An HPLC-based method of estimation of the total redox state of plastoquinone in chloroplasts, the size of the photochemically active plastoquinone-pool and its redox state in thylakoids of Arabidopsis

We have described a direct, high-performance liquid chromatography-based method of estimation of the total level of plastoquinone (PQ) in leaves, the redox state of total (photoactive and non-photoactive) PQ, as well as the redox state of the PQ-pool that is applicable to any illumination conditions. This method was applied to Arabidopsis thaliana leaves but it can be applied to any other plant species. The obtained results show that the level of total PQ was 25 ± 3 molecules/1000 chlorophyll (Chl) molecules in relation to foliar total Chl content. The level of the photoactive PQ, i.e., the PQ-pool, was about 31% of the total PQ present in Arabidopsis leaves that corresponds to about 8 PQ molecules/1000 Chl molecules. The reduction level of the non-photoactive PQ fraction, present outside thylakoids in chloroplasts, was estimated to account for about 49%. The measurements of the redox state of the PQ-pool showed that the pool was reduced during the dark period in about 24%, and during the light period (150 μmol/m²·s) the reduction of the PQ-pool increased to nearly 100%. The obtained results were discussed in terms of the activity of chlororespiration pathways in Arabidopsis and the regulatory role of the redox state of PQ-pool in various physiological and molecular processes in plants.     

Photosynthesis is improved by exogenous calcium in heat-stressed tobacco plants

Effects of exogenous calcium chloride (CaCl₂) (20 mM) on photosynthetic gas exchange, photosystem II photochemistry, and the activities of antioxidant enzymes in tobacco plants under high temperature stress (43 °C for 2 h) were investigated. Heat stress resulted in a decrease in net photosynthetic rate (P_n), stomatal conductance as well as the apparent quantum yield (AQY) and carboxylation efficiency (CE) of photosynthesis. Heat stress also caused a decrease of the maximal photochemical efficiency of primary photochemistry (F_v/F_m). On the other hand, CaCl₂ application improved P_n, AQY, and CE as well as F_v/F_m under high temperature stress. Heat stress reduced the activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), peroxidase (POD), whereas the activities of these enzymes either decreased less or increased in plants pretreated with CaCl₂; glutathione reductase (GR) activity increased under high temperature, and it increased more in plants pretreated with CaCl₂. There was an obvious accumulation of H₂O₂ and O₂⁻ under high temperature, but CaCl₂ application decreased the contents of H₂O₂ and O₂⁻ under heat stress conditions. Heat stress induced the level of heat shock protein 70 (HSP70), while CaCl₂ pretreatment enhanced it. These results suggested that photosynthesis was improved by CaCl₂ application in heat-stressed plants and such an improvement was associated with an improvement in stomatal conductance and the thermostability of oxygen-evolving complex (OEC), which might be due to less accumulation of reactive oxygen species.     

Spin-probes designed for measuring the intrathylakoid pH in chloroplasts

Nitroxide radicals are widely used as molecular probes in different fields of chemistry and biology. In this work, we describe pH-sensitive imidazoline- and imidazolidine-based nitroxides with pK values in the range 4.7-7.6 (2,2,3,4,5,5-hexamethylperhydroimidazol-1-oxyl, 4-amino-2,2,5,5-tetramethyl-2,5-dihydro-1H-imidazol-1-oxyl, 4-dimethylamino-2,2-diethyl-5,5-dimethyl-2,5-dihydro-1H-imidazol-1-oxyl, and 2,2-diethyl-5,5-dimethyl-4-pyrrolidyline-1-yl-2,5-dihydro-1H-imidazol-1-oxyl), which allow the pH-monitoring inside chloroplasts. We have demonstrated that EPR spectra of these spin-probes localized in the thylakoid lumen markedly change with the light-induced acidification of the thylakoid lumen in chloroplasts. Comparing EPR spectrum parameters of intrathylakoid spin-probes with relevant calibrating curves, we could estimate steady-state values of lumen pH_in established during illumination of chloroplasts with continuous light. For isolated bean (Vicia faba) chloroplasts suspended in a medium with pH_out = 7.8, we found that pH_in ≈ 5.4-5.7 in the state of photosynthetic control, and pH_in ≈ 5.7-6.0 under photophosphorylation conditions. Thus, ATP synthesis occurs at a moderate acidification of the thylakoid lumen, corresponding to transthylakoid pH difference ΔpH ≈ 1.8-2.1. These values of ΔpH are consistent with a point of view that under steady-state conditions the proton gradient ΔpH is the main contributor to the proton motive force driving the operation of ATP synthesis, provided that stoichiometric ratio H⁺/ATP is n ≥ 4-4.7.     

