Physiological functions of pyruvate:NADP+ oxidoreductase and 2-oxoglutarate decarboxylase in Euglena gracilis under aerobic and anaerobic conditions

In Euglena gracilis, pyruvate:NADP⁺ oxidoreductase, in addition to the pyruvate dehydrogenase complex, functions for the oxidative decarboxylation of pyruvate in the mitochondria. Furthermore, the 2-oxoglutarate dehydrogenase complex is absent, and instead 2-oxoglutarate decarboxylase is found in the mitochondria. To elucidate the central carbon and energy metabolisms in Euglena under aerobic and anaerobic conditions, physiological significances of these enzymes involved in 2-oxoacid metabolism were examined by gene silencing experiments. The pyruvate dehydrogenase complex was indispensable for aerobic cell growth in a glucose medium, although its activity was less than 1% of that of pyruvate:NADP⁺ oxidoreductase. In contrast, pyruvate:NADP⁺ oxidoreductase was only involved in the anaerobic energy metabolism (wax ester fermentation). Aerobic cell growth was almost completely suppressed when the 2-oxoglutarate decarboxylase gene was silenced, suggesting that the tricarboxylic acid cycle is modified in Euglena and 2-oxoglutarate decarboxylase takes the place of the 2-oxoglutarate dehydrogenase complex in the aerobic respiratory metabolism.     

Cadmium inhibitory action leads to changes in structure of ferredoxin:NADP+ oxidoreductase

This study deals with the influence of cadmium on the structure and function of ferredoxin:NADP(+) oxidoreductase (FNR), one of the key photosynthetic enzymes. We describe changes in the secondary and tertiary structure of the enzyme upon the action of metal ions using circular dichroism measurements, Fourier transform infrared spectroscopy and fluorometry, both steady-state and time resolved. The decrease in FNR activity corresponds to a gentle unfolding of the protein, caused mostly by a nonspecific binding of metal ions to multiple sites all over the enzyme molecule. The final inhibition event is most probably related to a bond created between cadmium and cysteine in close proximity to the FNR active center. As a result, the flavin cofactor is released. The cadmium effect is compared to changes related to ionic strength and other ions known to interact with cysteine. The complete molecular mechanism of FNR inhibition by heavy metals is discussed.Electronic supplementary material The online version of this article (doi:10.1007/s10867-012-9262-z) contains supplementary material, which is available to authorized users.     

Two isoforms of ferredoxin:NADP+ oxidoreductase from wheat leaves: purification and initial biochemical characterization

Ferredoxin:NADP⁺ oxidoreductase is an enzyme associated with the stromal side of the thylakoid membrane in the chloroplast. It is involved in photosynthetic linear electron transport to produce NADPH and is supposed to play a role in cyclic electron transfer, generating a transmembrane pH gradient allowing ATP production, if photosystem II is non-functional or no NADP⁺ is available for reduction. Different FNR isoforms have been described in non-photosynthetic tissues, where the enzyme catalyses the NADPH-dependent reduction of ferredoxin (Fd), necessary for some biosynthetic pathways. Here, we report the isolation and purification of two FNR isoproteins from wheat leaves, called FNR-A and FNR-B. These forms of the enzyme were identified as products of two different genes, as confirmed by mass spectrometry. The molecular masses of FNR-A and FNR-B were 34.3 kDa and 35.5 kDa, respectively. The isoelectric point of both FNR-A and FNR-B was about 5, but FNR-B appeared more acidic (of about 0.2 pH unit) than FNR-A. Both isoenzymes were able to catalyse a NADPH-dependent reduction of dibromothymoquinone and the mixture of isoforms catalysed reduction of cytochrome c in the presence of Fd. For the first time, the pH- and ionic strength dependent oligomerization of FNRs is observed. No other protein was necessary for complex formation. The putative role of the two FNR isoforms in photosynthesis is discussed based on current knowledge of electron transport in chloroplasts.     

