibrated over the different spectral forms and the excitation energy is efficiently trapped via charge separation in the reaction center. When the photochemistry in the reaction center is blocked, the excitations migrate to the LWC and are quenched either by P700 + or by the P700 triplet state depending on the state of P700.

Beta-carotene has been identified as an intermediate in a secondary electron transfer pathway that oxidizes Chl(Z) and cytochrome b(559) in Photosystem II (PS II) when normal tyrosine oxidation is blocked. To test the redox function of carotenoids in this pathway, we replaced the zeta-carotene desaturase gene (zds) or both the zds and phytoene desaturase (pds) genes of Synechocystis sp. PCC 6803 with the phytoene desaturase gene (crtI) of Rhodobacter capsulatus, producing carotenoids with shorter conjugated pi-electron systems and higher reduction potentials than beta-carotene. The PS II core complexes of both mutant strains contain approximately the same number of chlorophylls and carotenoids as the wild type but have replaced beta-carotene (11 double bonds), with neurosporene (9 conjugated double bonds) and beta-zeacarotene (9 conjugated double bonds and 1 beta-ionylidene ring). The presence of the ring appears necessary for PS II assembly. Visible and near-infrared spectroscopy were used to examine the light-induced formation of chlorophyll and carotenoid radical cations in the mutant PS II core complexes at temperatures from 20 to 160 K. At 20 K, a carotenoid cation radical is formed having an absorption maximum at 898 nm, an 85 nm blue shift relative to the beta-carotene radical cation peak in the WT, and consistent with the formation of the cation radical of a carotenoid with 9 conjugated double bonds. The ratio of Chl(+)/Car(+) is higher in the mutant core complexes, consistent with the higher reduction potential for Car(+). As the temperature increases, other carotenoids become accessible to oxidation by P(680)(+).

The regulatory Ni-Fe hydrogenase (RH) from Ralstonia eutropha functions as a hydrogen sensor. The RH consists of the large subunit HoxC housing the Ni-Fe active site and the small subunit HoxB containing Fe-S clusters. The heterolytic cleavage of H(2) at the Ni-Fe active site leads to the EPR-detectable Ni-C state of the protein. For the first time, the simultaneous but EPR-invisible reduction of Fe-S clusters during Ni-C state formation was demonstrated by changes in the UV-visible absorption spectrum as well as by shifts of the iron K-edge from x-ray absorption spectroscopy in the wild-type double dimeric RH(WT) [HoxBC](2) and in a monodimeric derivative designated RH(stop) lacking the C-terminal 55 amino acids of HoxB. According to the analysis of iron EXAFS spectra, the Fe-S clusters of HoxB pronouncedly differ from the three Fe-S clusters in the small subunits of crystallized standard Ni-Fe hydrogenases. Each HoxBC unit of RH(WT) seems to harbor two [2Fe-2S] clusters in addition to a 4Fe species, which may be a [4Fe-3S-3O] cluster. The additional 4Fe-cluster was absent in RH(stop). Reduction of Fe-S clusters in the hydrogen sensor RH may be a first step in the signal transduction chain, which involves complex formation between [HoxBC](2) and tetrameric HoxJ protein, leading to the expression of the energy converting Ni-Fe hydrogenases in R. eutropha.

