INHIBITION OF ELECTRON TRANSPORT IN CHLOROPLASTS BY CHAOTROPIC AGENTS AND THE USE OF MANGANESE AS AN ELECTRON DONOR TO PHOTOSYSTEM II*

Abstract— Incubating spinach chloroplasts with various chaotropic agents results in inhibition of photosynthetic electron transport between water and Photosystem II similar to the inhibition caused by washing chloroplasts with a high concentration of Tris buffer. Partial restoration of NADP photoreduction and fluorescence of variable yield is achieved by adding hydroquinone or Mn²⁺, either of which donates electrons to Photosystem II in the inhibited chloroplasts. The inhibitory treatments cause the release of Mn from its bound state in the chloroplast, thus allowing the measurement of the ESR signal of Mn²⁺. The ESR measurement is used to follow the photooxidation of Mn²⁺ as it donates electrons to photosystem II.     

LIMITING REACTIONS IN HYDROGEN PHOTOPRODUCTION BY CHLOROPLASTS AND HYDROGENASE

Abstract— Hydrogen was photoproduced from water in a system containing isolated chloroplasts, hy-drogenase, a coupling electron carrier (ferredoxin or methyl viologen), and an oxygen scavenger. The rate and extent of hydrogen production anaerobically was much less than the rate of aerobic electron-carrier reduction by chloroplasts and was not limited by hydrogenase. The limiting reaction in the coupled system was the extent of reduction of methyl viologen anaerobically rather than its oxidation by oxygen produced during the course of the reaction. Inhibition of photosystem II by 3-(3,4dichlorophenyl)-1,1-dimethylurea and addition of a photosystem 1 electron donor did not lead to photoproduction of hydrogen or photoreduction of methyl viologen. Extensive photosystem I hydrogen evolution was obtained when thiols were also present. Platinum asbestos or palladium asbestos replaced hydrogenase in a system coupled to chloroplasts.     

Artificial photosynthesis by using chloroplasts from spinach adsorbed on a nanocrystalline TiO2 electrode for photovoltaic conversion

Photovoltaic conversion has been achieved by use of chloroplasts (photosynthetic organs) from spinach adsorbed on a nanocrystalline TiO₂ film on an indium tin oxide (ITO) glass electrode (chloroplast/TiO₂ electrode). The shape of the absorption spectrum of the chloroplast/TiO₂ electrode is almost the same that of a dispersion of the chloroplasts. Absorption maxima of the chloroplast/TiO₂ electrode observed at 430, 475, and 670 nm were attributed to carotenoid and chlorophyll molecules, suggesting that chloroplasts have been adsorbed by the nanocrystalline TiO₂ film on the ITO electrode. The photocurrent responses of chloroplast/TiO₂ electrodes were measured by using a solution of 0.1 M tetrabutylammonium hexafluorophosphate in acetonitrile as redox electrolyte in the presence or absence of water and 100 mW cm^?2 irradiation. The photocurrent of the chloroplast/TiO₂ electrode was increased by adding water to the redox electrolyte. The photocurrent responses of chloroplast/TiO₂ electrodes irradiated with monochromatic light (680 nm, the absorption band of photosystem II complexed with evolved oxygen) were measured by use of a solution of 0.1 M tetrabutylammonium hexafluorophosphate in acetonitrile as redox electrolyte in the presence or absence of water. A chloroplast/TiO₂ electrode photocurrent was observed only when the redox electrolyte containing water was used, indicating that the oxygen evolved from water by photosystem II in chloroplasts adsorbed by a nanocrystalline TiO₂ film on an ITO electrode irradiated at 680 nm is reduced to water by the catalytic activity of the platinum electrode. The maximum incident photon-to-current conversion efficiency (IPCE) was 0.8 % on irradiation at 670 nm.     

Flavin-mediated photoinactivation of spinach leaf nitrate reductase involving superoxide radical and activating effect of hydrogen peroxide

Irradiation with white light of spinach leaf nitrate reductase (NR) in the presence of flavin mononucleotide (FMN) and ethylenediaminetetraacetic acid (EDTA) resulted in a gradual loss of the enzyme activity, measured with reduced methyl viologen as electron donor. Inactivation of NR was dependent on oxygen and was prevented by superoxide dismutase. On the contrary, the presence of catalase markedly enhanced the rate of inactivation. Nitrate showed a protective effect. NR previously inactivated by irradiation in the presence of FMN and EDTA was greatly reactivated by a short preincubation of the inactive enzyme with either ferricyanide or H₂O₂. These results suggest that spinach leaf NR is reversibly inactivated by photogenerated O₂ , while H₂O₂ has an activating effect.     