Non-photochemical quenching of chlorophyll a fluorescence by oxidised plastoquinone: new evidences based on modulation of the redox state of the endogenous plastoquinone pool in broken spinach chloroplasts

Twenty-five years ago, non-photochemical quenching of chlorophyll fluorescence by oxidised plastoquinone (PQ) was proposed to be responsible for the lowering of the maximum fluorescence yield reported to occur when leaves or chloroplasts were treated in the dark with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of electron flow beyond the primary quinone electron acceptor (Q_A) of photosystem (PS) II [C. Vernotte, A.L. Etienne, J.-M. Briantais, Quenching of the system II chlorophyll fluorescence by the plastoquinone pool, Biochim. Biophys. Acta 545 (1979) 519-527]. Since then, the notion of PQ-quenching has received support but has also been put in doubt, due to inconsistent experimental findings. In the present study, the possible role of the native PQ-pool as a non-photochemical quencher was reinvestigated, employing measurements of the fast chlorophyll a fluorescence kinetics (from 50 μs to 5 s). The about 20% lowering of the maximum fluorescence yield F_M, observed in osmotically broken spinach chloroplasts treated with DCMU, was eliminated when the oxidised PQ-pool was non-photochemically reduced to PQH₂ by dark incubation of the samples in the presence of NAD(P)H, both under anaerobic and aerobic conditions. Incubation under anaerobic conditions in the absence of NAD(P)H had comparatively minor effects. In DCMU-treated samples incubated in the presence of NAD(P)H fluorescence quenching started to develop again after 20-30 ms of illumination, i.e., the time when PQH₂ starts getting reoxidised by PS I activity. NAD(P)H-dependent restoration of F_M was largely, if not completely, eliminated when the samples were briefly (5 s) pre-illuminated with red or far-red light. Addition to the incubation medium of HgCl₂ that inhibits dark reduction of PQ by NAD(P)H also abolished NAD(P)H-dependent restoration of F_M. Collectively, our results provide strong new evidence for the occurrence of PQ-quenching. The finding that DCMU alone did not affect the minimum fluorescence yield F₀ allowed us to calculate, for different redox states of the native PQ-pool, the fractional quenching at the F₀ level (Q₀) and to compare it with the fractional quenching at the F_M level (Q_M). The experimentally determined Q₀/Q_M ratios were found to be equal to the corresponding F₀/F_M ratios, demonstrating that PQ-quenching is solely exerted on the excited state of antenna chlorophylls.     

The redox state of the plastoquinone pool directly modulates minimum chlorophyll fluorescence yield in Chlamydomonas reinhardtii

The effect of the plastoquionone (PQ) pool oxidation state on minimum chlorophyll fluorescence was studied in the green alga Chlamydomonas reinhardtii. In wild type and a mutant strain that lacks both photosystems but retains light harvesting complexes, oxygen depletion induced a rise in minimum chlorophyll fluorescence. An increase in minimum fluorescence yield is also observed when the PQ pool becomes reduced in the presence of oxygen and after application of an ionophore that collapses the transmembrane proton gradient. Together these results indicate that minimum chlorophyll fluorescence is modulated by the PQ oxidation state.     

