Subunit composition of CP43-less photosystem II complexes of Synechocystis sp. PCC 6803: implications for the assembly and repair of photosystem II.

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.

[Variability of light-induced circular dichroism spectra of photosystem I complexes of cyanobacteria].

Circular dichroism (CD) spectra of photosystem I (PSI) complexes of the cyanobacteria Thermosynechococcus elongatus, Arthrospira platensis and Synechocystis sp. PCC 6803 were studied. CD spectra of dark-adapted 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 complexes. 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 dark-adapted 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.

The primary electron donor of photosystem II of the cyanobacterium Acaryochloris marina is a chlorophyll d and the water oxidation is driven by a chlorophyll a/chlorophyll d heterodimer.

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.

Site-directed mutations at D1-His198 and D2-His197 of photosystem II in Synechocystis PCC 6803: sites of primary charge separation and cation and triplet stabilization.

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 temperatures > or =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) at < or =80 K but can be shared with P(A) at >80 K in a thermally activated process.

Time-resolved fluorescence emission measurements of photosystem I particles of various cyanobacteria: a unified compartmental model.

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.

Green plant photosystem I binds light-harvesting complex I on one side of the complex.

We report a structural characterization by electron microscopy of green plant photosystem I solubilized by the mild detergent n-dodecyl-alpha-D-maltoside. It is shown by immunoblotting that the isolated complexes contain all photosystem I core proteins and all peripheral light-harvesting proteins. The electron microscopic analysis is based on a large data set of 14 000 negatively stained single-particle projections and reveals that most of the complexes are oval-shaped monomers. The monomers have a tendency to associate into artificial dimers, trimers, and tetramers in which the monomers are oppositely oriented. Classification of the dimeric complexes suggests that some of the monomers lack a part of the peripheral antenna. On the basis of a comparison with projections from trimeric photosystem I complexes from cyanobacteria, we conclude that light-harvesting complex I only binds to the core complex at the side of the photosystem I F/J subunits and does not cause structural hindrances for the type of trimerization observed in cyanobacterial photosystem I.

How carotenoids protect bacterial photosynthesis.

The essential function of carotenoids in photosynthesis is to act as photoprotective agents, preventing chlorophylls and bacteriochlorophylls from sensitizing harmful photodestructive reactions in the presence of oxygen. Based upon recent structural studies on reaction centres and antenna complexes from purple photosynthetic bacteria, the detailed organization of the carotenoids is described. Then with specific reference to bacterial antenna complexes the details of the photoprotective role, triplet triplet energy transfer, are presented.

Conformational relaxation following reduction of the photoactive bacteriopheophytin in reaction centers from Balstochloris viridis. Influence of mutations at position M208.

The photochemically trapped bacteriopheophytin (BPh) b radical anion in the active branch (phi(*-)A) of reaction centers (RCs) from Blastochloris (formerly called Rhodopseudomonas) viridis is characterized by 1H-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 (BCh1), B(A), and the BPh, phiA, that are involved in the transmembrane electron transfer to the quinone, Q(A), in the RC. The analysis of the ENDOR spectra of (phi(*-)A at 160 K indicates that two distinct states of phi(*-)A are present in the wild type and the mutant YF(M208). Based on a comparison with phi(*-)A in RCs of Rhodobacter sphaeroides the two states are interpreted as torsional isomers of the 3-acetyl group of phiA. Only one phi(*-)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 phi(*-)AQ(*-)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 BCh1 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 phiA changes its orientation. It is concluded that this distinct structural relaxation of phiA can significantly affect the optical properties of B(A) and contribute to the light-induced absorption difference spectra.

Influence of the axial ligands on the spectral properties of P700 of photosystem I: a study of site-directed mutants.

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).

Conformational relaxation following reduction of the photoactive bacteriopheophytin in reaction centers from Blastochloris viridis. Influence of mutations at position M208.

