Protoporphyrinogen oxidation in chloroplasts and plant mitochondria,a step in heme and chlorophyll synthesis

The oxidation of protoporphyrinogen to protoporphyrin was demonstrated in greening plastids and mitochondria from greening barley shoots. The plastids, purified by sucrose gradient centrifugation, were essentially free of a mitochondrial marker enzyme. The plastid activity was destroyed by mild heating and was proportional to plastid concentration suggesting, an enzymatic reaction. Uroporphyrinogen I was not oxidized at an appreciable rate. Activity was also demonstrated in etioplasts and mitochondria from dark-grown barley, and in chloroplasts from commercial spinach leaves. The chelating agent 1,10-phenanthroline partially decreased activity in plant organelles, but cyanide did not. The plastid activity, like the activity in liver mitochondria, was readily demonstrable at pH 8.4 in the presence of glutathione as reducing agent. However, the plastid activity was markedly enhanced by assay at pH 7.0 and the absence of reducing agents. These properties distinguish the activity in plants from that previously described in mammalian mitochondria and photosynthetic bacteria.     

Oxidation of protoporphyrinogen to protoporphyrin, a step in chlorophyll and haem biosynthesis. Purification and partial characterization of the enzyme from barley organelles.

The protoporphyrinogen-oxidizing enzyme from Triton X-100 extracts of the mitochondrial and etioplast fractions of etiolated barley was purified by using ion-exchange and hydroxyapatite chromatography. The purified enzyme from both organelle fractions exhibited a Km of 5 microM and was labile to mild heat and acidification. The pH optimum (5-6) and the substrate-specificity (mesoporphyrinogen was oxidized as rapidly as protoporphyrinogen) revealed properties very different from the protoporphyrinogen-oxidizing enzyme of rat liver or yeast mitochondria, which is specific for protoporphyrinogen as substrate. The purest fractions showed a polypeptide band corresponding to an Mr of approx. 36,000 on SDS/polyacrylamide-gel electrophoresis. This is the first purification and characterization of the enzyme from a plant, and indicates no readily detectable differences between the enzyme isolated from mitochondrial or etioplast fractions, although only the latter organelle has the capacity for both haem and chlorophyll synthesis.     

Studies on ferrochelatase. The enzymic formation of haem in proplastids, chloroplasts and plant mitochondria

1. Ferrochelatase was demonstrated in the chloroplasts and proplastids isolated from the primary leaves of beans (a dicotyledon) and oats (a monocotyledon). It was also detected in chloroplasts from etiolated bean seedlings made green by illumination before being harvested. The specific activities of the three types of bean organelles are similar, as are the specific activities of the oat proplastids and chloroplasts. 2. Chloroplasts from young spinach leaves also contain ferrochelatase; these chloroplasts were tested for their ability to form magnesium tetrapyrroles and found unable to catalyse the insertion of Mg(2+) into mesoporphyrin IX. 3. Ferrochelatase was also detected in potato tuber mitochondria. 4. Ferrochelatase activity in these plant preparations is much less stable on storage than similar preparations from bacteria and animal tissues. 5. Temperature affects the activities of spinach chloroplast ferrochelatase and rat liver ferrochelatase differently. Activity of the chloroplast enzyme increases as the temperature rises from 20.6 degrees to 26 degrees , but becomes increasingly inactivated as the temperature rises further to 38 degrees . The initial velocity of the mammalian enzyme, however, increases as the temperature rises from 25.8 degrees to 65 degrees , but the enzyme is inactivated after several minutes at 65 degrees .     

The enzymic conversion of protoporphyrinogen IX to protoporphyrin IX. Protoporphyrinogen oxidase activity in mitochondrial extracts of Saccharomyces cerevisiae.