Characterisation of cryoinjury in Euglena gracilis using flow-cytometry and cryomicroscopy

Flow-cytometry and cryomicroscopy elucidated that the unicellular algal protist Euglena gracilis was undamaged by cryoprotectant added at 0 degree C, and super-cooling in the absence of ice. Cryoinjuries were however induced by: osmotic shock resulting from excessive cryodehydration, intracellular ice, and fracturing of the frozen medium on thawing. Suboptimal cooling at -0.3 degrees C min(-1) to -60 degrees C and osmotic shock invariably resulted in damage to the organism's pellicle and osmoregulatory system causing, a significant (P > 0.005) increase in cell size. Cell damage was not repairable and led to death. The responses of E. gracilis to cryopreservation as visualised by flow-cytometry and cryomicroscopy assisted the development of an improved storage protocol. This comprised: cryoprotection with methanol [10%(v/v)] at 0 degree C, cooling at 0.5 degrees C min(-1) to -60 degrees C, isothermal hold for 30 min, and direct immersion in liquid nitrogen. Highest post-thaw viability (>60%) was obtained using two-step thawing, which involved initial slow warming to -130 degrees C followed by relatively rapid warming (approximately 90 degrees C min(-1)) to ambient temperature (ca. 25 degrees C).     

Constitutive overexpression of immunoidentical forms of PCP-induced Euglena gracilis CYP-450

Environmental pollutants are classically associated with increased drug metabolism. In this report, antibodies that are able to detect mammalian CYP proteins, namely the CYP1A1, CYP1A2, CYP2B1/B2, and CYP3A4 proteins, were used to investigate the expression of CYP-related proteins in Euglena gracilis (EG) cells under normal and PCP-treated conditions and in a EG-cell line adapted to PCP. Compared to normal conditions, the presence of PCP in the culture medium induced elevated levels of EG CYP-like proteins. With the exception of CYP3A4, this overexpression was correlated with expression of additional forms of CYP proteins having, respectively, the same molecular weight but slightly different pIs. Even in EG cells which had lost their PCP-adapted property after having been cultured without PCP, these additional forms were continuously expressed. This observation raised the question about the definition of a biomarker of pollution.     

Low-resolution characterization of the 3D structure of the Euglena gracilis photoreceptor

This paper deals with the first characterization of the structure of the photoreceptive organelle of the unicellular alga Euglena gracilis (Euglenophyta). This organelle has a three-dimensional organization consisting of up to 50 closely stacked membrane lamellae. Ionically induced unstacking of the photoreceptor lamellae revealed ordered arrays well suited to structural analysis by electron microscopy and image analysis, which ultimately yielded a low-resolution picture of the structure. Each lamella is formed by the photoreceptive membrane protein of the cell assembled within the membrane layer in a hexagonal lattice. The first order diffraction spots in the calculated Fourier transform reveals the presence of 6-fold symmetrized topography (better resolution about 90A). The 2D and 3D structural data are very similar with those recently published on proteorodopsin, a membrane protein used by marine bacterio-plankton as light-driven proton pump. In our opinion these similarity indicate that a photoreceptive protein belonging to the same superfamily of proteorodopsin could form the Euglena photoreceptor.     

Effects of increased salinity on gravitaxis in Euglena gracilis

The unicellular freshwater flagellate Euglena gracilis regulates its position in the water column by means of phototactic and gravitactic behavior. Recent experiments have revealed that the cells switch between negative and positive gravitaxis depending upon environmental stimuli such as solar radiation. In this study, the effect of increased salinity on gravitaxis in Euglena gracilis was investigated. In some experiments it was found that salt concentrations up to 5 gL-1 (in some experiments 10 gL-1) increased the motility, velocity and precision of negative gravitactic orientation. Higher salt concentrations decreased all these parameters. At concentrations of about 15 gL-1, cells which did not become immobile, switched from negative to positive gravitaxis. Positive gravitaxis persisted for several hours or even days when the cells were transferred back to standard culture medium. Most of the cells in cultures exposed to salt concentrations above 20 gL-1 lost their motility (partial formation of palmella stages) but recovered when transferred back to standard medium or de-ionised water. Post recovery, the cells showed pronounced positive gravitaxis. Additional investigations on the pigmentation, revealed that the cells showed a complete loss of a carotenoid shoulder in the spectrum, which reappeared when the cells were brought back to standard medium.     

Kinetic and thermodynamic characterization of adenylyl cyclase from Euglena gracilis

Some kinetic and thermodynamic properties of the plasma membrane adenylyl cyclase (AC) from the protist Euglena gracilis were examined. The AC kinetics for Mg-ATP was hyperbolic with a K(m) value of 0.33-0.43 mM, whereas the inhibition exerted by 2('),5(')-dideoxyadenosine was of the mixed type with a K(i) of 80-147 microM. The V(m) value (0.9 or 1.8 nmol(mg protein)(-1)min(-1)) changed, depending upon the carbon source in the growth medium (lactic acid or glutamate plus malate). Lactic acid membrane AC was slightly more thermolabile (from 28 to 40 degrees C) and showed higher activation energy (range 15-25 degrees C). With lactate, the total and saturated fatty acid percentage content in the plasma membrane was significantly greater than with glutamate plus malate, whereas the percentage content of polyunsaturated (n-3) fatty acids was lower. The data suggest that the fatty acid composition, as changed by the carbon source in the growth medium, may modulate the AC activity in Euglena.     

