Two plant-type ferredoxins from a blue-green alga, Nostoc verrucosum

Two plant-type ferredoxins were isolated and purified from a blue-green alga, Nostoc verrucosum. They were separable by chromatography on a DEAE-cellulose column. The slow-moving band was designated ferredoxin I (Fd I) and the fast-moving band was ferredoxin II (Fd II). The ratio of the yield of ferredoxins I and II was about 1:0.84. Both ferredoxins had absorption spectra similar to those of plant-type ferredoxins. Two atoms of non-heme iron and two of labile sulfur were found per mol of both ferredoxin I and ferredoxin II. Their molecular weights were identical and estimated to be about 18 000 by a gel filtration method. The biochemical activities of these Nostoc ferredoxins were studied: the NADP photoreduction activity on one hand and the NADP-cytochrome c reductase activity on the other.     

Comparative studies on two ferredoxins from the cyanobacterium Nostoc strain MAC.

Two ferredoxins were isolated from the cyanobacterium Nostoc strain MAC grown autotrophically in the light or heterotrophically in the dark. In either case approximately three times as much ferredoxin I as ferredoxin II was obtained. Both ferredoxins had absorption maxima at 276, 282 (shoulder), 330, 423 and 465 nm in the oxidized state, and each possessed a single 2 Fe-2S active centre. Their isoelectric points were approx. 3.2. The midpoint redox potentials of the ferredoxins differed markedly; that of ferredoxin I was --350mV and that of ferredoxin II was --445mV, at pH 8.0. The midpoint potential of ferredoxin II was unusual in being pH dependent. Ferredoxin I was most active in supporting NADP+ photoreduction by chloroplasts, whereas ferredoxin II was somewhat more active in pyruvate decarboxylation by the phosphoroclastic system of Clostridum pasteurianum. Though the molecular weights of the ferredoxins determined by ultracentrifugation were the same within experimetnal error, the amino acid compositions showed marked differences. The N-terminal amino acid sequences of ferredoxins I and II were determined by means of an automatic sequencer. There are 11--12 differences between the sequences of the first 32 residues. It appears that the two ferredoxins have evolved separately to fulfil different roles in the organism.     

Two plant-type ferredoxins from a blue-green alga, Nostoc verrucosum.

Two plant-type ferredoxins were isolated and purified from a blue-green alga, Nostoc verrucosum. They were separable by chromatography on a DEAE-cellulose column. The slow-moving band was designated ferredoxin I (Fd I) and the fast-moving band was ferredoxin II (Fd II). The ratio of the yield of ferredoxins I and II was about 1 : 0.84. Both ferredoxins had absorption spectra similar to those of plant-type ferredoxins. Two atoms of non-heme iron and two of labile sulfur were found per mol of both ferredoxin I and ferredoxin II. Their molecular weights were identical and estimated to be about 18 000 by a gel filtration method. The biochemical activities of these Nostoc ferredoxins were studied: the NADP photoreduction activity on one hand and the NADP-cytochrome c reductase activity on the other.     

Isolation and properties of a ferredoxin from Anabaena Flos-Aquae

Ferredoxin was isolated from the blue-green alga Anabaena flos-aquae. Its homogeneity was shown by conventional and SDS-polyacrylamide gel electrophoresis, and isoelectric focusing on polyacrylamide gel columns, the latter indicating a pI at ca pH 3·7. The absorption spectrum had, in the oxidized state, maxima at 462, 421, 327 and 276 nm, with a shoulder at 284 nm, a spectrum characteristic of plant-type ferredoxins. The 421 : 276 nm absorbance ratio was typically 0.49. The ferredoxin effectively mediated the photoreduction of NADP⁺ by barley chloroplasts depleted of native ferredoxin. The MW obtained by sedimentation-equilibrium and sedimentation velocity-diffusion coefficient studies was ca 12 000 daltons, a value somewhat higher than suggested by amino acid composition data. The ferredoxin contained 2Fe and 2S per molecule.     

