Reaction Center Chlorophyll Research Articles

Deformation of Chlorin Rings in the Photosystem II Crystal Structure

The crystal structure of Photosystem II (PSII) analyzed at a resolution of 1.9 Å revealed deformations of chlorin rings in the chlorophylls for the first time. We investigated the degrees of chlorin ring deformation and factors that contributed to them in the PSII crystal structure, using a normal-coordinate structural decomposition procedure. The out-of-plane distortion of the P(D1) chlorin ring can be described predominantly by a large "doming mode" arising from the axial ligand, D1-His198, as well as the chlorophyll side chains and PSII protein environment. In contrast, the deformation of P(D2) was caused by a "saddling mode" arising from the D2-Trp191 ring and the doming mode arising from D2-His197. Large ruffling modes, which were reported to lower the redox potential in heme proteins, were observed in P(D1) and Chl(D1), but not in P(D2) and Chl(D2). Furthermore, as P(D1) possessed the largest doming mode among the reaction center chlorophylls, the corresponding bacteriochlorophyll P(L) possessed the largest doming mode in bacterial photosynthetic reaction centers. However, the majority of the redox potential shift in the protein environment was determined by the electrostatic environment. The difference in the chlorin ring deformation appears to directly refer to the difference in "the local steric protein environment" rather than the redox potential value in PSII.

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.

Janus-faced charge recombinations in photosystem II photoinhibition

Light-induced damage of the photosynthetic apparatus in plants is an important phenomenon that primarily affects the photosystem II complex. Here, we propose a new model of photoinhibition in which charge recombination processes have a double-faced role: first, photodamage is induced by singlet oxygen, which is produced via interaction with the triplet reaction center chlorophyll ((3)P(680)) arising from the recombination of the charge-separated state between P(680) and the pheophytin electron acceptor ((3)[P(680)(+)Phe(-)]). Second, photoprotection is provided by competition between (3)[P(680)(+)Phe(-)] formation and direct recombination of the (1)[P(680)(+)Phe(-)] and P(680)(+)Q(A)(-) states. The efficiency of these two pathways is under control of the redox potential of the Phe and Q(A) electron acceptors, which is utilized during adaptation to high light conditions.

A photoprotection mechanism involving the D2 branch in photosystem II cores with closed reaction centers

Nanosecond transient absorption spectroscopy has been used to study reaction centre (RC) chlorophyll triplet quenching by carotenoid in intact photosystem II cores from T. elongatus with closed RCs. We found a triplet beta-carotene ((3)Car) signal (absorption difference maximum at 530 nm) that is sensitized by the RC chlorophyll (Chl) triplet with a formation time of ca. 190 ns, has a decay time of 7 micros and is formed with a quantum yield between 10 and 20%. The (3)Car signal is assigned to the beta-carotene on the D(2) branch of the RC. We thus propose a new photoprotection mechanism operative in closed RCs where-as a consequence of the negative charge on the quinone Q(A)-the triplet chlorophyll ((3)Chl) is formed by the radical pair (RP) mechanism on the normally inactive D(2) branch where it can be subsequently quenched by the D(2) beta-carotene. We suggest that the D(2) branch becomes active when the RCs are closed under high light fluence conditions. Under these conditions the D(2) branch plays a photoprotective role. This interpretation allows combining many seemingly inconsistent observations in the literature and reveals the so far missing RC triplet quenching mechanism in photosystem II. The newly proposed mechanism also explains the reason why this RC triplet quenching is not observed in isolated D(1)-D(2)-cyt b(559) RCs. If Q(A) is either not present at all (as in the isolated RC) or is not charged (as in open RCs or with doubly reduced Q(A)) then the RC (3)Chl is formed on the D(1) branch. The D(1) branch (3)Chl can not be quenched due to the large distance to the beta-carotene. This interpretation is actually in line with the well-known (3)RC quenching mechanism in bacterial RCs, where also the carotenoid in the (analogous to the D(2) branch) B-branch of the RC becomes the quencher.