Pyruvate and acetate metabolism in termite mitochondria

Intact mitochondria have been successfully prepared from body tissues from the termites Nasutitermes walkeri and Coptotermes formosanus. This is the first report of the successful isolation of mitochondria from termites (Isoptera: Termitidae). Using an oxygen electrode, oxygen consumption by the mitochondria during the oxidation of various respiratory substrates was determined and their properties measured in terms of respiratory control index and ADP/O. ADP/O was as expected for substrates such as pyruvate, acetylcarnitine and acetyl-CoA and carnitine. Pyruvate and acetate were the major respiratory substrates in both species. The total activity of the pyruvate dehydrogenase complex (PDHc) in the mitochondria from N. walkeri and C. formosanus was determined to be 72.87+/-8.98 and 8.29+/-0.42 nmol/termite/h, respectively. Mitochondria isolated in the presence of inhibitors of PDHc interconversion were used to determine that about 60% of the PDHc was maintained in the active form in both N. walkeri and C. formosanus. The sufficient PDHc activity and high rate of pyruvate oxidation in mitochondria from N. walkeri suggest that pyruvate is rapidly metabolised, whereas the low mitochondrial PDHc activity of C. formosanus suggests that in this species more pyruvate is produced than can be oxidised in the termite tissues.     

A natural fusion of flavodiiron,rubredoxin, and rubredoxin oxidoreductase domains is a self-sufficient water-forming oxidase of Trichomonas vaginalis

Microaerophilic pathogens such as Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis have robust oxygen consumption systems to detoxify oxygen and maintain intracellular redox balance. This oxygen consumption results from H₂O-forming NADH oxidase (NOX) activity of two distinct flavin-containing systems: H₂O-forming NOXes and multicomponent flavodiiron proteins (FDPs). Neither system is membrane bound, and both recycle NADH into oxidized NAD⁺ while simultaneously removing O₂ from the local environment. However, little is known about the specific contributions of these systems in T. vaginalis. In this study, we use bioinformatics and biochemical analyses to show that T. vaginalis lacks a NOX–like enzyme and instead harbors three paralogous genes (FDPF1–3), each encoding a natural fusion product between the N-terminal FDP, central rubredoxin (Rb), and C-terminal NADH:Rb oxidoreductase domains. Unlike a “stand-alone” FDP that lacks Rb and oxidoreductase domains, this natural fusion protein with fully populated flavin redox centers directly accepts reducing equivalents of NADH to catalyze the four-electron reduction of oxygen to water within a single polypeptide with an extremely high turnover. Furthermore, using single-particle cryo-EM, we present structural insights into the spatial organization of the FDP core within this multidomain fusion protein. Together, these results contribute to our understanding of systems that allow protozoan parasites to maintain optimal redox balance and survive transient exposure to oxic conditions.     

Indications for acceleration-dependent changes of membrane potential in the flagellate Euglena gracilis

Summary. The effects of the calcium sequester EGTA on gravitactic orientation and membrane potential changes in the unicellular flagellate Euglena gracilis were investigated during a recent parabolic-flight experiment aboard of an Airbus A300. In the course of a flight parabola, an acceleration profile is achieved which yields subsequently about 20 s of hypergravity (1.8 g _n), about 20 s of microgravity, and another 20 s of hypergravity phases. The movement behavior of the cells was investigated with real-time, computer-based image analysis. Membrane potential changes were detected with a newly developed photometer which measures absorption changes of the membrane potential-sensitive probe oxonol VI. To test whether the data obtained by the oxonol device were reliable, the signal of non-oxonol-labelled cells was recorded. In these samples, no absorption shift was detected. Changes of the oxonol VI signals indicate that the cells depolarize during acceleration (very obvious in the step from microgravity to hypergravity) and slightly hyperpolarize in microgravity, which can possibly be explained with the action of Ca-ATPases. These signals (mainly the depolarization) were significantly suppressed in the presence of EGTA (5 mM). Gravitaxis in parallel was also inhibited after addition of EGTA. Initially, negative gravitaxis was inverted into a positive one. Later, gravitaxis was almost undetectable. Correspondence and reprints: Department of Plant Ecophysiology, University of Erlangen-Nürnberg, Staudtstrasse 5, 91058 Erlangen, Federal Republic of Germany.     

