The distribution of NADPH-protochlorophyllide oxidoreductase in relation to chlorophyll accumulation along the barley leaf gradient

The possible regulatory role of NADPH-protochlorophyllide oxidoreductase for chlorophyll accumulation has been investigated in barley plants. Within the primary leaf of etiolated plants the different maturation stages of etioplasts are found in a linear series with the youngest in cells near the base and the oldest in cells near the tip. This distribution of different plastid forms is paralleled by drastic differences in the NADPH-protochlorophyllide-oxidoreductase content of the plastids and their capacity to accumulate chlorophyll during illumination. The amount of enzyme and the rate of chlorophyll accumulation are highest in the mature etioplast in the tip of the leaf and both decline rapidly with decreasing age of the leaf tissue, being almost undetectable in the leaf base. The translatable mRNA coding for the enzyme shows a different distribution pattern within the leaf. The highest concentration is found in the middle part of the leaf while in the top part only traces of this mRNA are detectable. It is concluded that during leaf development the enzyme is synthesized rapidly only during a limited time period and that it is stored subsequently in the mature etioplast as a stable protein. The close correlation between the distribution of the enzyme within the barley leaf and that of the potential to accumulate chlorophyll during illumination would favour a control of chlorophyll accumulation by the amount of NADPH-protochlorophyllide oxidoreductase. Dark-grown plants which were exposed to far-red light were used to test this possibility. The far-red-absorbing form of phytochrome (P_fr) has an inverse effect on the kinetics of chlorophyll accumulation and the enzyme concentration. Our results indicate that the rate of chlorophyll accumulation in barley is not determined by the level of NADPH-protochlorophyllide oxidoreductase present in the leaves.     

Membrane-bound NADPH dehydrogenase- and ferredoxin: NADP oxidoreductase activity involved in electron transport during acetate oxidation to CO2 in Desulfobacter postgatei

Desulfobacter postgatei grows on acetate and sulfate as energy source. The oxidation of acetate to 2 CO₂ proceeds via the citric acid cycle involving membrane-bound succinate dehydrogenase and membrane-bound malate dehydrogenase. We report here that the organism contains membrane-bound NADPH dehydrogenase and ferredoxin: NADP oxidoreductase for the reoxidation of NADPH and reduced ferredoxin generated during isocitrate- and 2-oxoglutarate oxidation, respectively. The presence of proton translocating ATPase activity is also described.NADPH dehydrogenase and succinate dehydrogenase were found to be electrically connected within the membrane and electron transfer between these two enzymes was shown to be coupled with proton translocation. The membrane fraction catalyzed the oxidation of NADPH with fumarate and the reduction of NADP with succinate. NADPH oxidation with fumarate was stimulated by protonophores and inhibited by the proton translocating ATPase inhibitor dicyclohexylcarbodiimide (DCCD) and by heptylhydroxyquinoline-N-oxide (HQNO); inhibition by DCCD was relieved by protonophores. NADP reduction with succinate was dependent on ATP and inhibited by protonophores, DCCD, and HQNO. The membrane fraction also mediated the oxidation of NADPH with the water soluble menaquinone analogue dimethylnaphthoquinone (DMN) and the reduction of fumarate with DMNH₂. Only the former reaction was stimulated by protonophores and only the latter reaction was inhibited by HQNO. This suggests that the NADPH dehydrogenase reaction is the site of energy conservation and the succinate dehydrogenase is the site of HQNO inhibition.Non-standard abbreviations APS Adenosine 5-phosphosulfate - DCCD N,N-dicyclohexylcarbodiimide - DCPIP 2,6-dichloroindophenol - DMN 2,3-dimethyl-1,4-naphthoquinone - DTT DL-1,4-dithiothreitol - HQNO 2(n-heptyl)-4-hydroxyquinoline-N-oxide - TCS 3,5,3,4-tetrachlorosalicylanilide - Tricine N-tris-(hydroxymethyl)methylglycine - TTFB 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole - SF-6847 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile     