Substrate water exchange in photosystem II core complexes of the extremophilic red alga Cyanidioschyzon merolae

The binding affinity of the two substrate–water molecules to the water-oxidizing Mn₄CaO₅ catalyst in photosystem II core complexes of the extremophilic red alga Cyanidioschyzon merolae was studied in the S₂ and S₃ states by the exchange of bound ¹⁶O-substrate against ¹⁸O-labeled water. The rate of this exchange was detected via the membrane-inlet mass spectrometric analysis of flash-induced oxygen evolution. For both redox states a fast and slow phase of water-exchange was resolved at the mixed labeled m/z 34 mass peak: k_f = 52 ± 8 s^− 1 and k_s = 1.9 ± 0.3 s^− 1 in the S₂ state, and k_f = 42 ± 2 s^− 1 and k_slow = 1.2 ± 0.3 s^− 1 in S₃, respectively. Overall these exchange rates are similar to those observed previously with preparations of other organisms. The most remarkable finding is a significantly slower exchange at the fast substrate–water site in the S₂ state, which confirms beyond doubt that both substrate–water molecules are already bound in the S₂ state. This leads to a very small change of the affinity for both the fast and the slowly exchanging substrates during the S₂ → S₃ transition. Implications for recent models for water-oxidation are briefly discussed.     

H2O2 induces rapid biophysical and permeability changes in the plasma membrane of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the diffusion rate of hydrogen peroxide (H₂O₂) through the plasma membrane decreases during adaptation to H₂O₂ by means of a mechanism that is still unknown. Here, evidence is presented that during adaptation to H₂O₂ the anisotropy of the plasma membrane increases. Adaptation to H₂O₂ was studied at several times (15min up to 90min) by applying the steady-state H₂O₂ delivery model. For wild-type cells, the steady-state fluorescence anisotropy increased after 30min, or 60min, when using 2-(9-anthroyloxy) stearic acid (2-AS), or diphenylhexatriene (DPH) membrane probe, respectively. Moreover, a 40% decrease in plasma membrane permeability to H₂O₂ was observed at 15min with a concomitant two-fold increase in catalase activity. Disruption of the ergosterol pathway, by knocking out either ERG3 or ERG6, prevents the changes in anisotropy during H₂O₂ adaptation. H₂O₂ diffusion through the plasma membrane in S. cerevisiae cells is not mediated by aquaporins since the H₂O₂ permeability constant is not altered in the presence of the aquaporin inhibitor mercuric chloride. Altogether, these results indicate that the regulation of the plasma membrane permeability towards H₂O₂ is mediated by modulation of the biophysical properties of the plasma membrane.     

Non-photochemical reduction of thylakoid photosynthetic redox carriers in vitro: Relevance to cyclic electron flow around photosystem I?

Non-photochemical (dark) increases in chlorophyll a fluorescence yield associated with non-photochemical reduction of redox carriers (F_npr) have been attributed to the reduction of plastoquinone (PQ) related to cyclic electron flow (CEF) around photosystem I. In vivo, this rise in fluorescence is associated with activity of the chloroplast plastoquinone reductase (plastid NAD(P)H:plastoquinone oxidoreductase) complex. In contrast, this signal measured in isolated thylakoids has been attributed to the activity of the protein gradient regulation-5 (PGR5)/PGR5-like (PGRL1)-associated CEF pathway. Here, we report a systematic experimentation on the origin of F_npr in isolated thylakoids. Addition of NADPH and ferredoxin to isolated spinach thylakoids resulted in the reduction of the PQ pool, but neither its kinetics nor its inhibitor sensitivities matched those of F_npr. Notably, F_npr was more rapid than PQ reduction, and completely insensitive to inhibitors of the PSII Q_B site and oxygen evolving complex as well as inhibitors of the cytochrome b₆f complex. We thus conclude that F_npr in isolated thylakoids is not a result of redox equilibrium with bulk PQ. Redox titrations and fluorescence emission spectra imply that F_npr is dependent on the reduction of a low potential redox component (E_m about − 340 mV) within photosystem II (PSII), and is likely related to earlier observations of low potential variants of Q_A within a subpopulation of PSII that is directly reducible by ferredoxin. The implications of these results for our understanding of CEF and other photosynthetic processes are discussed.     