The photochemically trapped bacteriopheophytin (BPh) b radical anion in the active branch (Phi(A)(&z.rad;-)) of reaction centers (RCs) from Blastochloris (formerly called Rhodopseudomonas) viridis is characterized by 1H-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, Phi(A), that are involved in the transmembrane electron transfer to the quinone, Q(A), in the RC. The analysis of the ENDOR spectra of Phi(A)(&z.rad;-) at 160 K indicates that two distinct states of Phi(A)(&z.rad;-) are present in the wild type and the mutant YF(M208). Based on a comparison with Phi(A)(&z.rad;-) in RCs of Rhodobacter sphaeroides the two states are interpreted as torsional isomers of the 3-acetyl group of Phi(A). Only one Phi(A)(&z.rad;-) 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 Phi(A)(&z.rad;-)Q(A)(&z.rad;-) 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 Phi(A) changes its orientation. It is concluded that this distinct structural relaxation of Phi(A) can significantly affect the optical properties of B(A) and contribute to the light-induced absorption difference spectra.

Decay kinetics and quantum yields of fluorescence in photosystem I from Synechococcus elongatus with P700 in the reduced and oxidized state: are the kinetics of excited state decay trap-limited or transfer-limited?

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.

First photosystem II crystals capable of water oxidation.

Oxygen evolution and proton release of crystallised photosystem II core complexes isolated from Synechococcus elongatus were measured. The yields show that the crystals themselves are capable of highly active water oxidation. This opens the possibility for the structural analysis of the outstanding water-oxidising apparatus.

Stoichiometry of proton release from the catalytic center in photosynthetic water oxidation. Reexamination by a glass electrode study at ph 5.5-7.2.

The catalytic center (CC) of water oxidation in photosystem II passes through four stepwise increased oxidized states (S(0)-S(4)) before O(2) evolution takes place from 2H(2)O in the S(4) --> S(0) 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 S(0):S(1):S(2):S(3) 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 S(0) --> S(1) --> S(2) --> S(3) --> S(0) 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 approximately 5.7) that is deprotonated and reprotonated through electrostatic interaction with the oscillating net charges 0:0:1:1 in S(0):S(1):S(2):S(3). Possible water derivatives that circulate through the S states have been named.

Electron paramagnetic resonance studies of zinc-substituted reaction centers from Rhodopseudomonas viridis.

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].

Temperature dependence of forward and reverse electron transfer from A1-, the reduced secondary electron acceptor in photosystem I.

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.

Energy transfer and charge separation in photosystem I: P700 oxidation upon selective excitation of the long-wavelength antenna chlorophylls of Synechococcus elongatus.

Photosystem I of the cyanobacterium Synechococcus elongatus contains two spectral pools of chlorophylls called C-708 and C-719 that absorb at longer wavelengths than the primary electron donor P700. We investigated the relative quantum yields of photochemical charge separation and fluorescence as a function of excitation wavelength and temperature in trimeric and monomeric photosystem I complexes of this cyanobacterium. The monomeric complexes are characterized by a reduced content of the C-719 spectral form. At room temperature, an analysis of the wavelength dependence of P700 oxidation indicated that all absorbed light, even of wavelengths of up to 750 nm, has the same probability of resulting in a stable P700 photooxidation. Upon cooling from 295 K to 5 K, the nonselectively excited steady-state emission increased by 11- and 16-fold in the trimeric and monomeric complexes, respectively, whereas the quantum yield of P700 oxidation decreased 2.2- and 1.7-fold. Fluorescence excitation spectra at 5 K indicate that the fluorescence quantum yield further increases upon scanning of the excitation wavelength from 690 nm to 710 nm, whereas the quantum yield of P700 oxidation decreases significantly upon excitation at wavelengths longer than 700 nm. Based on these findings, we conclude that at 5 K the excited state is not equilibrated over the antenna before charge separation occurs, and that approximately 50% of the excitations reach P700 before they become irreversibly trapped on one of the long-wavelength antenna pigments. Possible spatial organizations of the long-wavelength antenna pigments in the three-dimensional structure of photosystem I are discussed.

Site-directed mutations affecting the spectroscopic characteristics and midpoint potential of the primary donor in photosystem I.

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.