Structure and function of the chloroplast cytochrome bf complex

The chloroplast cytochrome bf complex is an intrinsic multisubunit protein from the thylakoid membrane consisting of four polypeptides: cytochrome f, a two heme containing cytochrome b ₆, the Rieske iron-sulfur protein, and a 17 kD polypeptide of undefined function. The complex functions in electron transfer between PSII and PSI, where most mechanisms suggest that the transfer of a single reducing equivalent from plastoquinol to plastocyanin results in the translocation of two protons across the membrane. Primary sequence analyses, dichroism studies, and functional considerations allow the construction of an approximate structural model of a monomeric complex, although some evidence exists for a dimeric structure. Resolution of the properties of the two cytochrome b ₆ hemes has relied upon the availability of purified solubilized complex, while evidence in the thylakoid suggests the difference between the two hemes are not as great in situ. Such variability in the spectroscopic and electrochemical properties of the cytochrome b ₆ is a major concern during the experimental use of the purified complex. There is a general consensus that the complex contains a plastoquinol oxidizing (Q_z) site, although the evidence for a plastoquinone reduction (Q_c) site, called for in most mechanistic hypotheses, is less substantive. Probably the most severe challenge to the so called Q-cycle mechanism comes from experimental observations made with cytochrome b ₆ initially reduced, where proposed interpretations more closely resemble a b-cycle than a Q-cycle. Although functional during cyclic electron transfer, the role of the complex and its possible interaction with other proteins, has not been completely resolved.Abbreviations Cytochrome b _H high potential cytochrome b ₆ - Cytochrome b _L low potential cytochrome b ₆ - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DNP-INT 2-iodo-6-isopropyl-3-methyl-2,4,4-trinitrodiphenyl ether - FNR ferredoxin:NADP oxidoreductase - HQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - NQNO 2-n-nonyl-4-hydroxyquinoline-N-oxide - Q_c quinone binding site on the cytochrome bf complex near the outside of the thylakoid membrane, alternatively designated centre i or centre r - Q_z quinone binding site on the cytochrome bf complex near the inside of the thylakoid membrane, alternatively designated centre o     

Molecular and serological comparisons of purified xanthine dehydrogenases from root nodules of three ureide-producing legume species

Xanthine dehydrogenase (XDH, EC 1.2.1.37) was immunopurified from root nodules of three legume species, soybean [ Glycine max (L.) Merr. cv. Pella], cowpea [ Vigna unguiculata (L.) Walp. cv. California Black Eye], and lima bean [ Phaseolus lunatus L. Henderson]. Polyclonal antibodies raised against each enzyme and monoclonal antibodies raised against soybean XDH were used to compare the three enzymes serologically. Double diffusion and enzyme-linked immunosorbent assays with polyclonal and monoclonal antibodies showed that the cowpea and lima bean enzymes are very similar immunologically but both differ measurably from soybean. Amino acid compositions of the legume nodule XDHs are presented as well. Although relatedness between these enzymes can be detected by immunological crossreactivity, each XDH has unique epitopes that can be used to distinguish the three proteins.     

Properties of reformed prolamellar bodies from illuminated and redarkened etiolated wheat plants

Prolamellar bodies were isolated from etiolated leaves of wheat ( Triticum aestivum L. cv. Walde, Weibull), which were illuminated for 4 h and then grown in darkness for 16 h. The inner etiochloroplast membranes were isolated by differential centrifugation, and prolamellar bodies and thylakoids were separated on a 10–50% continuous sucrose density gradient. The reformed prolamellar bodies contained phototransformable protochlorophyllide as the main pigment as shown by low temperature fluorescence spectra and high performance liquid chromatography. After illumination with 3 flashes of white light almost all of the protochlorophyllide was transformed to chlorophyllide. In the thylakoids, however, most of the protochlorophyllide was not phototransformed. The reformed prolamellar bodies and the thylakoids showed a fluorescence emission ratio 657/633 nm of 5.6 and 0.5, respectively. Both membrane systems contained also chlorophyllide and chlorophyll synthesized during the illumination. Polyacrylamide gel electrophoresis showed the main chlorophyllide oxidoreductasse.
Teransmission and scanning electron micrographs indicated that the reformed prolamellar bodies are mainly of the "narrow" type and that the prolamellar body fraction had only a minor contamination with thylakoid membranes.
The results obtained showed that reformed prolamellar bodies isolated from illuminated redarkened etiolated wheat leaves had features very similar to the prolamellar bodies isolated from etiolated leaves. This provides support for the idea that prolamellar bodies are an important natural membrane system which plays a dynamic role in the development of the etio-chloroplasts in light.     

Involvement of transferrin in the reduction of iron by the transplasma membrane electron transport system

Nonpermeable electron acceptors can be reduced by a transplasma membrane electron transport system in suspensions of intact cells. Here we report that diferric transferrin is reduced by HeLa S3 cells. The reduction is recorded spectrophotometrically as the formation of the ferrous complex of bathophenanthroline disulfonate. Ferric ammonium citrate can also be used as an electron acceptor, and the presence of low concentrations of diferric transferrin greatly stimulates the reduction of trivalent iron under these conditions. Likewise very low concentrations of ferricyanide, which does not give rise to a ferrous bathophenanthroline disulfonate complex formation, have a strong stimulatory effect on the complex formation when ferric ammonium citrate is the source of ferric iron. Apotransferrin is a potent inhibitor of the reaction. The inhibition occurs at the concentration necessary for complete occupancy of the transferrin receptors. The inhibition can be demonstrated also when high concentrations of ferricyanide are used as electron acceptor. The possible mechanism behind the reported phenomena is discussed, and it is concluded that the transplasma membrane electron transport system can be involved in the process of cellular iron uptake.     