Energetics of Photosystem II charge recombination in Acaryochloris marina studied by thermoluminescence and flash-induced chlorophyll fluorescence measurements

We studied the charge recombination characteristics of Photosystem II (PSII) redox components in whole cells of the chlorophyll (Chl) d-dominated cyanobacterium, Acaryochloris marina, by flash-induced chlorophyll fluorescence and thermoluminescence measurements. Flash-induced chlorophyll fluorescence decay was retarded in the mus and ms time ranges and accelerated in the s time range in Acaryochloris marina relative to that in the Chl a-containing cyanobacterium, Synechocystis PCC 6803. In the presence of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea, which blocks the Q(B) site, the relaxation of fluorescence decay arising from S(2)Q(A)(-) recombination was somewhat faster in Acaryochloris marina than in Synechocystis PCC 6803. Thermoluminescence intensity of the so called B band, arising from the recombination of the S(2)Q(B)(-) charge separated state, was enhanced significantly (2.5 fold) on the basis of equal amounts of PSII in Acaryochloris marina as compared with Synechocystis 6803. Our data show that the energetics of charge recombination is modified in Acaryochloris marina leading to a approximately 15 meV decrease of the free energy gap between the Q(A) and Q(B) acceptors. In addition, the total free energy gap between the ground state and the excited state of the reaction center chlorophyll is at least approximately 25-30 meV smaller in Acaryochloris marina, suggesting that the primary donor species cannot consist entirely of Chl a in Acaryochloris marina, and there is a contribution from Chl d as well.

Site-directed Mutations at D1-Thr179 of Photosystem II in Synechocystis sp. PCC 6803 Modify the Spectroscopic Properties of the Accessory Chlorophyll in the D1-branch of the Reaction Center

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

Function of Chlorophyll d in Reaction Centers of Photosystems I and II of the Oxygenic Photosynthesis of Acaryochloris marina

Reaction center chlorophylls (Chls) in photosystems II and I were studied in the isolated thylakoid membranes of a cyanobacterium, Acaryochloris marina, which contains Chls d and a as the major and minor pigments, respectively. The membranes contained PS I and II complexes at a 1.8:1 molar ratio on the basis of the spin densities on the tyrosine D radical and the photo-oxidized PS I primary donor (P740+). In the presence of ferricyanide, laser excitation induced bleach at 725 nm that recovered with time constants of 25 micros and 1.2 ms. The signal, designated P725, was suppressed by PS II inhibitors DCMU and hydroxylamine. The P725 spectrum was tentatively assigned to the absorption changes of the special pair Chl d, the accessory Chl d, and the acceptor pheophytin a in PS II. The addition of ascorbate induced the additional signal with a slow decay time constant of 4.5 ms. This signal showed a broad bleach at 740 nm and shift-type absorption changes at around 707 and 685 nm, which were assigned to the absorption changes of PS I special pair of Chl d (P740), the accessory Chl d, and the primary acceptor Chl a (A0), respectively. Mechanisms and the evolution of the Chl-d based reaction centers using far-red light are discussed together with the amino acid sequences of PS II D1 and D2 proteins.

Assignment of the Qy Absorption Spectrum of Photosystem-I from Thermosynechococcus elongatus Based on CAM-B3LYP Calculations at the PW91-Optimized Protein Structure

The Qy absorption spectrum of Photosystem-I from Thermosynecochoccus elongatus (formerly Synecochoccus elongatus) is calculated using the CAM-B3LYP density functional and INDO schemes based on a quantum-mechanically refined structure for the entire photosystem obtained using the PW91 density functional. These methods present a priori predictions of the absorption and linear dichroism spectra and include protein electrostatic effects, short range inductive effects, long-range and short-range exciton couplings, and superexchange effects involving aromatic residues and carotenes. CAM-B3LYP is used as it is the only known density functional that correctly describes the Q bands of chlorophylls, all other methods contaminating them with erroneous charge-transfer excitations. A critical feature is found to be the use of fully optimized heavy-atom coordinates, with those obtained from just X-ray crystallography providing a poor description of the electronic properties of the chromophores. The result is a realistic first-principles prediction of the observed absorption band that identifies the nature of the red-shifted chlorophylls as well as the energies of the reaction-center chlorophylls and the exciton couplings acting between them. The "special pair" appears more like a dimer of dimers than a self-contained functional unit, with the exciton couplings between its members and the accessory chlorophylls exceeding the internal coupling.