PHOTOSYNTHETIC ACTIVITY AND CHLOROPHYLL ABSORPTION SPECTRA OF FRACTIONS FROM THE ALGA,DUNALIELLA*

Abstract— Dunaliella chloroplasts were fractionated according to C. Arntzen et al, Biochim. Biophys. Acta 256 , 85–107, 1972. The initial French-press treatment and differential centrifugation produced Fraction 1 (Fr 1) enriched in photosystem I activity and a heavier Fraction 2 (Fr 2). When Fr 2 was treated with digitonin followed by either gradient or differential centrifugation, two more fractions were recovered: Fr 1 g with a photosystem 1 activity similar to that of Fr 1, and Fr 2 g with very low photosystem II activity. Photosystem II activity was considerably lower in these Dunaliella chloroplasts and fractions than in spinach particles measured under the same conditions, but the relative activities between the fractions were similar to those for spinach. Fr 2 always had greater photosystem II activity than Fr 1, but the digitonin fractions were low and similar in photosystem II activity. Photosystem II activity was measured as the reduction of 2, 6–dichlorophenol indophenol (DCIP) with H₂O, diphenylcarbazide (DPC) or Mn²⁺ as electron donor. The results indicated that exogenous manganous ion competed with H₂O as an electron donor to photosystem II in broken chloroplasts initially, but after 10–15 s of illumination, the Mn³⁺ formed began to reoxidize DCIP and a cyclic reaction ensued. DPC and Mn²⁺ appeared to react at different sites. Computer-assisted curve analysis of the absorption spectrum of each fraction revealed four major component curves representing the absorbing forms of chlorophyll a at 663, 670, 679 and 684 nm seen in numerous other in vivo chlorophyll spectra (C. S. French et al., Plant Physiol. 49 , 421–429, 1972). However, Fr 2g had approx. 20 percent more of Ca663 and Ca670 and 10% more absorption by chl b than Fr 1 which correlated with the difference in photosystem II activity. On the long wavelength side, Fr 2 g had no Ca694 and almost no photosystem I activity. The results are not sufficient to answer the question of whether the photosystem I particle obtained from the original homogenate is significantly similar to or different from the corresponding fraction obtained from Fr 2 with digitonin.     

BASF 13.338 INDUCED CHANGES IN STRUCTURE and FUNCTION OF THE PHOTOSYNTHETIC APPARATUS OF WHEAT SEEDLINGS

Abstract— Growing wheat seedlings in the presence of BASF 13.338 [4-chloro-5-dimethylamino-2-phenyl-3(2H)pyridazinone], a PS II inhibitor of the pyridazinone group, brought about notable changes in the structure and functioning of photosynthetic apparatus. In BASF 13.338 treated plants, there was a decrease in the ratio of Chi a/Chl b, an increase in xanthophyll/carotene ratio and an increase in the content of Cyt b 559 (HP + LP). Chl/p700 ratio increased when measured with the isolated chloroplasts but not with the isolated PS I particles of the treated plants. The SDS-PAGE pattern of chloroplast preparations showed an increase in the CPII/CP I ratio. The F685/F740 ratio in the emission spectrum of chloroplasts at -196°C increased. The difference absorption spectrum of chloroplasts between the control and the treated plants showed a relative increase of a chlorophyll component with a peak absorption at 676 nm and a relative decrease of a chlorophyll component with a peak absorption at 692 nm for the treated plants. The excitation spectra of these chloroplast preparations were similar. Chloroplasts from the treated plants exhibited a greater degree of grana stacking as measured by the chlorophyll content in the 10 K pellet. The rate of electron transfer through photosystem II at saturating light intensity in chloroplast thylakoids isolated from the treated plants increased (by 50%) optimally at treatment of 125 μM BASF 13.338 as compared to the control. This increase was accompanied by an increase in (a) I₅₀ value of DCMU inhibition of photosystem II electron transfer; (b) the relative quantum yield of photosystem II electron transfer; (c) the magnitude of C550 absorbance change; and (d) the rate of carotenoid photobleaching. These observations were interpreted in terms of preferential synthesis of photosystem II in the treated plants. The rate of electron transfer through photosystems I and through the whole chain (H₂O → methyl viologen) also increased, due to an additional effect of BASF 13.338, namely, an increase in the rate of electron transfer through the rate limiting step (between plastoquinol and cytochrome f). This was linked to an enhanced level of functional cytochrome f. The increase in the overall rate of electron transfer occurred in spite of a decrease in the content of photosystem I relative to photosystem II. Treatment with higher concentrations (> 125 μM) of BASF 13.338 caused a further increase in the level of cytochrome f, but the rate of electron transfer was no greater than in the control. This was due to an inhibition of electron transfer at several sites in the chain.     