The oxidation of protoporphyrinogen IX to protoporphyrin IX in yeast cells is enzyme-dependent. The enzyme, protoporphyrinogen oxidase, associated with purified mitochondria isolated from Saccharomyces cerevisiae was solubilized by sonic treatment in the presence of detergent and partially purified. The molecular weight of the enzyme was 180,000 plus or minus 18,000. The purified preparation could be stored at -20 degrees in the presence of 20% glycerol for several months without loss of activity. Enzyme activity was destroyed by heating above 40 degrees and by proteolytic digestion and irreversible inactivation occurred outside the pH range of 4.0 to 9.5. The pH optimum of the enzymic reaction was 7.45 and the value of the Michaelis constant was approximately 4.8 muM. Protoporphyrinogen oxidase did not catalyse the oxidation of coproporphyrinogen I or III or uroporphyrinogen I or III and the rate of enzymic oxidation of mesoporphyrinogen IX was less than 20% of that observed with protoporphyrinogen IX. The presence of thiol groups in the enzyme system was indicated but no metal ion or other cofactor requirement was demonstrated. Enzyme activity was insensitive to cyanide, 2,4-dinitrophenol, and azide whereas it was inhibited in the presence of Cu-2+ or Co-2+ ions, high ionic strength, heme, or hemin.     

Effect of unsaturated fatty acids on protoporphyrinogen oxidation, a step in heme and chlorophyll synthesis in plant organelles

Oleic acid stimulates enzymatic protoporphyrinogen oxidation by extracts of barley mitochondria and etioplasts. Greater stimulation occurred with Triton X-100 extracts which had been passed over a Sephacryl S-200 column than with crude Triton extracts, suggesting that purification may have removed a lipid factor required for optimal enzymatic activity. Palmitic acid, various phospholipids and detergents, or esters and alcohols of oleic acid did not substitute for free oleic acid. Linoleic acid caused a greater stimulation of protoporphyrinogen oxidation in both crude and purified barley organelle extracts and also caused a slow chemical oxidation of protoporphyrinogen. The stimulating effect of unsaturated fatty acids on enzymatic protoporphyrinogen oxidation may indicate a lipid requirement for this membrane bound enzyme or may also indicate involvement of unsaturated lipid oxidation in plant protoporphyrinogen oxidation.     

Effect of diphenyl ether herbicides on oxidation of protoporphyrinogen to protoporphyrin in organellar and plasma membrane enriched fractions of barley

In barley (Hordeum vulgare L.) root cells, activity for oxidizing protoporphyrinogen to protoporphyrin (protoporphyrinogen oxidase), a step in chlorophyll and heme synthesis, was found both in the crude mitochondrial fraction and in a plasma membrane enriched fraction separated by a sucrose gradient technique utilized for preparing plasma membranes. The specific activity (expressed as nanomoles of protoporphyrin formed per hour per milligram protein) in the mitochondrial fraction was 8 and in the plasma membrane enriched fraction was 4 to 6. The plasma membrane enriched fraction exhibited minimal cytochrome oxidase activity and no carotenoid content, indicating little contamination with mitochondrial or plastid membranes. Etioplasts from etiolated barley leaves exhibited a protoporphyrinogen oxidase specific activity of 7 to 12. Protoporphyrinogen oxidase activity in the barley root mitochondrial fraction and etioplast extracts was more than 90% inhibited by assay in the presence of the diphenyl ether herbicide acifluorfen methyl, but the activity in the plasma membrane enriched fraction exhibited much less inhibition by this herbicide (12 to 38% inhibition) under the same assay conditions. Acifluorfen-methyl inhibition of the organellar (mitochondrial or plastid) enzyme was maximal upon preincubation of the enzyme with 4 mm dithiothreitol, although a lesser degree of inhibition was noted if the organellar enzyme was preincubated in the presence of other reductants such as glutathione or ascorbate. Acifluorfen-methyl caused only 20% inhibition if the enzyme was preincubated in buffer without reductants. Incubation of barley etioplast extracts with the earlier tetrapyrrole precursor coproporphyrinogen and acifluorfen-methyl resulted in the accumulation of protoporphyrinogen, which could be converted to protoporphyrin even in the presence of the herbicide by the addition of the plasma membrane enriched fraction from barley roots. These findings have implications for the toxicity of diphenyl ether herbicides, whose light induced tissue damage is apparently caused by accumulation of the photoreactive porphyrin intermediate, protoporphyrin, when the organellar protoporphyrinogen oxidase enzyme is inhibited by herbicides. Our results suggest that the protoporphyrinogen that accumulates as a result of herbicide inhibition of the organellar enzyme can be oxidized to protoporphyrin by a protoporphyrinogen oxidizing activity that is located at sites such as the plasma membrane, which is much less sensitive to inhibition by diphenylether herbicides.     