We applied optical spectroscopy, magnetic resonance techniques, and redox titrations to investigate the properties of the primary electron donor P700 in photosystem I (PS I) core complexes from cyanobacteria (Thermosynechococcus elongatus, Spirulina platensis, and Synechocystis sp. PCC 6803), algae (Chlamydomonas reinhardtii CC2696), and higher plants (Spinacia oleracea). Remarkable species-specific differences of the optical properties of P700 were revealed monitoring the (3P700-P700) and (P700+.-P700) absorbance and CD difference spectra. The main bleaching band in the Qy region differs in peak position and line width for the various species. In cyanobacteria the absorbance of P700 extends more to the red compared with algae and higher plants which is favorable for energy transfer from red core antenna chlorophylls to P700 in cyanobacteria. The amino acids in the environment of P700 are highly conserved with two distinct deviations. In C. reinhardtii a Tyr is found at position PsaB659 instead of a Trp present in all other organisms, whereas in Synechocystis a Phe is found instead of a Trp at the homologous position PsaA679. We constructed several mutants in C. reinhardtii CC2696. Strikingly, no PS I could be detected in the mutant YW B659 indicating steric constraints unique to this organism. In the mutants WA A679 and YA B659 significant changes of the spectral features in the (3P700-P700), the (P700+.-P700) absorbance difference and in the (P700+.-P700) CD difference spectra are induced. The results indicate structural differences among PS I from higher plants, algae, and cyanobacteria and give further insight into specific protein-cofactor interactions contributing to the optical spectra.

Photosystem I (PS I) is a large, membrane protein complex, consisting of 12–15 proteins and more than 100 bound cofactors, that catalyzes light-driven electron transfer across the photosynthetic membrane in cyanobacteria, green algae and plants. Photosystem I is unique compared to other photosynthetic systems because the majority of the antenna pigments and the cofactors of the electron transport system are bound to the same protein subunits. Photosystem I can therefore be regarded as a joint reaction center-core antenna system. In this chapter, PS I is described with respect to both structure and function. The kinetics of energy transfer and trapping are discussed in the light of the structural information. We also discuss models for the interaction of PS I with the external antenna complexes in cyanobacteria and plants.

The primary electron donor P700 of photosystem I is a dimer comprised of chlorophyll a (P(B)) and chlorophyll a' (P(A)). P(A) is involved in a hydrogen bond network with several surrounding amino acid residues and a nearby water molecule. To investigate the influence of hydrogen bond interactions on the properties of P700, the threonine at position A739, which donates a putative hydrogen bond to the 13(1)-keto group of P(A), was replaced with valine, histidine, and tyrosine in Chlamydomonas reinhardtii using site-directed mutagenesis. Growth of the mutants was not impaired. (i) The (P700(+)* - P700) FTIR difference spectra of the mutants lack a negative band at 1634 cm(-1) observed in the wild-type spectrum and instead exhibit a new negative band between 1658 and 1672 cm(-1) depending on the mutation. This band can therefore be assigned to the 13(1)-keto group of P(A) which is upshifted to higher frequencies upon removal of the hydrogen bond. (ii) The main bleaching band in the Q(y)() region of the (P700(+)* - P700) and ((3)P700 - P700) absorption difference spectra is blue shifted for the mutants by approximately 6 nm compared to that of the wild type. A blue shift is also observed for the main bleaching in the Soret region. (iii) The (P700(+)* - P700) CD difference spectrum of the wild type reveals two bands at 694 nm (positive CD) and 680 nm (negative CD) of approximately equal area. For each mutant, these two components are blue-shifted to the same extent. The results strongly suggest that a blue shift of the Q(y) absorption band of P(A) is responsible for a blue shift of the exciton bands. (iv) Redox titrations yielded a decrease in the midpoint potential for the oxidation of P700 by 32 mV for the exchange of Thr against Val. (v) ENDOR spectroscopy shows that the hfc of the methyl protons at position 12 of the spin-carrying Chl P(B) is decreased due to the removal of the hydrogen bond to P(A). This indicates a redistribution of spin density in P700(+)* compared to that in the wild type. This gives evidence for an electronic coupling between the two halves of the dimer in the wild type and mutants.