Contrasting modes of photosynthetic enzyme regulation in oxygenic and anoxygenic prokaryotes

Enzymes that are regulated by the ferredoxin/thioredoxin system in chloroplasts — fructose-1,6-bisphosphatase (FBPase), sedoheptulose-1,7-bisphosphatase purified from two different types of photosynthetic prokaryotes (cyanobacteria, purple sulfur bacteria) and tested for a response to thioredoxins. Each of the enzymes from the cyanobacterium Nostoc muscorum, an oxygenic organism known to contain the ferredoxin/thioredoxin system, was activated by thioredoxins that had been reduced either chemically by dithiothreitol or photochemically by reduced ferredoxin and ferredoxin-thioredoxin reductase. Like their chloroplast counterparts, N. muscorum FBPase and SBPase were activated preferentially by reduced thioredoxin f. SBPase was also partially activated by thioredoxin m. PRK, which was present in two regulatory forms in N. muscorum, was activated similarly by thioredoxins f and m. Despite sharing the capacity for regulation by thioredoxins, the cyanobacterial FBPase and SBPase target enzymes differed antigenically from their chloroplast counterparts. The corresponding enzymes from Chromatium vinosum, an anoxygenic photosynthetic purple bacterium found recently to contain the NADP/thioredoxin sytem, differed from both those of cyanobacteria and chloroplasts in showing no response to reduced thioredoxin. Instead, C. vinosum FBPase, SBPase, and PRK activities were regulated by a metabolite effector, 5-AMP. The evidence is in accord with the conclusion that thioredoxins function in regulating the reductive pentose phosphate cycle in oxygenic prokaryotes (cyanobacteria) that contain the ferredoxin/thioredoxin system, but not in anoxygenic prokaryotes (photosynthetic purple bacteria) that contain the NADP/thioredoxin system. In organisms of the latter type, enzyme effectors seem to play a dominant role in regulating photosynthetic carbon dioxide assimilation.     

Partial structure and properties of the ferredoxin from Rhodymenia palmata

A ferredoxin of MW 11 000 was isolated from the marine alga Rhodymenia palmata (Palmaria palmata). In its oxidised form the ferredoxin had absorption maxima at 276, sh 281, 328, 423 and 465 nm, and contained a single [2Fe-2S] cluster. The midpoint potential of the ferredoxin was ?400 mV and it effectively mediated electron transport in NADP⁺-photoreduction by higher plant chloroplasts, and pyruvate decarboxylation by the phosphoroclastic system of an anacrobic bacterium. The amino acid composition was Lys₃, His₁, Arg₁, Asx₁₂, Thr₉, Ser₈, Glx₁₃, Pro₄, Gly₈, Ala₇, Cys₅, Val₈, Ile₄, Leu₉, Tyr₄, Phe₂; tryptophan and methionine were absent from the molecule. The N-terminal amino acid region consisting of ca half the total amino acid sequence was determined using an automatic sequencer.     

Purification and Properties of Nitrite Reductase from Spinach Leaves

Nitrite reductase (EC 1.6.6.4) has been purified 730-fold from spinach leaves. The enzyme catalyzes the reduction of nitrite to ammonia, with the use of reduced form of methyl viologen and ferredoxin. A stoichiometry of one molecule of nitrite reduced per molecule of ammonia formed has been found. KCN at 2.5×10^-4 m inhibited nitrite reductase activity almost completely. Purified enzyme was almost homogeneous by disk electrophoresis with polyacrylamide gel. The molecular weight of the enzyme was estimated to be 61,000 from gel filtration. Nitrite reductase, in the oxidized form, has absorption maxima at 276, 388 and 573 mμ. Both methyl viologen and ferredoxin linked nitrite reductase activities of the enzyme were inactivated on exposure to low ionic strength.     

Plant-type and bacterial-type ferredoxins in a nitrogen-fixing cyanobacterium: Nostoc sp. strain PCC 73102

Abstract The cyanobacterium Nostoc sp. strain PCC 73102, cultured under nitrogen-fixing conditions, was investigated for the occurrence of ferrodoxins by SDS-PAGE/Western immunoblots using antisera directed against both a major plant-type and a bacterial-type ferredoxin purified from Anabaena variabilis . Immunocytological labelling and transmission electron microscopy were used to study the distribution of both types of ferredoxins in the Nostoc cells. SDS-PAGE/Western immunoblots revealed two proteins/polypeptides in the Nostoc strain, immunologically related to two soluble ferredoxins purified from Anabaena variabilis : the major plant-type ferredoxin (Fd I) and a bacterial-type ferredoxin (Fd III). Immunolocalization showed a uniform distribution of the plant-type and the bacterial-type ferredoxin in both the photosynthetic vegetative cells and in the nitrogen-fixing heterocysts, with no specific association with any subcellular inclusions. Using the particle analysis of an image processor, the labelling associated with the vegetative cells, expressed as number of gold particles per cell area, was found to be only slightly higher (1.2x) or almost twice as high (1.9x) compared to the heterocysts for the major plant-type and the bacterial-type ferredoxin, respectively.     