Effects of the photobleaching herbicide, acifluorfen-methyl, on protoporphyrinogen oxidation in barley organelles, soybean root mitochondria, soybean root nodules, and bacteria

The photobleaching herbicide, acifluorfen-methyl (AFM), has been reported to be an inhibitor of the heme and chlorophyll biosynthetic enzyme protoporphyrinogen oxidase (Protox) in several plant species. However, AFM had no effect on the levels of Protox activity measured in a mitochondrial fraction from soybean roots. In contrast, AFM inhibited Protox activity in etioplasts from barley leaves and in mitochondria from barley roots, but the extent of inhibition varied depending upon the assay conditions and was maximal only in the presence of 5 mM dithiothreitol (DTT). AFM inhibition was enhanced by preincubation of barley organelle extract in the presence of DTT. Preincubation of barley extract with DTT and AFM together (but not with AFM alone) caused extensive enzyme inhibition which was not reversible by dialysis. These findings have implications for the mechanism of AFM action and for the differential effect of these herbicides on crop and weed species. AFM had no effect on the Protox activity of membranes from free-living bacterial cell of Bradyrhizobium japonicum or Escherichia coli, or on the high levels of Protox activity associated with the plant-derived membrane surrounding the symbiotic bacteria within the soybean root nodule.     

Localization within chloroplasts of protoporphyrinogen oxidase, the target enzyme for diphenylether-like herbicides.

Plant protoporphyrinogen oxidase is of particular interest since it is the last enzyme of the common branch for chlorophyll and heme biosynthetic pathways. In addition, it is the target enzyme for diphenyl ether-type herbicides, such as acifluorfen. Two distinct methods were used to investigate the localization of this enzyme within Percoll-purified spinach chloroplasts. We first assayed the enzymatic activity by spectrofluorimetry and we analyzed the specific binding of the herbicide acifluorfen, using highly purified chloroplast fractions. The results obtained give clear evidence that chloroplast protoporphyrinogen oxidase activity is membrane-bound and is associated with both chloroplast membranes, i.e. envelope and thylakoids. Protoporphyrinogen oxidase specific activity was 7-8 times higher in envelope membranes than in thylakoids, in good agreement with the number of [3H]acifluorfen binding sites in each membrane system: 21 and 3 pmol/mg protein, respectively, in envelope membranes and thylakoids. On a total activity basis, 25% of protoporphyrinogen oxidase activity were associated with envelope membranes. The presence of protoporphyrinogen oxidase in chloroplast envelope membranes provides further evidence for a role of this membrane system in chlorophyll biosynthesis. In contrast, the physiological significance of the enzyme associated with thylakoids is still unknown, but it is possible that thylakoid protoporphyrinogen oxidase could be involved in heme biosynthesis.     

Glutathione and ascorbic acid in spinach (Spinacia oleracea) chloroplasts. The effect of hydrogen peroxide and of Paraquat.

The stroma of spinach chloroplasts contains ascorbic acid and glutathione at millimolar concentrations. [Reduced glutathione]/[oxidized glutathione] and [ascorbate]/[dehydroascorbate] ratios are high under both light and dark conditions and no evidence for a role of oxidized glutathione or dehydroascorbate in the dark-deactivation of fructose bisphosphatase could be obtained. Addition of H2O2 to chloroplasts in the dark decreases the above ratios, an effect that is reversed on illumination. Addition of Paraquat to illuminated chloroplasts caused a rapid oxidation of reduced glutathione and ascorbate, and apparent loss of dehydroascorbate. Paraquat rapidly inactivated fructose bisphosphatase activity, as assayed under physiological conditions.     