Hydrogen evolution from water by use of viologen polymers as electron transfer catalyst

The behavior of viologen polymer (P-V²⁺) as an electron transfer catalyst in the reaction of hydrogen generation was studied. In the photoirradiation system, which contains triethanolamine (TEA), Ru(bpy)³⁺₃, and P-V²⁺, the amount of hydrogen evolution was less than methyl viologen (MV²⁺); P-V²⁺, however, was more effective in sodium dithionite as the electron donor and showed higher initial rates than MV²⁺.     

Photo-Activating Biomimetic Polyoxomolybdate for Boosting Oxygen Evolution in Neutral Electrolytes

Inspired by the metal-oxo cluster structural feature and charge separation behaviour of the oxygen evolving center (OEC) in photosystem II (PS-II) under photoirradiation, a new crystalline photochromic polyoxomolybdate, MV₂[β-Mo₈O₂₆] ( 1 , MV=methyl viologen cation), is designed as a biomimetic oxygen evolution reaction (OER) catalyst in neutral electrolytes. After photoinduced electron transfer (PIET) with colour change from colourless to grey, it remains in an ultra-stable charge-separated state over a year under ambient conditions. The observed overpotential at 10 mA ⋅ cm⁻² and Tafel slope decrease by 49 mV and 62.8 mV ⋅ dec⁻¹ after coloration, respectively. The outstanding OER performance of the coloured state in neutral electrolytes even outperforms the commercial RuO₂ benchmark. Experimental and theoretical studies show that oxygen holes within polyanions after irradiation serve as sites for enhancing direct O−O coupling, thus effectively promoting OER. This is the first successful application of electron-transfer photochromism to realize OER activity gain.     

Comparative study of the photochemistry of chloroplast membranes and photosystem II particles

A comparative study of the photoreducing potentials of spinach thylakoid membranes and spinach photosystem II particles has been made. Hexachloroplatinate ions have been used as electron acceptors in a Hill-like assay for oxygen evolution measurements with both thylakoid membranes and photosystem II particles. However, unlike other Hill acceptors, such as ferricyanide, hexachloroplatinate can be fully reduced to metallic platinum that is catalytically active for hydrogen evolution. This is experimentally confirmed in the ability of chloroplast membranes to photoprecipitate platinum and photoproduce molecular hydrogen. Although similar experiments with photosystem II particles resulted in hexachloroplatinate-supported oxygen evolution, hydrogen evolution was not observed. Moreover, photosystem II particles coupled to ferredoxin and hydrogenase resulted in neither hydrogen nor oxygen evolution—a distinct contrast to the results obtained with chloroplast membranes.

     

Halocadmium Clusters with Different Linkage Modes Templated by Methyl Viologen: Structure,Photoluminescence, and Thermochromism

A new halocadmium/methyl viologen hybrid (MV)₄(Cd₂I₆)(Cd₂I₇)₂ (1) (MV²⁺ = methyl viologen) has been structurally determined. In 1, two kinds of dimeric halocadmium clusters constructed from edge/vertex-sharing CdI₄ tetrahedra respectively, i.e., Cd₂I₆ ^2? and Cd₂I₇ ^? dimmers were observed, which were templated by methyl viologen cation (MV²⁺). A highly interesting feature of 1 is the presence of mixed type dimeric halocadmium clusters in one crystal lattice templated by one kind of cation. 1 displays photoluminescences, and its thermochromism behavior is observed, whose mechanism was verified by theoretical calculations.     

