Effect of deuteration and cryosolvents on the energy transduction in primary processes of photosynthesis.

Effects of cryosolvents and D2O/H2O substitution on the reaction centres (RCs) isolated from photosynthetic bacteria were studied with respect to the role of intra-protein hydrogen bonds in the primary photosynthetic electron transfer. As a result of such treatment of RCs, the charge separation rate between the photoactive bacteriochlorophyll (P2 dimer) and bacteriopheophytin and the rate of electron transfer to the primary quinone slowed down. The energy migration rate from bacteriopheophytin (BPheM), inactive in electron transport, to P2 decreased as well. Although cryosolvents can shift the redox potential of the photoactive pigment, there is no direct correlation between the P2 potential and the effects of these modifying agents on the photosynthetic process in RCs occurring with participation of P2. The removal of H subunit from the pigment-protein complex results in the pronounced weakening of the dimethyl sulfoxide modifying effects on the RC hydrogen bonds. The role of structural and dynamic state in the functioning of the photosynthetic bacterial RCs is analyzed. Relaxation processes in purple bacteria RCs accompanying the primary picosecond steps of energy transformation proceed with the participation of small proton-containing molecular groups in the immediate surroundings of electron transfer carriers. In this paper, we present results concerning mechanisms of primary photosynthetic steps, which were initiated by A. A. Krasnovsky and have been studied for several years at the Department of Biophysics. This paper is dedicated to the memory of our teacher Prof. A. A. Krasnovsky.     

Characterization of target site of aluminum phytotoxicity in photosynthetic electron transport by fluorescence techniques in tobacco leaves

Aluminum (Al) toxicity limits crop yield in acidic soil through affecting diverse metabolic processes, especially photosynthesis. The aim of this work was to examine the effect of Al on photosynthetic electron transport in vivo as determined by chlorophyll fluorescence and delayed fluorescence of tobacco leaves. Results showed that Al treatment inhibited the photosynthetic rate and electron transfer, and decreased photosystem (PS) II photochemical activity in a time- and concentration-dependent manner, which could not be obviously alleviated by the addition of the reactive oxygen species (ROS) scavenger ascorbic acid (AsA). These results suggested that photosynthetic electron transfer chain components, especially PSII, might be directly damaged by Al instead of in an ROS-dependent manner. Furthermore, the fluorescence imaging and biochemical analysis exhibited that Al, after entering the cells, could accumulate in the chloroplasts, which paralleled the decreased content of Fe in the chloroplast. The changes in the chlorophyll fluorescence decay curve, the delayed fluorescence decay curve and the chlorophyll fluorescence parameters indicated that Al, through interacting with or replacing the non-heme iron between Q(A) and Q(B), caused the inhibition of electron transfer between Q(A) and Q(B), resulting in PSII photochemical damage and inhibition of the photosynthetic rate. In summary, our results characterized the target site of Al phytotoxicity in photosynthetic electron transport, providing new insight into the mechanism of Al phytotoxicity-induced chloroplast dysfunction and photosynthetic damage.     

Role of ascorbic acid in dopamine beta-hydroxylation. The endogenous enzyme cofactor and putative electron donor for cofactor regeneration

The role(s) of ascorbic acid in dopamine beta-hydroxylation was studied in primary cultures of bovine adrenomedullary chromaffin cells and in isolated bovine adrenomedullary chromaffin vesicles. Dopamine beta-hydroxylase activity was assessed by measuring the rate of conversion of tyramine to octopamine. The ascorbic acid content of chromaffin cells declined with time in culture and the dopamine beta-hydroxylase activity of ascorbate-depleted cells was low. Ascorbate additions to ascorbate-depleted cells increased both the intracellular ascorbate concentrations and the rates of dopamine beta-hydroxylation. Ascorbate uptake into the cells was rapid; however, the onset of enhanced octopamine synthesis by added ascorbate was delayed by several hours and closely followed the time course for accumulation of the newly taken up ascorbate into the chromaffin vesicle. The amount of octopamine synthesized by the chromaffin cells exceeded the intracellular ascorbate content and ascorbate levels were maintained during dopamine beta-hydroxylation in the absence of external ascorbate. This suggests an efficient recycling of ascorbate. In contrast to intact cells, ascorbic acid was depleted during octopamine synthesis in isolated chromaffin vesicles. The molar ratio of octopamine formed to ascorbate depleted was close to unity. Thus, the recycling of intravesicular ascorbate depends on an extravesicular factor(s). The depletion of intravesicular ascorbate during dopamine beta-hydroxylation was prevented by the addition of nonpermeant extravesicular electron donors such as ascorbate or glucoascorbate. This suggests that intravesicular ascorbate is maintained in the reduced state by electron transport across the vesicle membrane. These results are compatible with the hypothesis that both intra- and extravesicular ascorbate participate in the regulation of dopamine beta-hydroxylase. Intravesicular ascorbate is the cofactor for the enzyme. Cytosolic ascorbate is most likely the electron donor for the vesicle-membrane electron transport system which maintains the intravesicular cofactor concentration.     