Analysis of donors of electrons to photosystem I and cyclic electron flow by redox kinetics of P700 in chloroplasts of isolated bundle sheath strands of maize

Bundle sheath chloroplasts of NADP-malic enzyme (NADP-ME) type C4 species have a high demand for ATP, while being deficient in linear electron flow and oxidation of water by photosystem II (PSII). To evaluate electron donors to photosystem I (PSI) and possible pathways of cyclic electron flow (CEF1) in isolated bundle sheath strands of maize (Zea mays L.), an NADP-ME species, light-induced redox kinetics of the reaction center chlorophyll of PSI (P700) were followed under aerobic conditions. Donors of electrons to CEF1 are needed to compensate for electrons lost from the cycle. When stromal electron donors to CEF1 are generated during pre-illumination with actinic light (AL), they retard the subsequent rate of oxidation of P700 by far-red light. Ascorbate was more effective than malate in generating stromal electron donors by AL. The generation of stromal donors by ascorbate was inhibited by DCMU, showing ascorbate donates electrons to the oxidizing side of PSII. The inhibitors of NADPH dehydrogenase (NDH), amytal and rotenone, accelerated the oxidation rate of P700 by far-red light after AL, indicating donation of electrons to the intersystem from stromal donors via NDH. These inhibitors, however, did not affect the steady-state level of P700+ under AL, which represents a balance of input and output of electrons in P700. In contrast, antimycin A, the inhibitor of the ferredoxin-plastoquinone reductase-dependent CEF1, substantially lowered the level of P700+ under AL. Thus, the primary pathway of ATP generation by CEF1 may be through ferredoxin-plastoquinone, while function of CEF1 via NDH may be restricted by low levels of ferredoxin-NADP reductase. NDH may contribute to redox poising of CEF1, or function to generate ATP in linear electron flow to O2 via PSI, utilizing NADPH generated from malate by chloroplastic NADP-ME.

Influence of the Redox Potential of the Primary Quinone Electron Acceptor on Photoinhibition in Photosystem II

We report the characterization of the effects of the A249S mutation located within the binding pocket of the primary quinone electron acceptor, Q(A), in the D2 subunit of photosystem II in Thermosynechococcus elongatus. This mutation shifts the redox potential of Q(A) by approximately -60 mV. This mutant provides an opportunity to test the hypothesis, proposed earlier from herbicide-induced redox effects, that photoinhibition (light-induced damage of the photosynthetic apparatus) is modulated by the potential of Q(A). Thus the influence of the redox potential of Q(A) on photoinhibition was investigated in vivo and in vitro. Compared with the wild-type, the A249S mutant showed an accelerated photoinhibition and an increase in singlet oxygen production. Measurements of thermoluminescence and of the fluorescence yield decay kinetics indicated that the charge-separated state involving Q(A) was destabilized in the A249S mutant. These findings support the hypothesis that a decrease in the redox potential of Q(A) causes an increase in singlet oxygen-mediated photoinhibition by favoring the back-reaction route that involves formation of the reaction center chlorophyll triplet. The kinetics of charge recombination are interpreted in terms of a dynamic structural heterogeneity in photosystem II that results in high and low potential forms of Q(A). The effect of the A249S mutation seems to reflect a shift in the structural equilibrium favoring the low potential form.

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

Core antenna and reaction centre of photosytem 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 3P700 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.

A new fluorescence band F689 in photosystem II revealed by picosecond analysis at 4–77 K: Function of two terminal energy sinks F689 and F695 in PS II

We performed picosecond time-resolved fluorescence spectroscopy in spinach photosystem II (PS II) particles at 4, 40, and 77 K and identified a new fluorescence band, F689. F689 was identified in addition to the well-known F685 and F695 bands in both analyses of decay-associated spectra and global Gaussian deconvolution of time-resolved spectra. Its fast decay suggests the energy transfer directly from F689 to the reaction center chlorophyll P680. The contribution of F689, which increases only at low temperature, explains the unusually broad and variable bandwidth of F695 at low temperature. Global analysis revealed the three types of excitation energy transfer/dissipation processes: (1) energy transfer from the peripheral antenna to the three core antenna bands F685, F689, and F695 with time constants of 29 and 171 ps at 77 and 4 K, respectively; (2) between the three core bands (0.18 and 0.82 ns); and (3) the decays of F689 (0.69 and 3.02 ns) and F695 (2.18 and 4.37 ns). The retardations of these energy transfer rates and the slow F689 decay rate produced the strong blue shift of the PS II fluorescence upon the cooling below 77 K.