A non-photosynthetic ferredoxin gene is induced by ethylene in Citrus organs

The sequence and expression of mRNA homologous to a cDNA encoding a non-photosynthetic ferredoxin (Fd1) from Citrus fruit was investigated. The non-photosynthetic nature of this ferredoxin was deduced from: (1) amino acid sequence alignments showing better scores with non-photosynthetic than with photosynthetic ferredoxins, (2) higher expression in tissues containing plastids other than chloroplast such as petals, young fruits, roots and peel of fully coloured fruits, and (3) the absence of light-dark regulation characteristic of photosynthetic ferredoxins. In a phylogenetic tree constructed with higher-plant ferredoxins, Citrus fruit ferredoxin clustered together with root ferredoxins and separated from the photosynthetic ferredoxins. Non photosynthetic (root and fruit) ferredoxins, but not the photosynthetic ferredoxins, have their closest homologs in cyanobacteria. Analysis of ferredoxin genomic organization suggested that non-photosynthetic ferredoxins exist in Citrus as a small gene family. Expression of Fd1 is developmentally regulated during flower opening and fruit maturation, both processes may be mediated by ethylene in Citrus. Exogenous ethylene application also induced the expression of Fd1 both in flavedo and leaves. The induction of non-photosynthetic ferredoxins could be related with the demand for reducing power in non-green, but biosynthetically active, tissues.     

Consequences of the iron-dependent formation of ferredoxin and flavodoxin on photosynthesis and nitrogen fixation on Anabaena strains

Iron-dependent formation of ferredoxin and flavodoxin was determined in Anabaena ATCC 29413 and ATCC 29211 by a FPLC procedure. In the first species ferredoxin is replaced by flavodoxin at low iron levels in the vegetative cells only. In the heterocysts from Anabaena ATCC 29151, however, flavodoxin is constitutively formed regardless of the iron supply.Replacement of ferredoxin by flavodoxin had no effect on photosynthetic electron transport, whereas nitrogen fixation was decreased under low iron conditions. As ferredoxin and flavodoxin exhibited the same K_m values as electron donors to nitrogenase, an iron-limited synthesis of active nitrogenase was assumed as the reason for inhibited nitrogen fixation. Anabaena ATCC 29211 generally lacks the potential to synthesize flavodoxin. Under iron-starvation conditions, ferredoxin synthesis is limited, with a negative effect on photosynthetic oxygen evolution.     

Ferredoxin from Halobacterium of the Dead Sea — Mössbauer and EPR spectra and comparison with mössbauer spectrum of whole cells

Recoil-free measurements were carried out on a 2 Fe-ferredoxin, which was isolated and purified from an extreme halophile, Halobacterium of the Dead Sea. The spectrum of this ferredoxin in the oxidized state at 82 K is a superposition of two quadrupole doublets, representing two non-equivalent Fe³⁺ sites of equal intensity. The spectrum of the reduced ferredoxin is consistent with the presence of two pure classes of iron atoms, ferric (lower isomer shift) and ferrous (higher isomer shift). Interpretations of the recoil-free spectra are discussed. Mössbauer measurements were also carried out on frozen whole bacterial cells and the resulting spectrum was found to be quite different from that observed in the isolated ferredoxin. Tentative conclusions are reached concerning the localization of this ferredoxin in the cytosol of the Halobacteria.The EPR spectrum of the reduced ferredoxin obtained at 24 K exhibits rhombic symmetry with the following g values: 1.894, 1.984 and 2.07. These values are similar to those obtained with 2 Fe-ferredoxins of the plant type, except that the g _y and g _z values are somewhat higher. Both from the EPR and Mössbauer data, it is deduced that the spin relaxation times in reduced halophilic ferredoxins are faster than in the reduced plant ferredoxins.     