Radical‐Cation Dimerization Overwhelms Inclusion in [n]Pseudorotaxanes

Suppression of the dimerization of the viologen radical cation by cucurbit[7]uril ( CB7 ) in water is a well‐known phenomenon. Herein, two counter‐examples are presented. Two viologen‐containing thread molecules were designed, synthesized, and thoroughly characterized by ¹H DOSY NMR spectrometry, UV/Vis absorption spectrophotometry, square‐wave voltammetry, and chronocoulometry: BV ⁴⁺, which contains two viologen subunits, and HV ¹²⁺, which contains six. In both threads, the viologen subunits are covalently bonded to a hexavalent phosphazene core. The corresponding [3]‐ and [7]pseudorotaxanes that form on complexation with CB7 , that is, BV ⁴⁺?( CB 7)₂ and HV ¹²⁺?( CB 7)₆, were also analyzed. The properties of two monomeric control threads, namely, methyl viologen ( MV ²⁺) and benzyl methyl viologen ( BMV ²⁺), as well as their [2]pseudorotaxane complexes with CB7 ( MV ²⁺? CB7 and BMV ²⁺? CB7 ) were also investigated. As expected, the control pseudorotaxanes remained intact after one‐electron reduction of their viologen‐recognition stations. In contrast, analogous reduction of BV ⁴⁺?( CB 7)₂ and HV ¹²⁺?( CB 7)₆ led to host–guest decomplexation and release of the free threads BV ^{2( . +)} and HV ^{6( . +)}, respectively. ¹H DOSY NMR spectrometric and chronocoulometric measurements showed that BV ^{2( . +)} and HV ^{6( . +)} have larger diffusion coefficients than the corresponding [3]‐ and [7]pseudorotaxanes, and UV/Vis absorption studies provided evidence for intramolecular radical‐cation dimerization. These results demonstrate that radical‐cation dimerization, a relatively weak interaction, can be used as a driving force in novel molecular switches.     

PHOTOXIDATION OF ASCORBATE TO OXALATE AND THREONATE MEDIATED BY CHLOROPLASTS IN FAR RED LIGHT

Abstract— Chloroplasts illuminated with light absorbed predominantly by Photosystem I can mediate the photoxidation of ascorbic acid to threonic and oxalic acids. Two micromoles of oxygen are consumed for each micromole of ascorbic acid added. The kinetics of this endogenous chloroplast system closely resemble the kinetics of an artificial flavin-manganese-catalase system in which flavin is the photosensitizing dye.     

Amorphous manganese polyphosphates: preparation,characterization and incorporation of azo dyes

Amorphous Mn²⁺ polyphosphate materials were prepared at room temperature through the coacervation of polyphosphate solutions. These new materials display the typical Mn²⁺ red emission with a broad band centered at 15,290 cm⁻¹ (bandwidth—1,320 cm⁻¹). Excitation spectra display typical Mn²⁺ (d⁵) absorption bands assigned to transitions from the ⁶A_1g ground state to the ⁴T_2g, (⁴A_1g, ⁴E_g), ⁴T_2g, and ⁴T_1g Mn²⁺ excited states. Transition energies allow estimating the Mn²⁺ ligand field strength as intermediate between the one observed in Mn²⁺ aqueous solutions and the one observed in Mn²⁺ phosphate glasses. Azo dyes, methyl red and methyl orange, were incorporated into the manganese polyphosphate coacervates structure. Raman scattering and visible light reflectance results show azo dyes acid forms inside the hydrated polyphosphate structure and sensitivity to the atmosphere pH. Release of azo dyes species was observed from these new solid hybrid materials in aqueous medium.     