Mechanism of Linolenic Acid-induced Inhibition of Photosynthetic Electron Transport

The effect of linolenic acid on photosynthetic electron transport reactions in chloroplasts has been localized at a site on the donor side of photosystem I and at two functionally distinct sites in photosystem II.     

An alternate photosynthetic electron donor system for PSI supports light dependent nitrogen fixation in a non-heterocystous cyanobacterium,Plectonema boryanum

Plectonema boryanum exhibits temporal separation of photosynthesis and nitrogen fixation under diazotrophic conditions. During nitrogen fixation, the photosynthetic electron transport chain becomes impaired, which leads to the uncoupling of the PSII and PSI activities. A 30-40% increase in PSI activity and continuous generation of ATP through light-dependent processes seem to support the nitrogen fixation. The use of an artificial electron carrier that shuttles electrons between the plastoquinone pool and plastocyanin, bypassing cytochrome b/f complex, enhanced the photosynthetic electron transport activity five to six fold during nitrogen fixation. Measuring of full photosynthetic electron transport activity using methyl voilogen as a terminal acceptor revealed that the photosynthetic electron transport components beyond plastocyanin might be functional. Further, glycolate can act as a source of electrons for PSI for the nitrogen fixing cells, which have residual PSII activity. Under conditions when PSI becomes largely independent of PSII and glycolate provides electrons for PSI activity, the light-dependent nitrogen fixation also was stimulated by glycolate. These results suggest that during nitrogen fixation, when the photosynthetic electron transport from PSII is inhibited at the level of cytochrome b/f complex, an alternate electron donor system for PSI may be required for the cells to carry out light dependent nitrogen fixation.     

Oxidative damage to thylakoid proteins in water-stressed leaves of wheat (Triticum aestivum)

The production of reactive oxygen species in the chloroplast may increase under water deficit. To determine if this causes oxidative damage to the photosynthetic apparatus, we analyzed the accumulation of oxidatively damaged proteins in thylakoids of water-stressed wheat ( Triticum aestivum L.) leaves. Water stress was imposed on 4-week-old plants by withholding watering for 10 days to reach a soil water potential of about −2.0 MPa. In thylakoids of water-stressed leaves there was an increase in oxidative damage, particularly in polypeptides of 68, 54, 41 and 24 kDa. High molecular mass oxidized (probably cross-linked) proteins accumulated in chloroplasts of droughted leaves. Oxidative damage was associated with a substantial decrease in photosynthetic electron transport activity and photosystem II (PSII) efficiency (F_v/F_m). Treatment of stressed leaves with l -galactono-1,4-lactone (GL) increased their ascorbic acid content and enhanced photochemical and non-photochemical quenching of chlorophyll fluorescence. GL reduced oxidative damage to photosynthetic proteins of droughted plants, but it reverted the decrease in electron transport activity and PSII efficiency only partially, suggesting that other factors also contributed to loss of photosystem activity in droughted plants. Increasing the ascorbic acid content of leaves might be an effective strategy to protect thylakoid membranes from oxidative damage in water-stressed leaves.     

Kinetic studies on the hydroxylation of p-coumaric acid to caffeic acid by spinach-beet phenolase.

