Ferredoxin-Dependent Photoreduction of the Monodehydroascorbate Radical in Spinach Thylakoids

Thylakoid-bound and stromal ascorbate peroxidases scavenge thehydrogen peroxide that is photoproduced in PSI of chloroplastthylakoids. The primary oxidation product of ascorbate in thereaction catalyzed by ascorbate peroxidase, the monodehydroascorbate(MDA) radical, is photoreduced by thylakoids [Miyake and Asada(1992) Plant Cell Physiol. 33: 541]. We have now shown thatthe photoreduction of MDA radical in spinach thylakoids is largelydependent on ferredoxin (Fd), as determined by the monitoringthe MDA radical by electron paramagnetic resonance. Further,the reduced Fd generated by NADPH and Fd-NADP reductase couldreduce the MDA radical at a rate of over 10⁶ M^–1 s^–1,indicating that the photoreduced Fd in PSI directly reducesthe MDA radical to ascorbate. Photoreduction of NADP⁺ by spinach thylakoids was suppressedby the MDA radical and conversely that of MDA radical was suppressedby NADP⁺, indicating a competition between the MDA radical andNADP⁺ for the photoreduced Fd in PSI. The ratio of the rateconstant for the photoreduction of MDA radical to that for thephotoreduction of NADP⁺ was estimated to be more than 30 to1. Thus, MDA radical is preferentially photoreduced as comparedto NADP⁺. From these results, we propose that the thylakoid-boundascorbate peroxidase and the Fd-dependent photoreduction ofMDA radical in PSI are the primary system for the scavengingof the hydrogen peroxide that is photoproduced in the thylakoids. (Received December 9, 1993; Accepted February 16, 1994)     

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.     

Ferredoxin Isolated from Plant Non-Photosynthetic Tissues. Purficaition and Characterization

A ferredoxin was isolated from non-photosynthetic tissues ofthe lower storage root of radish (Raphanus sativus L. var. acantiformiscultivar Miyashige) in a pure form by conventional means. Itshowed the characteristic features in its absorption spectrumof chloroplast-type ferredoxin. However, amino acid compositionand amino (N)- terminal sequence were different from those ofradish leaf ferredoxin. Root ferredoxin was able to transferelectrons from dithionite to nitrite reductase [EC 1.7.7.1 [EC] ]isolated from mung bean seedling roots and also to mediate NADP⁺photoreduction in spinach broken chloroplasts. It therefore is suggested that a set of distinctive molecularspecies of ferredoxin is present in non-photosynthetic tissuesand functions as a redox mediator in ferredox-independent enzymesystems. (Received October 18, 1985; Accepted January 16, 1986)     

Comparative Studies on the Properties of Two Ferredoxins from Pisum sativum L.

Two ferredoxins in approximately equal amounts were isolatedfrom 3 week old Pisum sativum L. seedlings. Both ferredoxinshad identical absorption spectra with maxima at 276, 327, 424,and 468 nm in the oxidized state, and each possessed a single2Fe-2S active centre. The isoelectric points of the two ferredoxinswere both at pH 3·3, and mixtures could not be separatedby isoelectric focusing on polyacrylamide gels. The midpointredox potentials of the ferredoxins were close to –415mV, but they differed slightly in their biological activity.Ferredoxin I was slightly the more active of the two in catalysingNADP⁺ photoreduction by Pisum or Hordeum chloroplasts whereasferredoxin II was more active in catalysing the oxidative cleavageof pyruvate by extracts of Clostridium pasteurianum. Thoughthe molecular weights of the ferredoxins determined by ultracentrifugationwere the same within experimental error, the amino acid compositionsshowed marked differences. The N-terminal 40 amino acid residuesof ferredoxins I and II were determined by means of an automaticsequencer. There were 15 differences, suggesting that gene duplicationhad occurred early in evolutionary time. Ferredoxin I appearsto be more closely related to the other angiosperm ferredoxinssince it differed in only 6 positions compared with the correspondingsequence for Medicago sativa (alfalfa) ferredoxin. The ratioof the two ferredoxins in Pisum sativum was shown to be dependenton the age of the seedlings and environmental growth conditions.     