Iron transport and signal transduction inPseudomonas aeruginosa: crystal structures of the TonB-dependent outer membrane receptors FpvA and FptA

Crystal and NMR structures are essential and fundamental in performing almost all molecular modelling techniques. Three dimensions resolution of such structures is certainly one of the most crucial criteria of quality and credibility. Researchers made great effort to prepare crystals of photosystem II (PS II) from algae and higher plants in the last decades. However, till now there are only two experimental crystal structures resolved at adequate resolution. Both are from the same common organism Thermosynechococcus elongatus. First was obtained at 3.5 A (PDB code: 1S5L) [1] and second at 3.2 A (PDB code: 1W5C) [2] overall resolution. By performing series of molecular dynamics (MD) simulations at appropriate time scales also coupled partially with quantum-chemical calculations, it is possible to increase the model accuracy mainly in the regions, where the probability of spatial orientation of amino acid side chain lacks appropriate electron density or other sources of experimental data.We present heremore natural-like, geometrically optimised structures of extended reaction centre (RC) of PS II. Recently, changes in excitonic interactions of PS II RC pigments upon light-induced oxidation of primary donor (P680) or reduction of primary acceptor pheophytin a (Phe a), were analysed using absorption and circular dichroism (CD) spectra [3, 4]. In contrast to the oxidation of primary donor, the lightinduced change in the CD spectrum upon primary acceptor reduction was temperature-dependent. This suggests a hypothesis that at a room temperature the reduced Phe a induces conformational changes of the RC protein environment, which affects the excitonic interaction of the RC chlorophylls. Having optimised structural models of PS II RC we were able to elucidate and describe some of the details of these processes.

Ultrafast Transient Absorption Studies on Photosystem I Reaction Centers from Chlamydomonas reinhardtii. 2: Mutations near the P700 Reaction Center Chlorophylls Provide New Insight into the Nature of the Primary Electron Donor

The energy transfer and charge separation kinetics in several core Photosystem I particles of Chlamydomonas reinhardtii with point mutations around the P A and P B reaction center chlorophylls (Chls) have been studied using ultrafast transient absorption spectroscopy in the femtosecond to nanosecond time range to characterize the influence on the early electron transfer processes. The data have been analyzed in terms of kinetic compartment models. The adequate description of the transient absorption kinetics requires three different radical pairs in the time range up to ∼100 ps. Also a charge recombination process from the first radical pair back to the excited state is present in all the mutants, as already shown previously for the wild-type (Müller, M. G., J. Niklas, W. Lubitz, and A. R. Holzwarth. 2003. Biophys. J. 85:3899–3922; and Holzwarth, A. R., M. G. Müller, J. Niklas, and W. Lubitz. 2005. J. Phys. Chem. B. 109:5903–59115). In all mutants, the primary charge separation occurs with the same effective rate constant within the error limits as in the wild-type (»350 ns −1), which implies an intrinsic rate constant of charge separation of <1 ps −1. The rate constant of the secondary electron transfer process is slowed down by a factor of ∼2 in the mutant B-H656C, which lacks the ligand to the central metal of Chl P B. For the mutant A-T739V, which breaks the hydrogen bond to the keto carbonyl of Chl P A, only a slight slowing down of the secondary electron transfer is observed. Finally for mutant A-W679A, which has the Trp near the P A Chl replaced, either no pronounced effect or, at best, a slight increase on the secondary electron transfer rate constants is observed. The effective charge recombination rate constant is modified in all mutants to some extent, with the strongest effect observed in mutant B-H656C. Our data strongly suggest that the Chls of the P A and P B pair, constituting what is traditionally called the “primary electron donor P700”, are not oxidized in the first electron transfer process, but rather only in the secondary electron transfer step. We thus propose a new electron transfer mechanism for Photosystem I where the accessory Chl(s) function as the primary electron donor(s) and the A 0 Chl(s) are the primary electron acceptor(s). This new mechanism also resolves in a straightforward manner the difficulty with the previous mechanism, where an electron would have to overcome a distance of ∼14 Å in <1 ps in a single step. If interpreted within a scheme of single-sided electron transfer, our data suggest that the B-branch is the active branch, although parallel A-branch activity cannot be excluded. All the mutations do affect to a varying extent the energy difference between the reaction center excited state RC* and the first radical pair and thus affect the rate constant of charge recombination. It is interesting to note that the new mechanism proposed is in fact analogous to the electron transfer mechanism in Photosystem II, where the accessory Chl also plays the role of the primary electron donor, rather than the special Chl pair P680 (Prokhorenko, V. and A. R. Holzwarth. 2000. J. Phys. Chem. B. 104:11563–11578).