The enzymic conversion of protoporphyrinogen IX to protoporphyrin IX in mammalian mitochondria.

Protoporphyrinogen oxidase, an enzyme which catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX in yeast cells, has been found in several mammalian tissues. It has been extracted from rat liver mitochondria by sonication in the presence of salt and detergent and partially purified. The enzyme is similar in many respects to yeast protoporphyrinogen oxidase. Based on its behavior on Sephadex G-200 the molecular weight of the enzyme is approximately 35,000. Catalysis by protoporphyrinogen oxidase was specific for proteoporphyrinogen IX (apparent Km of 11 muM) and proceeded maximally at pH 8.6 to 8.7. The effect of temperature on enzyme activity plotted according to Arrhenius gave a value of E of 9,100 calories per mol. Enzyme activity was inhibited in the presence of high salt concentrations and temperatures above 45 degrees. Oxygen was essential for protoporphyrinogen oxidase activity and an alternative elevtron acceptor has not yet been found. No requirement for a metal or other cofactor could be demonstrated. The presence of monothiol groups was indicated; however, it is not known whether the thiol groups are involved directly in the binding of substrate to the enzyme.     

"Respirasome"-like supercomplexes in green leaf mitochondria of spinach

Higher plant mitochondria have many unique features compared with their animal and fungal counterparts. This is to a large extent related to the close functional interdependence of mitochondria and chloroplasts, in which the two ATP-generating processes of oxidative phosphorylation and photosynthesis, respectively, take place. We show that digitonin treatment of mitochondria contaminated with chloroplasts from spinach (Spinacia oleracea) green leaves at two different buffer conditions, performed to solubilize oxidative phosphorylation supercomplexes, selectively extracts the mitochondrial membrane protein complexes and only low amounts of stroma thylakoid membrane proteins. By analysis of digitonin extracts from partially purified mitochondria of green leaves from spinach using blue and colorless native electrophoresis, we demonstrate for the first time that in green plant tissue a substantial proportion of the respiratory complex IV is assembled with complexes I and III into "respirasome"-like supercomplexes, previously observed in mammalian, fungal, and non-green plant mitochondria only. Thus, fundamental features of the supramolecular organization of the standard respiratory complexes I, III, and IV as a respirasome are conserved in all higher eukaryotes. Because the plant respiratory chain is highly branched possessing additional alternative enzymes, the functional implications of the occurrence of respiratory supercomplexes in plant mitochondria are discussed.     

Induction by different thioredoxins of ATPase activity in coupling factor 1 from spinach chloroplasts

ATPase activity of the coupling factor 1, CF1, isolated from spinach chloroplasts, was enhanced by reduction with dithiothreitol. Reduced thioredoxins from spinach chloroplasts, Escherichia coli and human lymphocytes replaced dithiothreitol as reductant and activator of the ATPase. CF1 must be in an oxidized activated state to be further activated by reduced thioredoxin. This state was obtained either by heating CF1 or removing the inhibitory intrinsic epsilon subunit from CF1. Efficiency and primary structure of the different thioredoxins were compared. The progressive addition of KCl during ATPase activation by reduced thioredoxin increases then decreases this process. We proposed that three basic amino acids corresponding to arginine 73 and lysines 82 and 96 in Escherichia coli thioredoxin play an important role in the anchorage of the thioredoxin to the negatively charged surface of the CF1 and are involved in the dual effect of KCl. The variations in the screening effect of the negative charges of the CF1 surface by K+ ions can indeed explain the changes in the anchorage of these 3 basic amino acids with concomitant variation in ATPase activity. Human thioredoxin must be 10 times more concentrated than Escherichia coli or spinach chloroplast thioredoxin to exhibit the same activation effect on the ATPase. This fact was related to the properties of a sequence equivalent to the part from amino acid 59 to 72 in Escherichia coli thioredoxin. This part which joins the two lobes of the thioredoxin is more hydrophilic and more negatively charged in human thioredoxin than in Escherichia coli or spinach chloroplast thioredoxin. Although ATPase activation was obtained at a very low concentration of the reduced spinach chloroplast thioredoxin, the thioredoxin formed only a loose complex with CF1.     