Site-directed mutations were introduced to replace D1-His198 and D2-His197 of the D1 and D2 polypeptides, respectively, of the photosystem II (PSII) reaction center of Synechocystis PCC 6803. These residues coordinate chlorophylls P(A) and P(B) which are homologous to the special pair Bchlorophylls of the bacterial reaction centers that are coordinated respectively by histidines L-173 and M-200 (202). P(A) and P(B) together serve as the primary electron donor, P, in purple bacterial reaction centers. In PS II, the site-directed mutations at D1 His198 affect the P(+)--P-absorbance difference spectrum. The bleaching maximum in the Soret region (in WT at 433 nm) is blue-shifted by as much as 3 nm. In the D1 His198Gln mutant, a similar displacement to the blue is observed for the bleaching maximum in the Q(y) region (672.5 nm in WT at 80 K), whereas features attributed to a band shift centered at 681 nm are not altered. In the Y(Z*)--Y(Z)-difference spectrum, the band shift of a reaction center chlorophyll centered in WT at 433--434 nm is shifted by 2--3 nm to the blue in the D1-His198Gln mutant. The D1-His198Gln mutation has little effect on the optical difference spectrum, (3)P--(1)P, of the reaction center triplet formed by P(+)Pheo(-) charge recombination (bleaching at 681--684 nm), measured at 5--80 K, but becomes visible as a pronounced shoulder at 669 nm at temperaturesor =150 K. Measurements of the kinetics of oxidized donor--Q(A)(-) charge recombination and of the reduction of P(+) by redox active tyrosine, Y(Z), indicate that the reduction potential of the redox couple P(+)/P can be appreciably modulated both positively and negatively by ligand replacement at D1-198 but somewhat less so at D2-197. On the basis of these observations and others in the literature, we propose that the monomeric accessory chlorophyll, B(A), is a long-wavelength trap located at 684 nm at 5 K. B(A)* initiates primary charge separation at low temperature, a function that is increasingly shared with P(A)* in an activated process as the temperature rises. Charge separation from B(A)* would be potentially very fast and form P(A)(+)B(A)(-) and/or B(A)(+)Pheo(-) as observed in bacterial reaction centers upon direct excitation of B(A) (van Brederode, M. E., et al. (1999) Proc. Natl. Acad Sci. 96, 2054--2059). The cation, generated upon primary charge separation in PSII, is stabilized at all temperatures primarily on P(A), the absorbance spectrum of which is displaced to the blue by the mutations. In WT, the cation is proposed to be shared to a minor extent (approximately 20%) with P(B), the contribution of which can be modulated up or down by mutation. The band shift at 681 nm, observed in the P(+)-P difference spectrum, is attributed to an electrochromic effect of P(A)(+) on neighboring B(A). Because of its low-energy singlet and therefore triplet state, the reaction center triplet state is stabilized on B(A) ator =80 K but can be shared with P(A) at80 K in a thermally activated process.

Transfer and trapping of excitation energy in photosystem I (PS I) trimers isolated from Synechococcus elongatus have been studied by an approach combining fluorescence induction experiments with picosecond time-resolved fluorescence measurements, both at room temperature (RT) and at low temperature (5 K). Special attention was paid to the influence of the oxidation state of the primary electron donor P700. A fluorescence induction effect has been observed, showing a approximately 12% increase in fluorescence quantum yield upon P700 oxidation at RT, whereas at temperatures below 160 K oxidation of P700 leads to a decrease in fluorescence quantum yield ( approximately 50% at 5 K). The fluorescence quantum yield for open PS I (with P700 reduced) at 5 K is increased by approximately 20-fold and that for closed PS I (with P700 oxidized) is increased by approximately 10-fold, as compared to RT. Picosecond fluorescence decay kinetics at RT reveal a difference in lifetime of the main decay component: 34 +/- 1 ps for open PS I and 37 +/- 1 ps for closed PS I. At 5 K the fluorescence yield is mainly associated with long-lived components (lifetimes of 401 ps and 1.5 ns in closed PS I and of 377 ps, 1.3 ns, and 4.1 ns in samples containing approximately 50% open and 50% closed PS I). The spectra associated with energy transfer and the steady-state emission spectra suggest that the excitation energy is not completely thermally equilibrated over the core-antenna-RC complex before being trapped. Structure-based modeling indicates that the so-called red antenna pigments (A708 and A720, i.e., those with absorption maxima at 708 nm and 720 nm, respectively) play a decisive role in the observed fluorescence kinetics. The A720 are preferentially located at the periphery of the PS I core-antenna-RC complex; the A708 must essentially connect the A720 to the reaction center. The excited-state decay kinetics turn out to be neither purely trap limited nor purely transfer (to the trap) limited, but seem to be rather balanced.