Study on the heterogeneous degradation of chitosan with H2O2 catalyzed by a new supermolecular assembly crystal: [C6H8N2]6H3[PW12O40]·2H2O

A new supermolecular assembly crystal, [C₆H₈N₂]₆H₃[PW₁₂O₄₀]·2H₂O (DMB-PWA), was synthesized with phosphotungstic acid (PWA) and 1,2-diaminobenzene (DMB) under hydrothermal conditions and was characterized by Fourier-transform infrared spectra (FTIR) and single-crystal X-ray diffraction analysis. DMB-PWA could effectively catalyze oxidative degradation of chitosan with H₂O₂ in the heterogeneous phase. The optimum degradation conditions were determined by orthogonal tests as follows: amount of chitosan 1.00 g, 30% (wt %); H₂O₂, 3.0 mL; dosage of catalyst, 0.06 g; reaction temperature, 85 °C; and reaction time, 30 min. The water-soluble chitosan with a viscosity-average molecular weight (M_v) of 4900 was obtained under the optimum degradation conditions and was characterized by FTIR, ultraviolet-visible diffuse reflection spectra (UV-vis DRS), and X-ray powder diffraction analysis.     

Species-dependence of the redox potential of the primary quinone electron acceptor QA in photosystem II verified by spectroelectrochemistry

The redox potentials E_m(Q_A/) of the primary quinone electron acceptor Q_A in oxygen-evolving photosystem II complexes of three species were determined by spectroelectrochemistry. The E_m(Q_A/) values were experimentally found to be −162 ± 3 mV for a higher plant spinach, −171 ± 3 mV for a green alga Chlamydomonas reinhardtii and −104 ± 4 mV vs. SHE for a red alga Cyanidioschyzonmerolae. On the basis of possible deviations for the experimental values, as estimated to differ by 9-29 mV from each true value, plausible causes for such remarkable species-dependence of E_m(Q_A/) are discussed, mainly by invoking the effects of extrinsic subunits on the delicate structural environment around Q_A.     

Wavelength dependence of the fluorescence emission under conditions of open and closed Photosystem II reaction centres in the green alga Chlorella sorokiniana

The fluorescence emission characteristics of the photosynthetic apparatus under conditions of open (F₀) and closed (F_M) Photosystem II reaction centres have been investigated under steady state conditions and by monitoring the decay lifetimes of the excited state, in vivo, in the green alga Chlorella sorokiniana. The results indicate a marked wavelength dependence of the ratio of the variable fluorescence, F_V = F_M − F₀, over F_M, a parameter that is often employed to estimate the maximal quantum efficiency of Photosystem II. The maximal value of the F_V/F_M ratio is observed between 660 and 680 nm and the minimal in the 690–730 nm region. It is possible to attribute the spectral variation of F_V/F_M principally to the contribution of Photosystem I fluorescence emission at room temperature. Moreover, the analysis of the excited state lifetime at F₀ and F_M indicates only a small wavelength dependence of Photosystem II trapping efficiency in vivo.     

Kappaphycus alvarezii as a source of bioethanol

The present study describes production of bio-ethanol from fresh red alga, Kappaphycus alvarezii. It was crushed to expel sap - a biofertilizer - while residual biomass was saccharified at 100 °C in 0.9 N H₂SO₄. The hydrolysate was repeatedly treated with additional granules to achieve desired reducing sugar concentration. The best yields for saccharification, inclusive of sugar loss in residue, were 26.2% and 30.6% (w/w) at laboratory (250 g) and bench (16 kg) scales, respectively. The hydrolysate was neutralized with lime and the filtrate was desalted by electrodialysis. Saccharomyces cerevisiae (NCIM 3523) was used for ethanol production from this non-traditional bio-resource. Fermentation at laboratory and bench scales converted ca. 80% of reducing sugar into ethanol in near quantitative selectivity. A petrol vehicle was successfully run with E10 gasoline made from the seaweed-based ethanol. Co-production of ethanol and bio-fertilizer from this seaweed may emerge as a promising alternative to land-based bio-ethanol.     