Polarized site-selective fluorescence spectroscopy of the long-wavelength emitting chlorophylls in isolated Photosystem I particles of Synechococcus elongatus.

Isolated trimeric Photosystem I complexes of the cyanobacterium Synechococcus elongatus have been studied with absorption spectroscopy and site-selective polarized fluorescence spectroscopy at cryogenic temperatures. The 4 K absorption spectrum exhibits a clear and distinct peak at 710 nm and shoulders near 720, 698 and 692 nm apart from the strong absorption profile located at 680 nm. Deconvoluting the 4 K absorption spectrum with Gaussian components revealed that Synechococcus elongatus contains two types of long-wavelength pigments peaking at 708 nm and 719 nm, which we denoted C-708 and C-719, respectively. An estimate of the oscillator strengths revealed that Synechococcus elongatus contains about 4-5 C-708 pigments and 5-6 C-719 pigments. At 4 K and for excitation wavelengths shorter than 712 nm, the emission maximum appeared at 731 nm. For excitation wavelengths longer than 712 nm, the emission maximum shifted to the red, and for excitation in the far red edge of the absorption spectrum the emission maximum was observed 10-11 nm to the red with respect to the excitation wavelength, which indicates that the Stokes shift of C-719 is 10-11 nm. The fluorescence anisotropy, as calculated in the emission maximum, reached a maximal anisotropy of r=0.35 for excitation in the far red edge of the absorption spectrum (at and above 730 nm), and showed a complicated behavior for excitation at shorter wavelengths. The results suggest efficient energy transfer routes between C-708 and C-719 pigments and also among the C-719 pigments.

Charge recombination reactions in photosystem II. 2. Transient absorbance difference spectra and their temperature dependence.

Absorbance difference spectra of the transient states in photosystem II (PS II) have been examined in the Qv absorption region between 660 and 700 nm. The P680+Pheo-/P680Pheo, 3P680/P680, and P680+QA-/P680QA spectra were measured in O2-evolving PS II core complexes from Synechococcus and PS II-enriched membrane fragments from spinach. The low-temperature absorbance difference spectra vary only slightly between both PS II preparations. The 3P680/P680 spectrum is characterized by a bleaching at 685 nm at 25 K and indicates weak exciton coupling with neighboring pigment(s). We conclude that P680 absorbs at 685 nm in more intact PS II preparations at cryogenic temperature. The difference spectra of the radical pairs are strongly temperature dependent. At low temperature the P680+QA-/P680QA- spectrum exhibits the strongest bleaching at 675 nm whereas the P680+Phe-/P680Pheo spectra show two distinct bleaching bands at 674 and 684 nm. It is suggested that an electrochronic red shift resulting in a bleaching at 675 nm and an absorbance increase at about 682 nm dominates the spectral features of the charge-separated states. On the basis of the present results and those in the literature, we conclude that the interactions between the pigments and especially the organization of the primary donor must be quite different in PS II compared to bacterial reaction centers, although the basic structural arrangement of the pigments might be similar. Spectral data obtained with samples in the presence of singly and doubly reduced QA indicate that the primary photochemistry in PS II is not strongly influenced by the redox state of QA at low temperature and confirm the results of the accompanying paper [Van Mieghem, F. J. E., Brettel, K., Hillmann, B., Kamlowski, A., Rutherford, A. W., & Schlodder, E. (1995) Biochemistry 34, 4798-4813]. The spectra of the primary radical pair and the reaction center triplet obtained with more intact PS II preparations differ widely from those of D1/D2/cyt b-559 complexes. In the latter sample, where 3P680 formation results in a bleaching at 680 nm, the P680+Pheo-/P680Pheo spectrum shows only one broad bleaching band at about 680 nm, and the main bleaching due to photoaccumulation of Pheo- at 77 K appears at 682 nm instead of 685 nm in PS II core complexes. This indicates that the removal of the core antenna which is accompanied by the loss of QA causes also structural changes of the reaction center.

Charge recombination reactions in photosystem II. I. Yields, recombination pathways, and kinetics of the primary pair.

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)