Interaction Modes and Absolute Affinities of α‐Amino Acids for Mn2+: A Comprehensive Picture

Manganese is involved as a cofactor in the activation of numerous enzymes as well as the oxygen‐evolving complex of photosystem II. Full understanding of the role played by the Mn²⁺ ion requires detailed knowledge of the interaction modes and energies of manganese with its various environments, a knowledge that is far from complete. To bring detailed insight into the local interactions of Mn in metallopeptides and proteins, theoretical studies employing first‐principles quantum mechanical calculations are carried out on [Mn‐amino acid]²⁺ complexes involving all 20 natural α‐amino acids (AAs). Detailed investigation of [Mn‐serine]²⁺_, [Mn‐cysteine]²⁺, [Mn‐phenylalanine]²⁺, [Mn‐tyrosine]²⁺, and [Mn‐tryptophan]²⁺ indicates that with an electron‐rich side chain, the most stable species involves interaction of Mn²⁺ with carbonyl oxygen, amino nitrogen, and an electron‐rich section of the side chain of the AA in its canonical form. This is in sharp contrast with aliphatic side chains for which a salt bridge is formed. For aromatic AAs, complexation to manganese leads to partial oxidation as well as aromaticity reduction. Despite multisite binding, AAs do not generate strong enough ligand fields to switch the metal to a low‐ or even intermediate‐spin ground state. The affinities of Mn²⁺ for all AAs are reported at the B3LYP and CCSD(T) levels of theory, thereby providing the first complete series of affinities for a divalent metal ion. The trends are compared with those of other cations for which affinities of all AAs have been previously obtained.     

The role of excess Zn ions in improvement of red long lasting phosphorescence (LLP) performance of β-Zn3(PO4)2:Mn phosphor

Influences of excess Zn²⁺ ions and intrinsic defects on red (λ=616 nm) phosphorescence of β-Zn₃(PO₄)₂:Mn²⁺ are systematically investigated. It is clearly observed that red long lasting phosphorescence (LLP) properties of Mn²⁺, such as brightness and duration, are largely improved when excess Zn²⁺ ions are co-doped into the matrix. Photoluminescence (PL), LLP and thermoluminescence (TL) spectra indicate that Mn²⁺ ion acts as luminescent center whereas oxygen vacancy associated to Zn²⁺ ion plays a significant role in electron trap. The TL peak for oxygen vacancy is centered at 343 K, the depth of which is suitable for improvement in LLP performance of Mn²⁺ at room temperature. The possible mechanism for this phenomenon of red LLP of Mn²⁺ in β-Zn₃(PO₄)₂:Mn²⁺ with excess of Zn²⁺ is explained by means of a competitively trapping model.     

Binding of 1,5-bis(p-sulfonatophenyl)-3,7-diphenyl-1,5-diaza-3,7-diphosphacyclooctane with tetra(methyl viologen) calix[4]resorcinol

Binding of an amphiphilic dianion 1,5-bis(p-sulfonatophenyl)-3,7-diphenyl-1,5-diaza-3,7-diphosphacyclooctane (APCO^2?) with an amphiphilic octacation tetra(methyl viologen) calix[4]resorcinol (MVCA⁸⁺) in media containing different amounts of water and DMSO using NaClO₄ or NaCl as supporting electrolytes was shown for the first time by cyclic voltammetry and spectrophotometry. The stoichiometry of the complex depends on the MVCA⁸⁺: APCO^2? ratio, medium, and supporting electrolyte. A 1: 1 charge-transfer complex is mainly formed (λ_max = 480 nm) in 30% DMSO at a ratio of the compounds of 1: 1. A similar 1: 1 complex of APCO^2? with a model compound methyl viologen MV²⁺ (λ_max = 482 nm) is formed under these conditions. A donor-acceptor interaction occurs between the acceptor viologen units and nitrogen-centered electron-donating fragments of the APCO^2? dianion. An increase in the content of APCO^2? in the solution leads to an additional binding of one (30 vol.% DMSO, water, NaClO₄) or two (30 vol.% DMSO, water, NaCl) particles of APCO^2? with the hydrophobic fragments of MVCA⁸⁺. The complexes aggregate to form insoluble precipitates in aqueous and water-DMSO media. A selective reversible electroswitching from the bound to free state of one of the three bound APCO^2? particles was performed when reducing MVCA⁸⁺ to MVCA^4·+ in a 30 vol.% DMSO/NaCl medium.     