In vitro and in vivo pharmacology of CP-945,598, a potent and selective cannabinoid CB1 receptor antagonist for the management of obesity

Cannabinoid CB₁ receptor antagonists exhibit pharmacologic properties favorable for the treatment of metabolic disease. CP-945,598 (1-[9-(4-chlorophenyl)-8-(2-chlorophenyl)-9H-purin-6-yl]-4-ethylamino piperidine-4-carboxylic acid amide hydrochloride) is a recently discovered selective, high affinity, competitive CB₁ receptor antagonist that inhibits both basal and cannabinoid agonist-mediated CB₁ receptor signaling in vitro and in vivo. CP-945,598 exhibits sub-nanomolar potency at human CB₁ receptors in both binding (K_i = 0.7 nM) and functional assays (K_i = 0.2 nM). The compound has low affinity (K_i = 7600 nM) for human CB₂ receptors. In vivo, CP-945,598 reverses four cannabinoid agonist-mediated CNS-driven responses (hypo-locomotion, hypothermia, analgesia, and catalepsy) to a synthetic cannabinoid receptor agonist. CP-945,598 exhibits dose and concentration-dependent anorectic activity in two models of acute food intake in rodents, fast-induced re-feeding and spontaneous, nocturnal feeding. CP-945,598 also acutely stimulates energy expenditure in rats and decreases the respiratory quotient indicating a metabolic switch to increased fat oxidation. CP-945,598 at 10 mg/kg promoted a 9%, vehicle adjusted weight loss in a 10 day weight loss study in diet-induced obese mice. Concentration/effect relationships combined with ex vivo brain CB₁ receptor occupancy data were used to evaluate efficacy in behavioral, food intake, and energy expenditure studies. Together, these in vitro, ex vivo, and in vivo data indicate that CP-945,598 is a novel CB₁ receptor competitive antagonist that may further our understanding of the endocannabinoid system.     

Field-acclimated Gossypium hirsutum cultivars exhibit genotypic and seasonal differences in photosystem II thermostability

Previous investigations have demonstrated that photosystem II (PSII) thermostability acclimates to prior exposure to heat and drought, but contrasting results have been reported for cotton (Gossypium hirsutum). We hypothesized that PSII thermotolerance in G. hirsutum would acclimate to environmental conditions during the growing season and that there would be differences in PSII thermotolerance between commercially-available U.S. cultivars. To this end, three cotton cultivars were grown under dryland conditions in Tifton Georgia, and two under irrigated conditions in Marianna Arkansas. At Tifton, measurements included PSII thermotolerance (T₁₅, the temperature causing a 15% decline in maximum quantum yield), leaf temperatures, air temperatures, midday (1200 to 1400 h) leaf water potentials (Ψ_MD), leaf-air vapor pressure deficit (VPD), actual quantum yield (Φ_PSII) and electron transport rate through PSII (ETR) on three sample dates. At Marianna, T₁₅ was measured on two sample dates. Optimal air and leaf temperatures were observed on all sample dates in Tifton, but PSII thermotolerance increased with water deficit conditions (Ψ_MD = −3.1 MPa), and ETR was either unaffected or increased under water-stress. Additionally, T₁₅ for PHY 499 was ∼5 °C higher than for the other cultivars examined (DP 0912 and DP 1050). The Marianna site experienced more extreme high temperature conditions (20–30 days T_max ≥ 35 °C), and showed an increase in T₁₅ with higher average T_max. When average T₁₅ values for each location and sample date were plotted versus average daily T_max, strong, positive relationships (r² from .954 to .714) were observed between T_max and T₁₅. For all locations T₁₅ was substantially higher than actual field temperature conditions. We conclude that PSII thermostability in G. hirsutum acclimates to pre-existing environmental conditions; PSII is extremely tolerant to high temperature and water-deficit stress; and differences in PSII thermotolerance exist between commercially-available cultivars.     