Distribution of Pyruvate Dehydrogenase Complex Activities between Chloroplasts and Mitochondria from Leaves of Different Species

Protoplasts from barley (Hordeum vulgare), pea (Pisum sativum), wheat (Triticum aestivum), and spinach (Spinacia oleracea) leaves were fractionated into chloroplast- and mitochondrion-enriched fractions. Pyruvate dehydrogenase complex capacities in mitochondria (mtPDC) and chloroplasts (cpPDC) were measured in appropriate fractions under conditions optimal for each isozyme. The total cellular capacity of PDC was similar in barley and pea but about 50% lower in wheat and spinach. In pea a distribution of 87% mtPDC and 13% cpPDC was found on a cellular basis. In barley, wheat, and spinach the subcellular distribution was the opposite, with about 15% mtPDC and 85% cpPDC. cpPDC activity was constant at about 0.1 nmol cell-1 h-1 in cells from different regions along the developing barley leaf and showed no correlation with developmental patterns of photosynthetic parameters, such as increasing Chl and NADP-glyceraldehyde-3-phosphate dehydrogenase activity. Similarly, the capacity of the mitochondrial isoform did not change during barley leaf development and had a developmental pattern similar to that of citrate synthase and fumarase. Differences in subcellular distribution of PDCs in barley and pea are proposed to be due to differences in regulation, not to changes in isozyme proportions during leaf development or to species-specific differences in phosphorylation state of mtPDC after organelle separation.     

Immunogold localization of phytoene desaturase in higher plant chloroplasts

In situ location of phytoene desaturase, a key enzyme in the carotenoid biosynthesis pathway, has been investigated in chloroplasts from higher plants. For this purpose, an antiserum has been raised against the phytoene desaturase from the cyanobacterium Synechococcus PCC 7942 overexpressed in E. coli . The specifity of this antiserum was demonstrated by inhibition of the enzymatic desaturation reaction in vitro. The antiserum was further purified and immunoabsorbed with E. coli proteins. The resulting IgG-fraction was tested by western blotting against membrane proteins from chloroplasts of tobacco ( Nicotiana tabacum L. cv. Samsun) and spinach ( Spinacia oleracea L. cv. Atlanta). Apparent molecular masses of immunoreactive proteins were 62 and 64 kDa. A western blot of different membrane fractions of spinach chloroplasts (inner and outer envelopes, and thylakoids) indicated a localization of the phytoene desaturase in thylakoids. A post embedding immunogold microscopy procedure was employed. In these experiments the main labelling (79%) was associated with thylakoid membranes of tobacco chloroplasts. Of the counted colloidal gold particles, 16% were found in the stroma. Only 5% were detected in the envelope membranes. These results give clear evidence that at least the majority of phytoene desaturase molecules is localized within thylakoid membranes of higher plant chloroplasts and that the presence of the enzyme in the envelope is of minor significance.     

Formate oxidation and oxygen reduction by leaf mitochondria

Mitochondria isolated from the leaves of several plant species were investigated for the presence of NAD-linked formate dehydrogenase. The NADH produced was oxidized by the electron transport sequence and was coupled to ATP synthesis. The amounts of formate dehydrogenase, and, thereby, the capacity for formate-dependent O₂ uptake, varied greatly among species. While no activity was detectable in mitochondria from soybean leaves, the rate of formate oxidation by spinach mitochondria was about one-half the rate of malate oxidation. In spinach, only mitochondria from green tissues oxidized formate. These last two observations raise questions as to the role of this reaction and the possible sources of the formate metabolized.     

Comparison of the EPR properties of photosystem I iron-sulphur centres A and B in spinach and barley

The properties of Photosystem I iron-sulphur centres A and B from spinach and barley chloroplasts were investigated by electron paramagnetic resonance spectroscopy (EPR). Barley chloroplasts were shown to photoreduce significant amounts of centre B at cryogenic temperatures unlike those from spinach which only photoreduced centre A. Centre B in barley chloroplasts was also reduced by dithionite before centre A and the EPR spectrum of reduced centre B was obtained. Illumination of barley chloroplasts at 15 K where centre B was chemically reduced resulted in the reduction of centre A and the appearance of spectral features indicating interaction between the two reduced centres. The variation of behaviours of iron-sulphur centres A and B between species favours a scheme of electron flow for Photosystem I where either centre A or centre B act as parallel electron acceptors from the earlier acceptor X.     