1. A spectrophotometric assay is described that enables the hydroxylation of p-coumaric acid to caffeic acid, catalysed by spinach-beet phenolase, to be followed continuously. 2. Initial-velocity and inhibitor studies indicate that the order of substrate addition is oxygen, p-coumaric acid and electron donor, with an irreversible step separating the binding of each substrate. 3. Caffeic acid is most likely to act as electron donor at the active site; other electron donors, such as ascorbic acid, NADH and dimethyltetrahydropteridine, function mainly to recycle cofactor amounts of caffeic acid. 4. A reaction scheme, consistent with these data, is proposed.     

Inhibitors in the functional dissection of the photosynthetic electron transport system

The significance of inhibitors and artificial electron acceptor and donor systems as experimental tools for studying the photosynthetic system is described by reviewing early classical articles. The historical development in unravelling the role and sequence of electron carriers and energy conserving sites in the electron transport chain is acknowledged. Emphasis is given to inhibitors of the acceptor side of photosystem II and of the plastoquinol oxidation site in the cytochrome b6/f complex. Their role in regulatory processes under redox control is introduced.     

Involvement of ascorbic acid and ab-type cytochrome in plant plasma membrane redox reactions

Summary Higher plant plasma membranes contain ab-type cytochrome that is rapidly reduced by ascorbic acid. The affinity towards ascorbate is 0.37 mM and is very similar to that of the chromaffin granule cytochromeb ₅₆₁. High levels of cytochromeb reduction are reached when ascorbic acid is added either on the cytoplasmic or cell wall side of purified plasma membrane vesicles. This result points to a transmembrane organisation of the heme protein or alternatively indicates the presence of an effective ascorbate transport system. Plasma membrane vesicles loaded by ascorbic acid are capable of reducing extravesicular ferricyanide. Addition of ascorbate oxidase or washing of the vesicles does not eliminate this reaction, indicating the involvement of the intravesicular electron donor. Absorbance changes of the cytochromeb -band suggest the electron transfer is mediated by this redox component. Electron transport to ferricyanide also results in the generation of a membrane potential gradient as was demonstrated by using the charge-sensitive optical probe oxonol VI. Addition of ascorbate oxidase and ascorbate to the vesicles loaded with ascorbate results in the oxidation and subsequent re-reduction of the cytochromeb. It is therefore suggested that ascorbate free radical (AFR) could potentially act as an electron acceptor to the cytochrome-mediated electron transport reaction. A working model on the action of the cytochrome as an electron carrier between cytoplasmic and apoplastic ascorbate is discussed.Abbreviations AFR ascorbate free radical - AO ascorbate oxidase - DTT dithiothreitol - FCCP carbonylcyanidep-trifluorome-thoxyphenylhydrazon - Hepes N-(2-hydroxyethyl)-piperazine-N-(2-ethanesulfonic acid) - Oxonol VI bis(3-propyl-5-oxoisoxazol-4-yl) penthamethine oxonol - PMSF phenylmethylsulfluoride     

An electron transfer dependent membrane potential in chromaffin-vesicle ghosts

Adrenal medullary chromaffin-vesicle membranes contain a transmembrane electron carrier that may provide reducing equivalents for intravesicular dopamine beta-hydroxylase in vivo. This electron transfer system can generate a membrane potential (inside positive) across resealed chromaffin-vesicle membranes (ghosts) by passing electrons from an internal electron donor to an external electron acceptor. Both ascorbic acid and isoascorbic acid are suitable electron donors. As an electron acceptor, ferricyanide elicits a transient increase in membrane potential at physiological temperatures. A stable membrane potential can be produced by coupling the chromaffin-vesicle electron-transfer system to cytochrome oxidase by using cytochrome c. The membrane potential is generated by transferring electrons from the internal electron donor to cytochrome c. Cytochrome c is then reoxidized by cytochrome oxidase. In this coupled system, the rate of electron transfer can be measured as the rate of oxygen consumption. The chromaffin-vesicle electron-transfer system reduces cytochrome c relatively slowly, but the rate is greatly accelerated by low concentrations of ferrocyanide. Accordingly, stable electron transfer dependent membrane potentials require cytochrome c, oxygen, and ferrocyanide. They are abolished by the cytochrome oxidase inhibitor cyanide. This membrane potential drives reserpine-sensitive norepinephrine transport, confirming the location of the electron-transfer system in the chromaffin-vesicle membrane. This also demonstrates the potential usefulness of the electron transfer driven membrane potential for studying energy-linked processes in this membrane.     