Photoinactivation of Photosystem II by flashing light

Inhibition of Photosystem II (PS II) activity by single turnover visible light flashes was studied in thylakoid membranes isolated form spinach. Flash illumination results in decreased oxygen evolving activity of PS II, which effect is most pronounced when the water-oxidizing complex is in the S2 and S3 states, and increases with increasing time delay between the subsequent flashes. By applying the fluorescent spin-trap DanePy, we detected the production of singlet oxygen, whose amount was increasing with increasing flash spacing. These findings were explained in the framework of a model, which assumes that recombination of the S2QB - and S3QB - states generate the triplet state of the reaction center chlorophyll and lead to the production of singlet oxygen.

The Formation of Zn-Chl a in Chlorella Heterotrophically Grown in the Dark with an Excessive Amount of Zn2+

Chlorella, when heterotrophically cultivated in the dark, is able to grow with Zn2+ at 10-40 mM, which is 10 times the concentration lethal to autotrophically grown cells. However, the lag phase is prolonged with increasing concentrations of Zn2+; for example, in this study, 1 d of the control lag phase was prolonged to about 16 d with Zn2+ at 16.7 mM (x2,000 of the control). Once the cells started to grow, the log phase was finished within 4-6 d regardless of Zn concentration, which was almost the same as that of the control. The photosysystem I reaction center chlorophyll, P700, and the far-red fluorescence were detected only after the late log phase of the growth curve, suggesting that chlorophyll-protein complexes can be organized after cell division has ceased. Interestingly, at more than 16.7 mM of Zn2+, Zn-chlorophyll a was accumulated and finally accounted for about 25% of the total chlorophyll a in the late stationary phase. We found that the Zn-chlorophyll a was present in the thylakoid membranes and not in the soluble fractions of the cells. The rather low fluorescence yield at around 680 nm in the stationary phase suggests that Zn-chlorophyll a can transfer its excitation energy to other chlorophylls. Before accumulation of Zn-chlorophyll a, a marked amount of pheophytin a was temporally accumulated, suggesting that Zn-chlorophyll a could be chemically synthesized via pheophytin a.

LdpA: a component of the circadian clock senses redox state of the cell

The endogenous 24-h (circadian) rhythms exhibited by the cyanobacterium Synechococcus elongatus PCC 7942 and other organisms are entrained by a variety of environmental factors. In cyanobacteria, the mechanism that transduces environmental input signals to the central oscillator of the clock is not known. An earlier study identified ldpA as a gene involved in light-dependent modulation of the circadian period, and a candidate member of a clock-entraining input pathway. Here, we report that the LdpA protein is sensitive to the redox state of the cell and exhibits electron paramagnetic resonance spectra consistent with the presence of two Fe4S4 clusters. Moreover, LdpA copurifies with proteins previously shown to be integral parts of the circadian mechanism. We also demonstrate that LdpA affects both the absolute level and light-dependent variation in abundance of CikA, a key input pathway component. The data suggest a novel input pathway to the circadian oscillator in which LdpA is a component of the clock protein complex that senses the redox state of a cell.

Pigment Binding, Fluorescence Properties, and Oligomerization Behavior of Lhca5, a Novel Light-harvesting Protein

A new potential light-harvesting protein, named Lhca5, was recently detected in higher plants. Because of the low amount of Lhca5 in thylakoid membranes, the isolation of a native Lhca5 pigment-protein complex has not been achieved to date. Therefore, we used in vitro reconstitution to analyze whether Lhca5 binds pigments and is actually an additional light-harvesting protein. By this approach we could demonstrate that Lhca5 binds pigments in a unique stoichiometry. Analyses of pigment requirements for light-harvesting complex formation by Lhca5 revealed that chlorophyll b is the only indispensable pigment. Fluorescence measurements showed that ligated chlorophylls and carotenoids are arranged in a way that allows directed energy transfer within the light-harvesting complex. Reconstitutions of Lhca5 together with other Lhca proteins resulted in the formation of heterodimers with Lhca1. This result demonstrates that Lhca5 is indeed a protein belonging to the light-harvesting antenna of photosystem I. The properties of Lhca5 are compared with those of previously characterized Lhca proteins, and the consequences of an additional Lhca protein for the composition of the light-harvesting antenna of photosystem I are discussed in view of the recently published photosystem I structure of the pea.