Evidence for a lack of phospholipid transfer protein in the stroma of spinach chloroplasts

The possible activity of phospholipid transfer protein in stroma extracts from spinach leaf has been investigated. Stroma, prepared from purified intact chloroplasts, was dialyzed and passed through various chromatography columns. None of the protein fractions eluted was able to stimulate the transfer of phosphatidylglycerol (PG) or phosphatidylcholine (PC) from liposomes to mitochondria, suggesting the lack of phospholipid transfer protein in the stroma from mature spinach chloroplasts.     

Mechanism of KCN inhibition of photosystem I.

Experiments with chloroplasts and purified spinach plastocyanin suggest a mechanism for KCN inhibition of Photosystem I. KCN inhibition can be bypassed by a detergent or reversed by replacement of the inactive plastocyanin. KCN bleaches and inactivates purified plastocyanin. KCN releases copper from chloroplast membranes and from purified plastocyanin. Cyanide does not bind to the apoprotein produced when plastocyanin is treated with KCN, and KCN-produced apoplastocyanin has a N-ethylmaleimide-reactive sulfhydryl group not found in holoplastocyanin. Apoplastocyanin is not active in restoring Photosystem I activity to plastocyanin-depleted membranes. Holoplastocyanin restores Photosystem I activities to plastocyanin-depleted membranes prepared from either control or KCN-treated chloroplasts to about the same extent. KCN-treated chloroplast membranes are found to have higher amounts of apoplastocyanin than do control chloroplast membranes. These results offer evidence that KCN removes the copper from plastocyanin in the chloroplast membrane, leaving the inactive apoplastocyanin which is unable to transfer electrons to Photosystem I.     

Plant pyruvate dehydrogenase complex: Inactivation and reactivation by phosphorylation and dephosphorylation

Pyruvate dehydrogenase complex activity from spinach leaf mitochondria was inhibited up to 90% within 2 min of incubation with 1 mm ATP at 27 °C. The inhibition was time, temperature and ATP concentration dependent. The inhibition was partially prevented with 3.0 mm dichloroacetate, a known inhibitor of mammalian pyruvate dehydrogenase kinases. Optimum pH for ATP-dependent inactivation was between 8.0 and 9.0 The inactivated complex was reactivated with 10 to 20 mm MgCl₂. Complete reactivation occurs within 10 min after MgCl₂ addition. Reactivation was inhibited by fluoride, a known inhibitor of mammalian pyruvate dehydrogenase phosphatase. Optimum pH for Mg²⁺-dependent reactivation was 8.0. It is concluded that the inactivation and reactivation process of pyruvate dehydrogenase complex from spinach leaf mitochondria is due to phosphorylation and dephosphorylation.     

Cytochrome f from spinach and cyanobacteria. Purification and characterization

Cytochrome f has been purified from spinach chloroplasts and from the photosynthetic membranes of the cyanobacterium Spirulina maxima. The spinach protein has an isoelectric point of 5.2 and gives a single band on isoelectric focusing gels. The S. maxima cytochrome shows a major band with a pI of 4.01 and a minor band with a pI of 3.97. S. maxima cytochrome f has a molecular weight approximately 38,000 and is monomeric, while the spinach protein is slightly smaller, approximately 36,000 daltons, and aggregates to form an octamer. S. maxima cytochrome f has an E'0 of +339 mV which is close to that of cytochromes f from higher plants. The NH2-terminal amino acid sequences of the cytochromes show striking similarities. Spinach cytochrome f shows a clear preference for oxidation by spinach plastocyanin and S. maxima cytochrome f is more readily oxidized by its in vivo reaction partner, cytochrome c553.