Role of auxin and gibberellin in the synthesis of Ascorbic Acid and growth of tissue explants

Coleoptile and root tips ofTriticum aestivum cv. Arnej 624 and those ofAvena sativa cv. Victory (Svalöf) as well as dry excised embryos ofTriticum aestivum cv. Rival (Svalöf) and those ofArachis hypogaea cv. 34 3A. H. were cultivated in media containing various concentrations of sucrose and growth regulators, like ascorbic acid, indole-3-acetic acid and gibberellin. Growth, differentiation and water uptake of the various explants were determined at regular time intervals. Further, the concentration of the endogenous ascorbic acid in mg./g. fresh weight, as well as the amount of this growth regulator utilized as per cent of the total were determined. Although all the three growth regulators promote growth in the explants, their effect is best felt when sucrose of a higher concentration (1.0 per cent) is added to the medium. In fact, the response to 1.0 per cent sucrose is sometimes as good as a combination of a growth regulator with sucrose, especially in the case of root explants. The results clearly indicate that the biosynthesis of ascorbic acid in the explants is catalyzed by the addition of indole-3-acetic acid as well as gibberellin. Simultaneously, the utilization of ascorbic acid is also appreciably increased by the presence of these growth regulators. Addition of 1.0 per cent sucrose to the medium containing the above mentioned growth regulators augments to a considerable extent not only the concentration of ascorbic acid, but also steps up its utilization. Enhancement of ascorbic acid as well as its increased utilization are correlated with rapid imbibition of water, growth and differentiation. The role of ascorbic acid in growth is discussed; and on the basis of the data presented here it is postulated that: (1) auxin and gibberellin function in the growth process by catalyzing the biosynthesis of ascorbic acid; and (2) that ascorbic acid not only participates in activation of various enzyme systems, but also stimulates the production of adenosine triphosphate by acting as an electron donor in photosynthetic phosphorylation as well as oxidative phosphorylation; (3) that the above action of ascorbic acid creates a favourable redox balance for synthesis of nucleic acids, proteins, enzymeproteins, and cell-wall constituents, thus enabling the processes of cell division and enlargement to proceed at a fast rate; and (4) that the relative rates of cell division and cell enlargement as well as “ageing” will determine the pattern of plant development.     

Two pathways of electron transport to nitrogenase in Rhodospirillum rubrum: the major pathway is dependent on the fix gene products

In the photosynthetic bacterium Rhodospirillum rubrum, as in many other diazotrophs, electron transport to nitrogenase has not been characterized in great detail. In this study, we show that there are two pathways operating in R. rubrum. The products of the fix genes constitute the major pathway operating under heterotrophic conditions, whereas a pyruvate:ferredoxin oxidoreductase, encoded by the nifJ gene, may play a central role under anaerobic conditions in the dark. In both systems, ferredoxin N is the main direct electron donor to dinitrogenase reductase. Furthermore, we suggest from studying mutants lacking components in one or both systems under different conditions, that the Fix system operates most efficiently under conditions when a proton motive force is generated. A model for our current view of the electron transfer pathways in R. rubrum is presented.     

Modeling chlorophyll a fluorescence transient: Relation to photosynthesis

To honor Academician Alexander Abramovitch Krasnovsky, we present here an educational review on the relation of chlorophyll a fluorescence transient to various processes in photosynthesis. The initial event in oxygenic photosynthesis is light absorption by chlorophylls (Chls), carotenoids, and, in some cases, phycobilins; these pigments form the antenna. Most of the energy is transferred to reaction centers where it is used for charge separation. The small part of energy that is not used in photochemistry is dissipated as heat or re-emitted as fluorescence. When a photosynthetic sample is transferred from dark to light, Chl a fluorescence (ChlF) intensity shows characteristic changes in time called fluorescence transient, the OJIPSMT transient, where O (the origin) is for the first measured minimum fluorescence level; J and I for intermediate inflections; P for peak; S for semi-steady state level; M for maximum; and T for terminal steady state level. This transient is a real signature of photosynthesis, since diverse events can be related to it, such as: changes in redox states of components of the linear electron transport flow, involvement of alternative electron routes, the build-up of a transmembrane pH gradient and membrane potential, activation of different nonphotochemical quenching processes, activation of the Calvin-Benson cycle, and other processes. In this review, we present our views on how different segments of the OJIPSMT transient are influenced by various photosynthetic processes, and discuss a number of studies involving mathematical modeling and simulation of the ChlF transient. A special emphasis is given to the slower PSMT phase, for which many studies have been recently published, but they are less known than on the faster OJIP phase.     