Characterization of photosynthetic electron transport in bundle sheath cells of maize. I. Ascorbate effectively stimulates cyclic electron flow around PSI.

Redox changes of the reaction-center chlorophyll of photosystem I (P700) and chlorophyll fluorescence yield were measured in bundle sheath strands (BSS) isolated from maize (Zea mays L.) leaves. Oxidation of P700 in BSS by actinic light was suppressed by nigericin, indicating the generation of a proton gradient across the thylakoid membranes of BSS chloroplasts. Methyl viologen, which transfers electrons from photosystem I (PSI) to O2, caused a considerable decrease in the reduction rate of P700+ in BSS after turning off actinic light, showing that electron flow from the acceptor side of PSI to stromal components is critical for this reduction. Ascorbate (Asc), and to a lesser extent malate (Mal), caused a lower level of P700+ in BSS under aerobic conditions in far-red light, implying electron donation from these substances to the intersystem carriers. When Asc or Mal was added to BSS during pre-illumination under anaerobic conditions in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), the far-red-induced level of P700+ was lowered. The results suggest Asc and Mal can cause reduction of stromal donors, which in turn establishes conditions for rapid PSI-driven P700+ reduction. Addition of these metabolites also strongly stimulated the development of a proton gradient in thylakoids under aerobic conditions in the absence of DCMU, i.e. under conditions analogous to those in vivo. Ascorbate was a much more effective electron donor than Mal, suggesting it has a physiological role in activation of cyclic electron flow around PSI.

Excitation Dynamics in Eukaryotic PS I from Chlamydomonas reinhardtii CC 2696 at 10 K. Direct Detection of the Reaction Center Exciton States

Excitation energy transfer in PS I particles from the green alga Chlamydomonas reinhardtiiCC 2696 was studied at 10 K by femtosecond transient absorption spectroscopy. Five-nm wide excitation pulses at 670, 680, 695, and 700 nm were applied to selectively excite different spectral forms contributing to the wide Q Y transition band of chlorophyll a. Absorbance changes between 630 and 770 nm, up to 100 ps after excitation, were collected with a time step of 54 fs during the first 5 ps. Excitation at 700 nm leads to a structured initial absorbance difference spectra with four positive bands clearly resolved at 634, 645, 652, and 661 nm, and four negative bands at 667, 675, 684, and 695 nm. These spectra are interpreted in terms of excitonic coupling between the six electron-transfer chlorophyll a molecules: a special pair, two accessory and two A0 chlorophylls. The negative bands were ascribed to photobleaching of the four one-exciton states in line with theoretical predictions (Beddard, G. S. J. Phys. Chem. B. 1998, 102, 10 966), and the positive ones to excited-state absorption. The significance of the broad absorbance changes is proposed to be the introduction of spectral overlap between the reaction center and different spectral forms of the antenna chlorophylls that is expected to increase the efficiency of energy flow to the reaction center. Excitation at different wavelengths shows indeed that trapping can occur from different spectral pools of chlorophylls with similar efficiency (trapping time 29-44 ps). Following the excitation at 670 and 680 nm, trapping was shown to occur from the same pool as at room temperature centered at 682-685 nm, containing apparently only a minority of antenna molecules located close to the reaction center. The trapping time was found to be only slightly longer compared to that at room temperature (20-23 ps at RT). At 10 K, a significant amount of chlorophylls cannot exchange excitation energy with their neighbors. Our results are consistent with previous reports that at cryogenic temperatures, charge separation is possible in 50% of PS I particles and that excitation quenching by the oxidized and reduced primary donor is equally effective. As was observed at room temperature, there is no indication of red chlorophylls absorbing above 700 nm. This lack of red chlorophylls makes it possible to directly excite reaction center chlorophylls and study interaction between them in wild type and, in future, mutant PS I from Chlamydomonas.