Bifunctional chelates with mixed aromatic and aliphatic amine donors for labeling of biomolecules with the {Tc(CO)3} and {Re(CO)3} cores

A series of tridentate ligands consisting of mixed aromatic and aliphatic amine derivatives of single amino acid chelates and phenylpiperazine have been developed, and their reactions with [NEt₄]₂[ReBr₃(CO)₃] have been investigated. The compounds [Re(CO)₃{(NC₅H₄CH₂)NCH₃(C₂H₄)NHCH₃}]Br (4), [Re(CO)₃{(NC₅H₄CH₂)NCH₃(C₂H₄)NCH₃(CH₂)_xCOOC₂H₅}]Br (x = 1, 5; x = 4, 6) [Re(CO)₃{(NC₅H₄CH₂)NH(C₂H₄)N(CH₃)₂}]Br (7), [Re(CO)₃{(NC₅H₄CH₂)N(CH ₂COOC₂H₅)(C₂H₄)N(CH₃)₂}]Br (8) and [Re(CO)₃(NC₅H₄CH₂)(C₂H₄NH₂)N(CH₂)₃-CH₃Ophenpip]Br (9) (phenpip: phenylpiperazine, -C₆H₄-(CH₂CH₂)₂N-) were prepared and characterized by elemental analysis, NMR, IR, HSMS and X-ray crystallography. All complexes exhibit fac-{Re(CO)₃N₃} coordination geometry in the cationic molecular unit. Crystal data for C₁₃H₁₇BrN₃O₃Re (4): orthorhombic, Pbca, a = 13.4510(8) Å, b = 10.5728(6) Å, c = 22.5378(13) Å, V = 3205.2(3) ų, Z = 8; C₁₇H₂₃BrN₃O₅Re (5): orthorhombic, Pcca, a = 16.5907(7) Å,b = 14.8387(6) Å, c = 16.7075(7) Å, V = 4113.1(3) ų, Z = 8; C₁₃H₂₅BrN₃O₇Re (7 · 4H₂O): monoclinic, P2₁/n, a = 14.0698(17) Å, b = 9.6760(12) Å, c = 15.6099 (19) Å, β = 114.930(2)°, V = 1927.1(4) ų, Z = 4; C₁₇H₂₃BrN₃O₅Re (8): monoclinic, P2₁/n, a = 7.5312(5) Å, b = 16.0366(10) Å, c = 16.8741(10) Å, β = 98.9990(10)°, V = 2012.9(2) ų, Z = 4.     

ATP binding and hydrolysis steps of the uni-site catalysis by the mitochondrial F1-ATPase are affected by inorganic phosphate

The effect of inorganic phosphate (P_i) on uni-site ATP binding and hydrolysis by the nucleotide-depleted F₁-ATPase from beef heart mitochondria (ndMF₁) has been investigated. It is shown for the first time that P_i decreases the apparent rate constant of uni-site ATP binding by ndMF₁ 3-fold with the K_d of 0.38 ± 0.14 mM. During uni-site ATP hydrolysis, P_i also shifts equilibrium between bound ATP and ADP + P_i in the direction of ATP synthesis with the K_d of 0.17 ± 0.03 mM. However, 10 mM P_i does not significantly affect ATP binding during multi-site catalysis.     

Kinetic studies of the reduction of hexaaquacobalt(III) perchlorate by a cobaloxime, [Co(dmgBF2)2(H2O)2]

The reaction of with Co(dmgBF₂)₂(H₂O)₂ in 1.0 M HClO₄/LiClO₄ was found to be first-order in both reactants and the [H⁺] dependence of the second-order rate constant is given by k_2obs = b/[H⁺], b at 25 °C is 9.23 ± 0.14 × 10² s⁻¹. The [H⁺] dependence at lower temperatures shows some saturation effect that allowed an estimate of the hydrolysis constant for as K_a = 9.5 × 10⁻³ M at 10 and 15 °C. Marcus theory and the known self-exchange rate constant for Co(OH₂)₅OH^2+/+ were used to estimate an electron self-exchange rate constant of k₂₂ = 1.7 × 10⁻⁴ M⁻¹ s⁻¹ for .     