Continuous spectrophotometric assays for dopamine beta-monooxygenase based on two novel electron donors: N,N-dimethyl-1,4-phenylenediamine and 2-aminoascorbic acid.

Based on the novel chromophoric electron donors, N,N-dimethyl-1,4-phenylenediamine (DMPD) and 2-amino-2-deoxy-L-ascorbic acid (2-aminoascorbic acid), two sensitive, convenient, and continuous spectrophotometric assays for dopamine beta-monooxygenase (EC 1.14.17.1) are described. Both, DMPD and 2-aminoascorbic acid are kinetically and stoichiometrically well-behaved electron donors for dopamine beta-monooxygenase with kinetic parameters comparable to the most efficient physiological electron donor, ascorbic acid. During dopamine beta-monooxygenase turnover, DMPD is converted to its chromophoric cation radical which is stable under the standard assay conditions. The rate of the enzyme-dependent formation of DMPD cation radical under standard assay conditions could easily be followed at 515 nm with high accuracy and reproducibility. Similarly, dopamine beta-monooxygenase-mediated oxidation of 2-aminoascorbic acid results in the formation of the known, stable chromophoric product, 2,2'-nitrilodi-2(2')-deoxy-L-ascorbic acid (red pigment), which has a very strong absorption maximum at 385 nm. Both the above assays are superior to the existing assays in their convenience, reproducibility, and sensitivity for routine kinetic analysis of dopamine beta-monooxygenase and may be adopted as a simple color test for the enzyme. We propose that the above assays could also be adopted to design continuous and sensitive spectrophotometric assays for ascorbate oxidase, peptidyl alpha-amidating monooxygenase, and the chromaffin granule electron transport protein, cytochrome b561, due to their remarkable similarity to dopamine beta-monooxygenase in the chemistry of catalysis with regard to the electron donor.     

Is carbonic anhydrase activity of photosystem II required for its maximum electron transport rate?

It is known, that the multi-subunit complex of photosystem II (PSII) and some of its single proteins exhibit carbonic anhydrase activity. Previously, we have shown that PSII depletion of HCO₃^?/CO₂ as well as the suppression of carbonic anhydrase activity of PSII by a known inhibitor of α?carbonic anhydrases, acetazolamide (AZM), was accompanied by a decrease of electron transport rate on the PSII donor side. It was concluded that carbonic anhydrase activity was required for maximum photosynthetic activity of PSII but it was not excluded that AZM may have two independent mechanisms of action on PSII: specific and nonspecific. To investigate directly the specific influence of carbonic anhydrase inhibition on the photosynthetic activity in PSII we used another known inhibitor of α?carbonic anhydrase, trifluoromethanesulfonamide (TFMSA), which molecular structure and physicochemical properties are quite different from those of AZM. In this work, we show for the first time that TFMSA inhibits PSII carbonic anhydrase activity and decreases rates of both the photo-induced changes of chlorophyll fluorescence yield and the photosynthetic oxygen evolution. The inhibitory effect of TFMSA on PSII photosynthetic activity was revealed only in the medium depleted of HCO₃^?/CO₂. Addition of exogenous HCO₃^? or PSII electron donors led to disappearance of the TFMSA inhibitory effect on the electron transport in PSII, indicating that TFMSA inhibition site was located on the PSII donor side. These results show the specificity of TFMSA action on carbonic anhydrase and photosynthetic activities of PSII. In this work, we discuss the necessity of carbonic anhydrase activity for the maximum effectiveness of electron transport on the donor side of PSII.     