Evidence from directed mutagenesis that positively charged amino acids are necessary for interaction of nitrogenase with the [2Fe-2S) heterocyst ferredoxin (FdxH) from the cyanobacterium Anabaena sp., PCC7120

Sequence comparison of the heterocyst-type ferredoxin (FdxH) from Anabaena 7120 and type-I ferredoxins (PetF) from the same organism and other cyanobacteria revealed a group of positively charged residues characteristic for FdxH. Molecular modeling showed that these basic amino acids are clustered on the surface of FdxH. The corresponding domain of PetF contained acidic or nonpolar residues instead. To identify amino acids that are important for interaction with nitrogenase, we generated site-directed mutations in the fdxH gene and assayed the in vitro activity of the resulting recombinant proteins isolated from Escherichia coli. In addition to the point mutants, two chimeric proteins, FdxH : PetF and PetF : FdxH, were constructed containing the 58 N-terminal amino acids of one ferredoxin fused to the 40 C-terminal amino acids of the other. Exchange of lysines 10 and 11 of FdxH for the corresponding residues of PetF (glutamate 10 and alanine 11) resulted in a ferredoxin with greatly decreased affinity to nitrogenase. This indicates an important function of these basic amino acids in interaction with dinitrogenase reductase (NifH) from Anabaena. In addition we checked the reactivity of the recombinant ferredoxins with ferredoxin-NADP⁺ oxidoreductase (FNR) and photosystem I. The experiments with both the chimeric and point mutated ferredoxins showed that the C-terminal part of this protein determines its activity in NADP⁺ photoreduction.     

Evidence from directed mutagenesis that positively charged amino acids are necessary for interaction of nitrogenase with the [2Fe-2S) heterocyst ferredoxin (FdxH) from the cyanobacterium Anabaena sp., PCC7120

Sequence comparison of the heterocyst-type ferredoxin (FdxH) from Anabaena 7120 and type-I ferredoxins (PetF) from the same organism and other cyanobacteria revealed a group of positively charged residues characteristic for FdxH. Molecular modeling showed that these basic amino acids are clustered on the surface of FdxH. The corresponding domain of PetF contained acidic or nonpolar residues instead. To identify amino acids that are important for interaction with nitrogenase, we generated site-directed mutations in the fdxH gene and assayed the in vitro activity of the resulting recombinant proteins isolated from Escherichia coli. In addition to the point mutants, two chimeric proteins, FdxH : PetF and PetF : FdxH, were constructed containing the 58 N-terminal amino acids of one ferredoxin fused to the 40 C-terminal amino acids of the other. Exchange of lysines 10 and 11 of FdxH for the corresponding residues of PetF (glutamate 10 and alanine 11) resulted in a ferredoxin with greatly decreased affinity to nitrogenase. This indicates an important function of these basic amino acids in interaction with dinitrogenase reductase (NifH) from Anabaena. In addition we checked the reactivity of the recombinant ferredoxins with ferredoxin-NADP⁺ oxidoreductase (FNR) and photosystem I. The experiments with both the chimeric and point mutated ferredoxins showed that the C-terminal part of this protein determines its activity in NADP⁺ photoreduction.     

Effects of copper on photosynthetic electron transport systems in spinach chloroplasts

The effects of copper on photosynthetic electron transfer systemsin isolated spinach chloroplasts were studied. Two differentinhibitions were observed. First, copper markedly inhibitedferredoxin-catalyzed reactions such as NADP⁺ photoreduction.The concentration required for 50% inhibition was about 2 µMof cupric sulfate. However, electron flow from reduced 2,6-dichloroindophenol(DCIP) to methyl viologen was not affected. The dissociationconstant between ferredoxin and ferredoxin-NADP⁺ reductase wasunchanged in the presence of 2.5 µM of cupric sulfate.In enzymic reaction systems, the ferredoxin-dependent electronflow from NADPH to cytochrome c was also strongly inhibitedin the presence of cupric sulfate, while DCIP reduction withNADPH as the electron donor was not affected. Second, DCIP photoreductionwas weakly blocked by copper and the lost activity could notbe recovered by adding 1,5-diphenylcarbazide (DPC). It can be concluded that copper directly interacted with ferredoxincausing inhibition of ferredoxin-dependent reactions. Further,copper caused weak inactivation between the oxidizing side ofthe reaction center of photosystem II and the electron donatingsite of DPC. (Received August 8, 1977; )     

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.     