Conversion of the g = 4.1 EPR signal to the multiline conformation during the S2 to S3 transition of the oxygen evolving complex of Photosystem II

The oxygen evolving complex of Photosystem II undergoes four light-induced oxidation transitions, S₀-S₁,…,S₃-(S₄)S₀ during its catalytic cycle. The oxidizing equivalents are stored at a (Mn)₄Ca cluster, the site of water oxidation. EPR spectroscopy has yielded valuable information on the S states. S₂ shows a notable heterogeneity with two spectral forms; a g = 2 (S = 1/2) multiline, and a g = 4.1 (S = 5/2) signal. These oscillate in parallel during the period-four cycle. Cyanobacteria show only the multiline signal, but upon advancement to S₃ they exhibit the same characteristic g = 10 (S = 3) absorption with plant preparations, implying that this latter signal results from the multiline configuration. The fate of the g = 4.1 conformation during advancement to S₃ is accordingly unknown. We searched for light-induced transient changes in the EPR spectra at temperatures below and above the half-inhibition temperature for the S₂ to S₃ transition (ca 230 K). We observed that, above about 220 K the g = 4.1 signal converts to a multiline form prior to advancement to S₃. We cannot exclude that the conversion results from visible-light excitation of the Mn cluster itself. The fact however, that the conversion coincides with the onset of the S₂ to S₃ transition, suggests that it is triggered by the charge-separation process, possibly the oxidation of tyr Z and the accompanying proton relocations. It therefore appears that a configuration of (Mn)₄Ca with a low-spin ground state advances to S₃.     

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

Absorbance difference spectroscopy and redox titrations have been applied to investigate the properties of photosystem I from the chlorophyll d containing cyanobacterium Acaryochloris marina. At room temperature, the (P740⁺ − P740) and (F_A/B⁻ − F_A/B) absorbance difference spectra were recorded in the range between 300 and 1000 nm while at cryogenic temperatures, (P740⁺A₁⁻ − P740A₁) and (³P740 − 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 (A₁⁻), 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:~ 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.     

Replacement of the methionine axial ligand to the primary electron acceptor A0 slows the A0 reoxidation dynamics in Photosystem I

The recent crystal structure of photosystem I (PSI) from Thermosynechococcus elongatus shows two nearly symmetric branches of electron transfer cofactors including the primary electron donor, P₇₀₀, and a sequence of electron acceptors, A, A₀ and A₁, bound to the PsaA and PsaB heterodimer. The central magnesium atoms of each of the putative primary electron acceptor chlorophylls, A₀, are unusually coordinated by the sulfur atom of methionine 688 of PsaA and 668 of PsaB, respectively. We [Ramesh et al. (2004a) Biochemistry 43:1369-1375] have shown that the replacement of either methionine with histidine in the PSI of the unicellular green alga Chlamydomonas reinhardtii resulted in accumulation of A₀⁻ (in 300-ps time scale), suggesting that both the PsaA and PsaB branches are active. This is in contrast to cyanobacterial PSI where studies with methionine-to-leucine mutants show that electron transfer occurs predominantly along the PsaA branch. In this contribution we report that the change of methionine to either leucine or serine leads to a similar accumulation of A₀⁻ on both the PsaA and the PsaB branch of PSI from C. reinhardtii, as we reported earlier for histidine mutants. More importantly, we further demonstrate that for all the mutants under study, accumulation of A₀⁻ is transient, and that reoxidation of A₀⁻ occurs within 1-2 ns, two orders of magnitude slower than in wild type PSI, most likely via slow electron transfer to A₁. This illustrates an indispensable role of methionine as an axial ligand to the primary acceptor A₀ in optimizing the rate of charge stabilization in PSI. A simple energetic model for this reaction is proposed. Our findings support the model of equivalent electron transfer along both cofactor branches in Photosystem I.