The primary quinone acceptor radical anion Q(A)(-)(*) (a menaquinone-9) is studied in reaction centers (RCs) of Rhodopseudomonas viridis in which the high-spin non-heme Fe(2+) is replaced by diamagnetic Zn(2+). The procedure for the iron substitution, which follows the work of Debus et al. [Debus, R. J., Feher, G., and Okamura, M. Y. (1986) Biochemistry 25, 2276-2287], is described. In Rps. viridisan exchange rate of the iron of approximately 50% +/- 10% is achieved. Time-resolved optical spectroscopy shows that the ZnRCs are fully competent in charge separation and that the charge recombination times are similar to those of native RCs. The g tensor of Q(A)(-)(*) in the ZnRCs is determined by a simulation of the EPR at 34 GHz yielding g(x) = 2.00597 (5), g(y) = 2.00492 (5), and g(z) = 2.00216 (5). Comparison with a menaquinone anion radical (MQ(4)(-)(*)) dissolved in 2-propanol identifies Q(A)(-)(*) as a naphthoquinone and shows that only one tensor component (g(x)) is predominantly changed in the RC. This is attributed to interaction with the protein environment. Electron-nuclear double resonance (ENDOR) experiments at 9 GHz reveal a shift of the spin density distribution of Q(A)(-)(*) in the RC as compared with MQ(4)(-)(*) in alcoholic solution. This is ascribed to an asymmetry of the Q(A) binding site. Furthermore, a hyperfine coupling constant from an exchangeable proton is deduced and assigned to a proton in a hydrogen bond between the quinone oxygen and surrounding amino acid residues. By electron spin-echo envelope modulation (ESEEM) techniques performed on Q(A)(-)(*) in the ZnRCs, two (14)N nuclear quadrupole tensors are determined that arise from the surrounding amino acids. One nitrogen coupling is assigned to a N(delta)((1))-H of a histidine and the other to a polypeptide backbone N-H by comparison with the nuclear quadrupole couplings of respective model systems. Inspection of the X-ray structure of Rps. viridis RCs shows that His(M217) and Ala(M258) are likely candidates for the respective amino acids. The quinone should therefore be bound by two H bonds to the protein that could, however, be of different strength. An asymmetric H-bond situation has also been found for Q(A)(-)(*) in the RC of Rhodobacter sphaeroides. Time-resolved electron paramagnetic resonance (EPR) experiments are performed on the radical pair state P(960)(+) (*)Q(A)(-)(*) in ZnRCs of Rps. viridis that were treated with o-phenanthroline to block electron transfer to Q(B). The orientations of the two radicals in the radical pair obtained from transient EPR and their distance deduced from pulsed EPR (out-of-phase ESEEM) are very similar to the geometry observed for the ground state P(960)Q(A) in the X-ray structure [Lancaster, R., Michel, H. (1997) Structure 5, 1339].

The interaction of the primary donor (P), a dimer of bacteriochlorophylls (BChls), denoted PL and PM, with the protein environment in reaction centers (RCs) of Rhodobacter (Rb.) sphaeroides is studied in a series of mutants, in which His M202 is exchanged to either Glu, Asn, Ser, as well as in HL(M202) [1] and HG(M202) [2]. Mutant RCs were isolated and investigated by optical spectroscopy, redox titrations of P and EPR/ENDOR spectroscopy of the light-induced radical cation P*+. The aim of this study is to assess the ability of different amino acid residues to serve as axial ligand to PM and to influence the pigment composition of the RCs.