EPR study of electron transport in the cyanobacterium Synechocystis sp. PCC 6803: oxygen-dependent interrelations between photosynthetic and respiratory electron transport chains

In this work, we investigated electron transport processes in the cyanobacterium Synechocystis sp. PCC 6803, with a special emphasis focused on oxygen-dependent interrelations between photosynthetic and respiratory electron transport chains. Redox transients of the photosystem I primary donor P700 and oxygen exchange processes were measured by the EPR method under the same experimental conditions. To discriminate between the factors controlling electron flow through photosynthetic and respiratory electron transport chains, we compared the P700 redox transients and oxygen exchange processes in wild type cells and mutants with impaired photosystem II and terminal oxidases (CtaI, CydAB, CtaDEII). It was shown that the rates of electron flow through both photosynthetic and respiratory electron transport chains strongly depended on the transmembrane proton gradient and oxygen concentration in cell suspension. Electron transport through photosystem I was controlled by two main mechanisms: (i) oxygen-dependent acceleration of electron transfer from photosystem I to NADP(+), and (ii) slowing down of electron flow between photosystem II and photosystem I governed by the intrathylakoid pH. Inhibitor analysis of P700 redox transients led us to the conclusion that electron fluxes from dehydrogenases and from cyclic electron transport pathway comprise 20-30% of the total electron flux from the intersystem electron transport chain to P700(+).     

Oxidative stress and innate immunity status in chickens exposed to high dose of ascorbic acid

The effects of high dose ascorbic acid (10 000 mg· kg^–1 in the diet) and the transition metal on the presence of oxidative stress in the internal organs of growing chicks, as well as on the innate immune system status, were investigated. Supplementation with a high dose of ascorbic acid had pro‐inflammatory effects on the intestinal mucosa, and lysozyme levels were decreased significantly in all organs studied. High‐dose ascorbic acid caused an imbalance between prooxidative and antioxidative activities and was associated with the generation of semiquinone radicals. We observed that ascorbic acid increased iron and cadmium absorption. When a high dose of ascorbic acid was applied, elevated kidney and intestinal mucosa iron concentrations were observed. The amount of free malondialdehyde in the above organs has increased as well. These data have important implications for the mechanism of the oxidative stress development under the influence of high dose of ascorbic acid, indicating the importance of the side reactions of the mitochondrial electron transport chain with the formation of semiquinone radicals and the role of transition metals in this process. Copyright © 2013 John Wiley & Sons, Ltd.     

Mechanism of photoinactivation in photosynthetic systems II. The occurrence and properties of two different types of photoinactivation

Photoinactivation of the activity of NADP photoreduction withreduced DPIP or with reduced TMPD as the electron donor wasinhibited by the absence of oxygen in the atmosphere or by thepresence of photosynthetic inhibitors (CMU, DCMU, o-phenanthroline)in the preillumination mixture. Photoinactivation of the photoreductionof NADP or DPIP with water as the electron donor was not affected,or even accelerated, by these conditions of preillumination.The concentrations of inhibitors required for maximum inhibitionin the former case corresponded to those required for inhibitionof photosynthetic electron transport. The results indicatedthe occurrence of 2 different types of photoinactivation, eachspecifically affecting photosystems I and II, and differingin behaviours; including their requirement for oxygen in theatmosphere and their responses toward the presence of photosyntheticinhibitors during the preillumination period. (Received July 30, 1969; )     

Modulation of chloroplast membrane lipids by homogeneous catalytic hydrogenation

A method is reported for the modification of lipids in situ in chloroplast membrane by which a homogeneous, water-soluble catalyst Pd(QS)2 (QS, sulphonated alizarine; C14H6O7NaS) is incorporated into the thylakoids of isolated chloroplast. The catalyst itself did not affect the photosynthetic activity but caused an extensive loss of unsaturated fatty acids in the presence of hydrogen gas. The polyunsaturated fatty acids were hydrogenated at a faster rate than the monoenoic acids. During hydrogenation the orientational ordering of membrane lipids, as measured with the C-12 positional isomer of spin-labelled stearic acid, displayed a slight increase in agreement with the alterations in membrane composition. Progressive saturation of double bonds of lipids primarily inhibits electron transport between the photosystems followed by the inhibition of electron flow around photosystem II. Photosystem I electron transport was not inhibited even by 50% fatty acid hydrogenation. We suggest that using Pd(QS)2 catalyst for thylakoid hydrogenation offers an excellent technique to study the role of various unsaturated fatty acids in the regulation of membrane fluidity and photosynthetic processes.