Spectroscopic and Biochemical Analyses of UV Effects on Phycobiliproteins of Anabaena sp. and Nostoc carmium

The effects of UV (280–400 nm) irradiation on phycobiliprotein composition have been studied in two N₂-fixing cyanobacteria, Anabaena sp. and Nostoc carmium, isolated from rice paddy fields in India. Phycobiliproteins were isolated and separated by sucrose density gradient centrifugation. After UV exposure the top fraction mainly contained carotenoids (absorption maximum at 485 nm), which first showed an increase in intensity and absorption and then a gradual decrease with increasing UV exposure in Anabaena sp., whereas, in Nostoc carmium this fraction showed a steady increase over the whole exposure time. The bottom fraction of both organisms mainly contained phycocyanin (absorption peak at 620 nm) which showed a steady decline in intensity, as well as absorption. Fluorescence excitation at 620 nm resulted in an emission at 650 nm which underwent a shift towards shorter wave-lengths with increasing UV-exposure time, indicating a disassembly of the phycobilisomal complex and of impaired energy transfer from accessory pigments to the reaction centers. SDS PAGE analysis of the fractions revealed a loss of high molecular mass linker proteins and low molecular mass (αβ monomers indicating that the phycobiliproteins, which function as accessory pigments for the operation of photosystem II, disassemble during UV irradiation.     

Response of N2-Fixing Cyanobacteria to Salt

The effect of salt on photosynthetic activity, acetylene reduction, and related activities was examined in two species of cyanobacteria, Nostoc muscorum and Calothrix scopulorum. Photosynthesis was more resistant to high salt concentration than was N₂ fixation. The salt resistance of both activities increased after a period of exposure of the cells to salinity. The transfer of electrons via ferredoxin and ferredoxin-nicotinamide adenine dinucleotide phosphate reductase was found to be extremely sensitive to salt. In comparison, the transfer of reducing power by glucose-6-phosphate dehydrogenase, isocitric dehydrogenase, and photosystem 1 was less affected by NaCl, whereas glutamine synthetase exhibited higher tolerance to salt.     

X-ray effects on ferredoxin fromClostridium pasteurianum

Summary X-ray irradiation of aqueous ferredoxin solutions isolated fromClostridium pasteurianum causes a rapid destruction of the ferredoxin molecule.The destruction is manifested by the decrease of the absorption at 390 nm, the liberation of ferric iron and hydrogen sulfide, and concommittant loss of biological activity in the phosphoroclastic reaction ofC. pasteurianum. The biological activity decreases parallel with the iron liberation, and was found to be dose and pH dependent.The yield of biological inactivation (G-value) and the yield of iron liberation showed the same value of 0.8. OH-radical scavengers like p-amino-benzoic acid and cysteine in low concentrations of 2×10^–3 M, protect ferredoxin effectively against radiation, suggesting that OH-radicals are mainly responsible for the inactivation.     

Action Spectra and the Role of Carotenoids in Photosynthesis

The action spectrum of photosynthetic (Vprochtetion was determined with ehloro-plast suspensions and whole leaves. Owing to a dominating influence of light scattering the action spectrum shows a much more uniform absorption of the incident light than would be expected from the absorption spectrum in direct light (0° deviation). The comparatively strong photosynthesis in green is probably further aided by the light reaction between cytochrome f and chlorophyll in which also the cyto-chromes act as pigments. The whole region 400 to about 900 mn is photosynthetically active with an obvious tendency to linear relation between the intensity of incident radiation ami photosynthetic Co-production, certain deviations hereby caused by specific absorption or activation. The results illustrate the participation of carotenoids in the primary energy conversion of photons to activated electrons and the role of at least two light reactions in the photosynthetic cycle. The reducing power of illuminated (3-carotene was demonstrated in vitro in its effect on ferredoxin. The steady state situation of pigments and enzymes is discussed.     

X-ray inactivation studies of nonheme iron proteins the action of X-rays on Spinach ferredoxin in aqueous solutions

Summary Exposure of aqueous spinach ferredoxin solutions to X-rays results in a rapid and irreversible denaturation of the molecule. The denaturation is manifested by a decrease of the characteristic absorption of spinach ferredoxin at 320 and 416 nm, and by the concomitant liberation of ferric iron and hydrogen sulfide. The absorption decrease at 320 and 416 nm and the iron liberation are found to parallel the activity decrease in functioning as electron transfer factor in the noncyclic electron transport system in spinach chloroplasts.X-ray inactivated spinach ferredoxin does not contain iron or free SH-groups, and can be regarded as anapo-form of the native protein. This X-ray-inactivated apoprotein, however, showed a higher molar extinction coefficient at 275 nm than the apoferredoxin, and was not reconstitutable.Spinach ferredoxin was found to be even more radiosensitive than clostridial ferredoxin. AG- value of 1.25 for biological inactivation and iron liberation was found, as compared to aG- value of 0.8 for clostridial ferredoxin.     