Purification of membrane-bound ferredoxin: NADP+ oxidoreductase and of plastocyanin from a detergent extract of washed thylakoids

A method is described for the isolation and purification of ferredoxin-NADP⁺ oxidoreductase (FNR, E.C. 1.18.1.2) and plastocyanin from spinach thylakoids. FNR is recovered from pools which are loosely and tightly bound to the membrane, with minimal disruption of pigment-protein complexes; yields can thus be higher than from procedures which extract only the loosely bound enzyme.Washed thylakoid membranes were incubated with the dipolar ionic detergent CHAPS (3-(3-cholamidopropyl-dimethylammonio)-1-propane-sulfonate). This provided an extract containing FNR and PC as its principal protein components, which could be rapidly separated from one another by chromatography on an anion-exchange column. FNR was purified to homogeneity (as judged from sodium dodecyl sulfate gel electrophoresis and the ratio between protein and flavin absorption maxima), using chromatography on phosphocellulose followed by batchwise adsorption to, and elution from hydroxylapatite. Plastocyanin was further purified on a Sephadex G-75 molecular sieve column.A typical yield, obtained in 3–4 days from 1 kg of deveined spinach leaves, was 7 mg of pure FNR (a single protein of M_r=37,000) and 3.5 mg of plastocyanin.Abbreviations CHAPS- 3-(3-cholamidopropyl-dimethylammonio)-1-propanesulfonate) - Chl- chlorophyll - FNR- ferredoxin-NADP⁺ oxidoreductase - Mops- 3-(N-morpholino) propanesulfonic acid - PC- plastocyanin - PMSF- phenylmethanesulfonylfluoride - SDS- sodium dodecyl sulfate - SDS-PAGE- sodium dodecyl sulfate polyacrylamide gel electrophoresis - Tricine- N-tris (hydroxymethyl) methylglycine     

Adaptive modifications of the photosynthetic apparatus in Euglena gracilis Klebs exposed to manganese excess

Summary. Asynchronous cultures of wild-type Euglena gracilis were tested for their morphophysiological response to 10mM MnSO₄. Growth was only moderately slowed (15%), while oxygen evolution was never compromised. Inductively coupled plasma analyses indicated that the Mn cell content doubled with respect to controls, but no signs of localised accumulation were detected with X-ray microanalysis. Evident morphological alterations were found at the plastid level with transmission electron microscopy and confocal laser scanning microscopy. An increase in the plastid mass, accompanied by frequent aberrations of chloroplast shape and of the organisation of the thylakoid system, was observed. These aspects paralleled a decrease in the molar ratio of chlorophyll a to b and an increase in the fluorescence emission ratio of light-harvesting complex II to photosystem II, the latter evaluated by in vivo single-cell microspectrofluorimetry. These changes were observed between 24 and 72h of treatment. However, the alterations in the pigment pattern and photosystem II fluorescence were no longer observed after 96h of Mn exposure, notwithstanding the maintenance of the large plastid mass. The response of the photosynthetic apparatus probably allows the alga to limit the photooxidative damage linked to the inappropriately large peripheral antennae of photosystem II. On the whole, the resistance of Euglena gracilis to Mn may be due to an exclusion–tolerance mechanism since most Mn is excluded from the cell, and the small amount entering the organism is tolerated by means of morphophysiological adaptation strategies, mainly acting at the plastid level.Correspondence and reprints: Dipartimento delle Risorse Naturali e Culturali, Università degli Studi di Ferrara, Corso Porta Mare 2, 44100 Ferrara, Italy.     

FAD semiquinone stability regulates single- and two-electron reduction of quinones by Anabaena PCC7119 ferredoxin:NADP+ reductase and its Glu301Ala mutant