Forms of oxygen in La1 − x Ca x MnO3 + δ (x = 0–1) perovskites and their reactivities in oxidation reactions

The effects of substitution in the cationic sublattice and of the synthesis procedure on the reactivity of different forms of oxygen in La_{1 ? x} Ca _x MnO_{3 + δ} perovskites synthesized by mechanochemical and ceramic processing was studied by temperature-programmed reduction (TPR) with hydrogen. As the calcium content of the perovskite is raised, the maxima of the TPR peaks shift to lower temperatures and the extent of reduction of the perovskite increase, implying an increase in the reactivity of the system. Conversely, raising the calcination temperature or extending the calcination time shifts the maxima of the peaks to higher temperatures and diminishes the extent of reduction of the sample. TPR data for the intermediate-composition samples can be explained in terms of the dependence of microstructure on the synthesis procedure (near-surface calcium segregation in the mechanochemically synthesized samples and the microheterogeneous structure of the ceramic samples). The reduction process Mn⁴⁺ → Mn²⁺ takes place in the low- and medium-temperature regions. According to the literature, the bulk reduction process Mn³⁺ → Mn²⁺ occurs at high temperatures. The activity of the system in CO oxidation is correlated with the amount of the most reactive surface oxygen, which is eliminated in hydrogen TPR runs below 250–300°C.     

Visible‐Light‐Driven Water Oxidation by a Molecular Manganese Vanadium Oxide Cluster

Photosynthetic water oxidation in plants occurs at an inorganic calcium manganese oxo cluster, which is known as the oxygen evolving complex (OEC), in photosystem II. Herein, we report a synthetic OEC model based on a molecular manganese vanadium oxide cluster, [Mn₄V₄O₁₇(OAc)₃]^3?. The compound is based on a [Mn₄O₄]⁶⁺ cubane core, which catalyzes the homogeneous, visible‐light‐driven oxidation of water to molecular oxygen and is stabilized by a tripodal [V₄O₁₃]^6? polyoxovanadate and three acetate ligands. When combined with the photosensitizer [Ru(bpy)₃]²⁺ and the oxidant persulfate, visible‐light‐driven water oxidation with turnover numbers of approximately 1150 and turnover frequencies of about 1.75 s^?1 is observed. Electrochemical, mass‐spectrometric, and spectroscopic studies provide insight into the cluster stability and reactivity. This compound could serve as a model for the molecular structure and reactivity of the OEC and for heterogeneous metal oxide water‐oxidation catalysts.     

Use of ring-disc electrodes and viologens for the titration of cytochrome C and oxygen and for the study of their reduction kinetics

The use of ring-disc electrodes enable the electrogeneration of viologen cation radicals V⁺ when the disc potential is controlled in the range ?0.3 to ?0.65 and ?0.2 to ?0.45 V/NHE respectively in MV²⁺ and BV²⁺ solutions. We confirmed the occurrence of very fast reactions between V^.+ and horse-heart cytochrome c or oxygen and of slow or no reaction between V^.+ and β-nicotinamide adenine dinucleotide with xanthine oxidase, riboflavine and ferricyanide. Ring current versus dise current curves enable the titration of cytochrome c and oxygen and the estimation of the reaction rate between cytochrome c and MV^.+ [(6.2±2)10⁵M^?1 s^?1].     

POLAROGRAPHIC DETECTION OF AN ENDOGENOUS REDUCTANT GENERATED DURING FLASH ILLUMINATION OF CHLOROPLASTS

A Joliot-type polarographic electrode with the platinum surface biased 200 mV positive with respect to the Ag/AgCl junction was found to give a signal upon flash illumination of chloroplasts. The presence of either methyl viologen or ferricyanide as electron acceptors greatly enhanced the positivebias signal. By varying the electrode bias, a polarogram is obtained of this positive-bias signal. A half-wave reduction potential of 340 mV indicates that this signal is not due directly to superoxide (O^-₂) nor the direct interaction of reduced methyl viologen or ferrocyanide with the electrode. Instead, the reduced exogenous acceptors act as intermediates in the generation of superoxide, and superoxide reduces an unidentified acceptor which is endogenous to chloroplasts, has a 340 mV redox potential, and gives rise to the positive bias polarographic signal. The extent of intersystem electron carrier oxidation can be changed by varying electrode bias with ferricyanide present. As the platinum is made more positive than 250 mV with respect to the Ag/AgCl junction, P₇₀₀ becomes largely oxidized during 5 min dark adaptation.