Light- and thiosulfate-dependent reduction of nicotinamide adenine dinucleotides in whole cells of Chromatium vinosum

The light-driven, thiosulfate-dependent reduction of nicotinamideadenine dinucleotides under acrobic conditions in whole cellsof Chromatium vinosum was investigated. The total concentration of pyridine nucleotides in whole cellswas about 50 nmoles per µmole of bacteriochlorophyll.Under dark aerobic conditions, the majority of the nucleotidespresent was NAD⁺ with about 20% as NADP⁺. About 40% of the total NAD was reduced under continuous illumination.Thiosulfate or sulfide was needed for the photoreduction, whileorganic acids such as succinate or malate were not. The initialrate of NAD⁺ photoreduction in the presence of thiosulfate wasapproximately 100 nmoles per µmole of bacteriochlorophyllper min. The NAD⁺ photoreduction was strongly inhibited by uncouplersand electron transfer inhibitors. In contrast, an energy transferinhibitor, N, N'-dicyclohexylcarbodiimide, did not affect NAD⁺photoreduction at a concentration at which the light-inducedATP formation was inhibited. A transmembrane electrochemicalH⁺ gradient generated by cyclic electron transfer may be theenergy source for reduction of NAD⁺ in Chromatium vinosum. (Received April 2, 1980; )     

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     

Crystalline Ferredoxin from a Blue-Green Alga, Nostoc sp.

Ferredoxin isolated from a blue-green alga, Nostoc sp., was purified and crystallized. The absorption spectrum of Nostoc ferredoxin had, in the oxidized state, peaks at 276, 331, 423, and 470 nm, a pattern characteristic of chloroplast-type ferredoxin. The 423:276 absorption ratio was 0.57. The midpoint oxidation-reduction potential of Nostoc ferredoxin was found to be –406 mV, at pH 7.5. Nostoc ferredoxin mediated the photoreduction of NADP by isolated Nostoc chromato-phores and spinach chloroplasts from which the native ferredoxin was removed. The molar ratio of Nostoc ferredoxin to chlorophyll a was about 1:50, a ratio higher than usually found in photosynthetic cells. The possible evolutionary significance of the properties of Nostoc ferredoxin compared with those of ferredoxins from other photosynthetic organisms is discussed.     

Primary structure of the two (4 Fe-4 S) clusters ferredoxin from Desulfovibrio desulfuricans (strain Norway 4)

The primary structure of a ferredoxin isolated from D. desulfuricans Norway strain, which we called ferredoxin II (Fd II) has been elucidated. This ferredoxin is a dimer constituted of two identical subunits of molecular weight 6000. In ferredoxin II two (4 Fe-4 S) centers are present per subunit instead of one (Fe-S) center as is the case for the other ferredoxins isolated from Desulfovibrio and for Fd I from the same organism. The comparison of amino-acid sequences shows that ferredoxin II presents more homologies with clostridial type ferredoxin than with the ferredoxins from D. gigas and D. africanus.     

Effects of magnesium and chloride ions on light-induced electron transport in membrane fragments from a blue-green alga

The effects of magnesium and chloride ions on photosynthetic electron transport were investigated in membrane fragments of a blue-green alga, Nostoc muscorum (Strain 7119), noted for their stability and high rates of electron transport from water or reduced dichlorophenolindophenol to NADP⁺. Magnesium ions were required not only for light-induced electron transport from water to NADP⁺ but also for protection in the dark of the integrity of the water-photooxidizing system (Photosystem II). Membrane fragments suspended in the dark in a medium lacking Mg²⁺ lost the capacity to photoreduce NADP⁺ with water on subsequent illumination. Chloride ions could substitute, but less effectively, for each of these two effects of magnesium ions. By contrast, the photoreduction of NADP⁺ by DCIPH₂ was independent of Mg²⁺ (or Cl^?) for the protection of the electron transport system in the dark or during the light reaction proper. Furthermore, high concentrations of MgCl₂ produced a strong inhibition of NADP⁺ photoreduction with DCIPH₂ without significantly affecting the rate of NADP⁺ photoreduction with water. The implications of these findings for the differential involvement of Photosystem I and Photosystem II in the photoreduction of NADP⁺ with different electron donors are discussed.     

Differential activities of heterocyst ferredoxin,vegetative cell ferredoxin,and flavodoxin as electron carriers in nitrogen fixation and photosynthesis in Anabaena sp.