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     

Cloning and transcription analysis of the ndh(A-I-G-E) gene cluster and the ndhD gene of the cyanobacterium Synechocystis sp. PCC6803

The plastid DNA of higher plants contains eleven reading frames that are homologous to subunits of the mitochondrial NADH-ubiquinone oxidoreductase (complex I). The genes are expressed, but a plastid NAD(P)H dehydrogenase has not yet been isolated and the function of the enzyme in plastid metabolism is unknown. Cyanobacteria also contain a NADH dehydrogenase that is homologous to the mitochondrial complex I. The enzyme is sensitive to rotenone and is located on the cytoplasmic and the thylakoid membrane. We report here the sequence of five subunits (ndhA, -I, G, -E and -D) of the NADH dehydrogenase from the unicellular cyanobacterium Synechocystis sp. PCC6803. As in plastid DNA, the genes ndh(A-I-G-E) are clustered and probably constitute an operon. The ndhD gene is associated with a gene encoding an iron-sulphur protein of photosystem I (psaC) as in plastid DNA. In contrast to the situation in plastids, psaC and ndhD are not cotranscribed but transcribed from opposite strands. The deduced amino acid sequence of the cyanobacterial polypeptides is more similar to the corresponding plastid (40-68% identity) than to the corresponding mitochondrial subunits (17-39% identity). Thus, the cyanobacterial NADH-dehydrogenase provides a prokaryotic model system which is more suitable to genetic analysis than the enzyme of plastids.     

Fermentation of mandelate to benzoate and acetate by a homoacetogenic bacterium

A strictly anaerobic, Gram-positive, rod-shaped bacterium, strain AmMan1, was isolated from freshwater sediment with mandelate (-hydroxy-phenylacetate) as sole carbon and energy source, and was assigned to the genus Acetobacterium. Only the d-enantiomer of mandelate was degraded, and was fermented to acetate and benzoate. Non-aromatic growth substrates (pyruvate, lactate, malate, glycerol, ethylene glycol, and H₂/CO₂) were fermented to acetate as sole product. Methoxylated aromatics were demethoxylated to the corresponding phenols. The guanine-plus-cytosine content of the DNA was 36.5±1.5%. Carbon monoxide dehydrogenase, dichlorophenol indophenol-reducing lactate dehydrogenase, NAD-dependent mandelate dehydrogenase, phosphate acetyl transferase, acetate kinase, and pyruvate- or phenylglyoxylate-dependent benzylviologen reductase were measured in mandelate-and/or lactate-grown cells, respectively. A pathway of the homoacetogenic fermentation of mandelate is suggested as another example of incomplete substrate oxidation by homoacetogenic bacteria.     

EPR and Mössbauer spectroscopic studies on the tetrameric,NAD-linked hydrogenase of Nocardia opaca 1b and its two dimers: 1. The βδ-dimer—a prototype of a simple hydrogenase

The cytoplasmic, tetrameric NAD-linked hydrogenase from Nocardia opaca 1b can be separated in two dimeric substructures, an -dimer with NADH:electron acceptor oxidoreductase (diaphorase) activity and a -dimer which displays hydrogenase activity with artificial electron carriers. These two dimers were preparatively isolated by a FPLC Mono Q procedure in the absence of nickel and at alkaline pH values. The hydrogenase-active -dimer contained, as analyzed by inductively coupled plasma mass spectrometry (ICP-MS), 3.5–3.9 iron atoms and 1.3–1.7 nickel atoms per dimer molecule. EPR and Mössbauer spectra indicated the presence of a [4Fe-4S] cluster. This center turned out to be extremely labile towards oxidants. Oxidation led to irreversible convertion into a [3Fe-4S] form, thus representing an artifact and not a regulatory state of the cluster. The midpoint redox potential of the [4Fe-4S] cluster was determined to be -385 mV. Very weak EPR Ni signals of the -dimer were detectable in the oxidized as well as in the reduced state. The diaphorase-active -dimer was free of nickel and the iron content corresponded to 11.2–12.8 Fe atoms per dimer molecule. From EPR and Mössbauer measurements it was concluded that this dimer contained two [4Fe-4S] clusters, one [2Fe-2S] and one [3Fe-4S] cluster. In accordance with the results obtained for the dimer proteins, for the whole enzyme an iron content of 15.8–16.2 atoms per enzyme molecule have been determined. EPR spectra and spectrum simulations of the native hydrogenase corroborate the cluster assignments of the two dimers: in total the enzyme contains one [2Fe-2S] cluster, one [3Fe-4S] cluster and three [4Fe-4S] clusters.