Photochemical system for regenerating NADPH from NADP with use of immobilized chloroplasts

Spinach chloroplasts were immobilized in 2% agar gel. Crude ferredoxin and NADP–ferredoxin oxidoreductase isolated from spinach were used as electron carriers. The activity of the NADP reduction by immobilized chloroplasts increased with increasing ferredoxin concentration and the maximum activity was obtained at 8μM ferredoxin. The saturation of NADP reduction was observed at a light intensity of over 1000 lx. The optimum pH and temperature of NADP reduction were 8 and 25°C, respectively. The reduced NADP in a reaction medium increased linearly with increasing reaction time under illumination. NADP was continuously reduced for 2 hr with a hollow-fiber reactor containing immobilized chloroplasts. NADPH and NADP were separated with a hollow-fiber dialyzer from ferredoxin and NADP–ferredoxin oxidoreductase, which were reused. The conversion ratio of NADP to NADPH was from 40 to 80%.     

Purification and molecular properties of ferredoxin-glutamate synthase from Chlamydomonas reinhardii

Ferredoxin-glutamate synthase (EC 1.4.7.1) from Chlamydomonas reinhardii has been purified to electrophoretic homogeneity, with a specific activity of 10.4 units mg^-1 protein, by a method which included chromatography on diethylaminoethyl sephacel and hydroxylapatite, and ferredoxin-sepharose affinity treatment. The enzyme is a single polypeptide chain of M _r 146000 dalton which shows an absorption spectrum with maxima at 278, 377 and 437 nm, and an A₂₇₆/A₄₃₇ absorptivity ratio of 7.0. The anaerobic addition of dithionite results in the loss of the absorption peak at 437 nm, which is restored upon reoxidation of the enzyme with an excess of 2-oxoglutarate, alone or in the presence of glutamine. This indicates the presence in the enzyme of a flavin prosthetic group, which is functional during the catalysis. The ferredoxin-glutamate synthase can be assayed with methyl viologen, chemically reduced with dithionite, but it is unable to use reduced pyridine nucleotide. Azaserine, 6-diazo-5-oxo-norleucine, bromocresol green and p-hydroxymercuribenzoate are potent inhibitors of this activity, which, on the other hand, is stable upon heating at 45°C for 10 min.Abbreviations DEAE-sephacel diethylaminoethyl sephacel - Fd ferredoxin - GOGAT glutaniate synthase (glutamine: -ketoglutarate aminotransferase) - SDS sodium dodecyl sulfate     

Interaction of flavodoxin with cyanobacterial thylakoids

Flavodoxin from the cyanobacterium Anabaena PCC 7119 has been shown to mediate, under illumination, the transfer of electrons from the thylakoidal membranes that were isolated from the same organism, to both the enzyme ferredoxin-NADP⁺ reductase and cytochrome c. Chemical cross-linking of ferredoxin or flavodoxin to the photosynthetic membranes provides a preparation that is active in cytochrome c photoreduction without the addition of external protein carrier. NADP⁺ photoreduction, albeit diminished, was observed only after addition of exogenous electron carrier protein. Immunoblotting analysis of the chemical adduct reveals that flavodoxin binds to a 10 kDa polypeptide subunit in the cyanobacterial Photosystem I which appears to act as its physiological partner in the electron transfer process.Abbreviations Fd ferredoxin - Fld flavodoxin - cyt c cytochrome c - EDC 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide - PS I Photosystem I     

On a water soluble factor neutralizing antibodies against the primary acceptor in photosynthetic electron transport of chloroplasts

Summary Rabbits immunized against the thylakoid system of chloroplasts form two different antibodies against the reducing site of photosystem I. One inhibits photosynthetic NADP⁺ and ferredoxin dependent cytochrome c reduction, but not photosynthetic anthraquinone reduction by isolated spinach chloroplasts. The other inhibits all three reductions. Both do not inhibit a Hill reaction with ferricyanide. The presumed antigen moiety against these antibodies was isolated as a water soluble factor released from lyophilized chloroplasts after treatment with diethylether. The heat stable, nondialysable factor has absorption peaks at 262 and 315 m. It is autoxidizable and has the property of a cytochrome c reducing substance. The factor neutralizes the antibody inhibition of photosynthetic anthraquinone and ferredoxin reduction. It is proposed that the soluble factor is the prosthetic group of the primary acceptor of photosystem I.Abbreviations CRS cytochrome c reducing substance - FRS ferredoxin reducing substance - DAD diaminodurene - TMPD N-tetramethyl-p-phenylene-diamine. The work reported on here was supported by Verband der chemischen Industrie — Fonds der chemischen Industrie