In cyanobacteria an increasing number of low potential electron carriers is found, but in most cases their contribution to metabolic pathways remains unclear. In this work, we compare recombinant plant-type ferredoxins from Anabaena sp. PCC 7120, encoded by the genes petF and fdxH, respectively, and flavodoxin from Anabaena sp. PCC 7119 as electron carriers in reconstituted in vitro assays with nitrogenase, Photosystem I, ferredoxin-NADP⁺ reductase and pyruvate-ferredoxin oxidoreductase. In every experimental system only the heterocyst ferredoxin catalyzed an efficient electron transfer to nitrogenase while vegetative cell ferredoxin and flavodoxin were much less active. This implies that flavodoxin is not able to functionally replace heterocyst ferredoxin. When PFO-activity in heterocyst extracts was reconstituted under anaerobic conditions, both ferredoxins were more efficient than flavodoxin, which suggested that this PFO was of the ferredoxin dependent type. Flavodoxin, synthesized under iron limiting conditions, replaces PetF very efficiently in the electron transport from Photosystem I to NADP⁺, using thylakoids from vegetative cells.Abbreviations BSA bovine serum albumin - FdxH heterocyst ferredoxin - Fld flavodoxin - FNR ferredoxin-NADP⁺ reductase - MV methyl viologen - PetF vegetative cell ferredoxin - PFO pyruvate-ferredoxin oxidoreductase - Pyr piruvate - PS I Photosystem I     

The electron transport to nitrogenase in Mycobacterium flavum

1. Two ferredoxin-type iron-sulfur proteins have been isolated from Mycobacterium flavum 301 grown under nitrogen-fixing, iron-sufficient conditions. No flavodoxin was observed. 2. These ferredoxins are apparently soluble: they were present in the supernatant fraction after disrupting by decompression. Only small amounts were present in particulate fractions. 3. The two ferredoxins were separated by chromatography on DEAE-cellulose, Sephadex or electrophoresis. 4. Both ferredoxins mediated the transfer of electrons from illuminated spinach chloroplasts to a nitrogenase preparation to reduce acetylene. Ferredoxin II was specifically about five times more active than ferredoxin I. Ferredoxin II was also active in the photosynthetic NADP+-reduction whereas ferredoxin I was not. 5. Both ferredoxins were reversibly reduced by either sodium dithionite, illuminated spinach chloroplasts or hydrogen plus hydrogenase from Clostridium pasteurianum. 6. Attempts to determine the primary electron donor for nitrogen fixation in Mycobacterium flavum were unsuccessful. Acetylene reduction in Mycobacterium extracts was obtained only with sodium dithionite or illuminated spinach chloroplasts as electron donors. The reduction of the electron carrier (e.g. ferredoxin) rather than the transfer of electrons from the reduced carrier to nitrogenase was rate-limiting.     

Evaluation of the participation of ferredoxin in oxygen reduction in the photosynthetic electron transport chain of isolated pea thylakoids

The contribution to reduction of oxygen by ferredoxin (Fd) to the overall reduction of oxygen in isolated pea thylakoids was studied in the presence of Fd versus Fd + NADP⁺. The overall rate of electron transport was measured using a determination of Photosystem II quantum yield from chlorophyll fluorescence parameters, and the rate of oxidation of Fd was measured from the light-induced redox changes of Fd. At low light intensity, increasing Fd concentration from 5 to 30 μM in the absence of NADP⁺ increased the proportion of oxygen reduction by Fd from 25–35 to 40–60% in different experiments. This proportion decreased with increasing light intensity. When NADP⁺ was added in the presence of 15 μM Fd, which was optimal for the NADP⁺ reduction rate, the participation of Fd in the reduction of oxygen was low, no more than 10%, and it also decreased with increasing light intensity. At high light intensity, the overall oxygen reduction rates in the presence of Fd + NADP⁺ and in the presence of Fd alone were comparable. The significance of reduction of dioxygen either by water-soluble Fd or by the membrane-bound carriers of the photosynthetic electron transport chain for redox signaling under different light intensities is discussed.     