Flavoenzymes may reduce quinones in a single-electron, mixed single- and two-electron, and two-electron way. The mechanisms of two-electron reduction of quinones are insufficiently understood. To get an insight into the role of flavin semiquinone stability in the regulation of single- vs. two-electron reduction of quinones, we studied the reactions of wild type Anabaena ferredoxin:NADP(+)reductase (FNR) with 48% FAD semiquinone (FADH*) stabilized at the equilibrium (pH 7.0), and its Glu301Ala mutant (8% FADH* at the equilibrium). We found that Glu301Ala substitution does not change the quinone substrate specificity of FNR. However, it confers the mixed single- and two-electron mechanism of quinone reduction (50% single-electron flux), whereas the wild type FNR reduces quinones in a single-electron way. During the oxidation of fully reduced wild type FNR by tetramethyl-1,4-benzoquinone, the first electron transfer (formation of FADH*) is about 40 times faster than the second one (oxidation of FADH*). In contrast, the first and second electron transfer proceeded at similar rates in Glu301Ala FNR. Thus, the change in the quinone reduction mechanism may be explained by the relative increase in the rate of second electron transfer. This enabled us to propose the unified scheme of single-, two- and mixed single- and two-electron reduction of quinones by flavoenzymes with the central role of the stability of flavin/quinone ion-radical pair.     

Photosensory transduction in Euglena gracilis: Effect of some metabolic drugs on the photophobic response

We have investigated the effect of some metabolic drugs, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 2,4-dinitrophenol (DNP), sodium azide (NaN₃), on the photobehavior of single cells of Euglena gracilis, in order to clarify the relevance of different metabolic pathways in the process of photoperception and sensory transduction in this alga. The results obtained show that the photophobic response of Euglena is not affected by the action of these drugs. This suggests that neither the photosynthetic process nor oxidative phosphorylation play a significant role in the phenomenon of photosensory transduction in Euglena.List of Abbreviations DNP 2,4-dinitrophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PSI Photosystem I - PSII Photosystem II     

Plastoquinones are effectively reduced by ferredoxin:NADP+ oxidoreductase in the presence of sodium cholate micelles. Significance for cyclic electron transport and chlororespiration

The effect of sodium cholate and other detergents (Triton X-100, sodium dodecyl sulphate, octyl glucoside, myristyltrimethylammonium bromide) on the reduction of plastoquinones (PQ) with a different length of the side-chain by spinach ferredoxin:NADP(+) oxidoreductase (FNR) in the presence of NADPH has been studied. Both NADPH oxidation and oxygen uptake due to plastosemiquinone autoxidation were highly stimulated only in the presence of sodium cholate among the used detergents. Sodium cholate at the concentration of 20 mM was found to be the most effective on both PQ-4 and PQ-9-mediated oxygen uptake. The FNR-dependent reduction of plastoquinones incorporated into sodium cholate micelles was stimulated by spinach ferredoxin but inhibited by Mg(2+) ions. It was concluded that the structure of sodium cholate micelles facilitates contact of plastoquinone molecules with the enzyme and creates favourable conditions for the reaction similar to those found in thylakoid membranes for PQ-9 reduction. The obtained results were discussed in terms of the function of FNR as a ferredoxin:plastoquinone reductase both in cyclic electron transport and chlororespiration.     

Pyruvate:NADP+ oxidoreductase from Euglena gracilis: mechanism of O2-inactivation of the enzyme and its stability in the aerobe

O2-inactivation of pyruvate:NADP+ oxidoreductase from mitochondria of Euglena gracilis was studied in vitro, and a mechanism which consists of two sequential stages was proposed. Initially, the enzyme is inactivated by the direct action of O2 in a process obeying second-order kinetics. Although the catalytic activity for pyruvate oxidation is lost by this initial inactivation, NADPH oxidation with artificial electron acceptors still occurs. Subsequently, a secondary, O2-independent inactivation occurs, rendering the enzyme completely inactive. Pyruvate stimulates the O2-inactivation while CoA and NADP+ protect the enzyme from O2. The O2-inactivation is accelerated by reduction of the enzyme with pyruvate and CoA. Reactivation of the O2-inactivated enzyme was studied in Ar by incubation with Fe2+ in the presence of some other reducing reagent such as dithiothreitol. The evidence obtained indicates that the partially inactivated enzyme, which retains catalytic activity for NADPH oxidation, can be reactivated, but the completely inactivated enzyme is not. When Euglena cells were exposed to 100% O2 the enzyme in the cells was inactivated by O2, but the rate was quite slow compared with that observed in vitro. The enzyme inactivated by O2 in the cells was almost completely reactivated in vitro by incubation with Fe2+ and other reducing reagents in Ar, suggesting that the secondary, O2-independent inactivation does not occur in situ. When the cells were returned to air, reactivation of the O2-inactivated enzyme in the cells began immediately. The enzyme, kept in isolated, intact mitochondria, was stable in air; however, the enzyme was inactivated by O2 when the mitochondria were incubated with a high concentration of pyruvate.