Inactivation of Ascorbate Peroxidase in Spinach Chloroplasts on Dark Addition of Hydrogen Peroxide: Its Protection by Ascorbate

Dark addition of hydrogen peroxide to intact spinach chloroplastsresulted in the inactivation of ascorbate peroxidase accompaniedby a decrease in ascorbate contents. This was also the casein reconstituted chloroplasts containing ascorbate, NADP⁺, NAD⁺and ferredoxin. The addition of hydrogen peroxide during light,however, showed little effect on ascorbate contents and ascorbateperoxidase activity in either the intact or reconstituted chloroplasts.In contrast to ascorbate peroxidase, the enzymes participatingin the regeneration of ascorbate in chloroplasts (monodehydroascorbatereductase, dehydroascorbate reductase and glutathione reductase)were not affected by the dark addition of hydrogen peroxide.Ascorbate contents increased again by illumination of the chloroplastsafter the dark addition of hydrogen peroxide. These resultsshow that the inactivation of the hydrogen peroxide scavengingsystem on dark addition of hydrogen peroxide [Anderson et al.(1983) Biochim. Biophys. Acta 724: 69, Asada and Badger (1984)Plant & Cell Physiol. 25: 1169] is caused by the loss ofascorbate peroxidase activity. Ascorbate peroxidase activitywas rapidly lost in ascorbate-depleted medium, and protectedby its electron donors, ascorbate, isoascorbate, guaiacol andpyrogallol, but not by GSH, NAD(P)H and ferredoxin. (Received June 14, 1984; Accepted August 15, 1984)     

Purification and properties of the photoactive particle corresponding to photosystem II

1. 1) Purification of the photoactive particle corresponding tophotosystem II (particle II) was achieved using a combinationof procedures including digitonin- and Triton X-100-treatments,sonication, and differential- and density-gradient centrifugations.
2. 2) The "purified" particle II preparation showed only oxygenevolution activity and showed no NADP⁺ photoreduction activityeven when an electron donor couple was added.
3. 3) The chemicalcompositions of this particle and of the particlecorrespondingto photosystem I (particle I) were compared withrespect tothe composition and contents of their chlorophylls,carotenoids,cytochromes, plastoquinones, protein and lipid.Marked differenceswere found.
4. 4) Using the two photoactive particles I and IIlacking therespective counterpart activity, a partial successwas attainedin reconstituting a system, in which photoinducedelectron flowfrom water to NADP⁺ was observed, provided thatsuitable intermediateelectron carriers, e. g. plastoquinoneand plastocyanin, wereadded.
5. 5) The nature of the two photoactiveparticles obtained andtheir relationship to particles so farreported are discussed.

¹This study was aided by grants from the Ministry of Education(407160-1965, 91402-1966, 1967). Financial support from SanyoBroadcasting Scientific Foundation is also acknowledged withcordial thanks. (Received October 23, 1968; )     

Formation of Radioactive Citrulline during Photosynthetic C14O2-Fixation by Blue-Green Algae

Citrulline has been isolated and identified from extracts ofNostoc muscorum. All members of the Cyanophyceae hitherto investigatedshow a relatively large amount of the CO₂ fixed during photosynthesisin citrulline (ranging as high as 20 per cent. in Nostoc) whencompared to the trace amounts found in the Chlorophyceae. Nostocalso has the ability to fix C¹⁴ in citrulline during dark fixation,but at a rate slower than in light. As no free urea or arginine was found in Nostoc, it is likelythat citrulline is functioning in reactions other than thoseleading to arginine and urea synthesis. Other possible functions for citrulline are briefly discussed.     

Ferredoxin Cross-Links to a 22 kD Subunit of Photosystem I

We have used a cross-linking approach to study the interaction of ferredoxin (Fd) with photosystem I (PSI). The cross-linking reagent N-ethyl-3-(3-dimethylaminopropyl) carbodiimide was found to cross-link spinach Fd to a 22 kilodalton subunit of PSI in both isolated spinach (Spinacia oleracea) PSI complexes and spinach thylakoid membranes. The product had an apparent molecular weight of 38 kilodaltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was identified as a cross-linked product using specific antibodies to Fd and the 22 kilodalton subunit. In both a native PSI complex (200 Chl/P700) and a PSI core complex (100 Chl/P700), a second cross-linked product at 36 kilodaltons was seen. The latter cross-reacted with an antibody to Fd but did not cross-react with antibodies directed against the 24.3, 22, 19, 17.3 or 8.5 kilodalton, or psaC subunits of PSI. Its composition remains to be determined. In thylakoids only the 38 kilodalton product was observed along with a cross-linked complex of Fd and Fd:NADP⁺ reductase.