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PubMed1990

Metal-ion-center assembly of ferredoxin and plastocyanin in isolated chloroplasts.

Most chloroplastic proteins are cytosolically synthesized and posttranslationally transported to their proper locations. Two examples of this group of proteins are ferredoxin and plastocyanin, both of which are metal-containing components of the photosynthetic electron-transport chain. The import process for these two proteins includes the insertion of the metal ions to produce the holo forms of the proteins. We show here that in vitro translated precursor proteins of ferredoxin and plastocyanin are synthesized as apo forms and are assembled into their respective holo forms after being imported into isolated chloroplasts. We also provide evidence that only mature-sized proteins are competent to be assembled into holo forms.

Journal Article↗
PubMed1995

Glyceraldehyde-3-phosphate ferredoxin oxidoreductase, a novel tungsten-containing enzyme with a potential glycolytic role in the hyperthermophilic archaeon Pyrococcus furiosus.

The archaeon Pyrococcus furiosus grows optimally at 100 degrees C by the fermentation of carbohydrates to yield acetate, CO2, and H2. Cell-free extracts contain very low activity of the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase, but extremely high activity of glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR). GAPOR was purified under strictly anaerobic conditions. It is a monomeric, O2-sensitive protein of M(r) approximately 63,000 which contains pterin and approximately 1 tungsten and 6 iron atoms per molecule. The enzyme oxidized glyceraldehyde-3-phosphate (Km 28 microM) to 3-phosphoglycerate and reduced P. furiosus ferredoxin (Km 6 microM), but it did not oxidize formaldehyde, acetaldehyde, glyceraldehyde, benzaldehyde, glucose, glucose 6-phosphate, or glyoxylate, nor did it use NAD(P) as an electron acceptor. It is proposed that GAPOR has a glycolytic role and functions in place of glyceraldehyde-3-phosphate dehydrogenase and possibly phosphoglycerate kinase.

Amino Acid Sequence↗
PubMed1999

Rubredoxin from the green sulfur bacterium Chlorobium tepidum functions as an electron acceptor for pyruvate ferredoxin oxidoreductase.

Rubredoxin (Rd) from the moderately thermophilic green sulfur bacterium Chlorobium tepidum was found to function as an electron acceptor for pyruvate ferredoxin oxidoreductase (PFOR). This enzyme, which catalyzes the conversion of pyruvate to acetyl-CoA and CO(2), exhibited an absolute dependence upon the presence of Rd. However, Rd was incapable of participating in the pyruvate synthase or CO(2) fixation reaction of C. tepidum PFOR, for which two different reduced ferredoxins are employed as electron donors. These results suggest a specific functional role for Rd in pyruvate oxidation and provide the initial indication that the two important physiological reactions catalyzed by PFOR/pyruvate synthase are dependent on different electron carriers in the cell. The UV-visible spectrum of oxidized Rd, with a monomer molecular weight of 6500, gave a molar absorption coefficient at 492 nm of 6.89 mM(-1) cm(-1) with an A(492)/A(280) ratio of 0.343 and contained one iron atom/molecule. Further spectroscopic studies indicated that the CD spectrum of oxidized C. tepidum Rd exhibited a unique absorption maximum at 385 nm and a shoulder at 420 nm. The EPR spectrum of oxidized Rd also exhibited unusual anisotropic resonances at g = 9.675 and g = 4.322, which is composed of a narrow central feature with broader shoulders to high and low field. The midpoint reduction potential of C. tepidum Rd was determined to be -87 mV, which is the most electronegative value reported for Rd from any source.

Chlorobi↗
PubMed2005

Thermococcus profundus 2-ketoisovalerate ferredoxin oxidoreductase, a key enzyme in the archaeal energy-producing amino acid metabolic pathway.

2-Ketoisovalerate ferredoxin oxidoreductase (VOR) is a key enzyme in hyperthermophiles catalyzing the coenzyme A-dependent oxidative decarboxylation of mainly aliphatic amino acid-derived 2-keto acids. The very oxygen-labile enzyme purified under anaerobic conditions from a hyperthermophilic archaeon, Thermococcus profundus, is a hetero-octamer (alphabetagammadelta)(2) consisting of four different subunits, alpha = 45,000, beta = 31,000, gamma = 22,000 and delta = 13,000, respectively. Electron paramagnetic resonance and resonance Raman spectra of the purified enzyme indicate the presence of approximately three [4Fe-4S] clusters per alphabetagammadelta-protomer, although one of the clusters has a tendency to be converted to a [3Fe-4S] form during purification. The optimal temperature for the enzyme activity is 93 +/- 2 degrees C and the cognate [4Fe-4S] ferredoxin serves as an electron acceptor of the enzyme. The purified enzyme is highly oxygen-labile (t(1/2), approximately 5 min at 25 degrees C), and is partly protected in the presence of magnesium ions, thiamine pyrophosphate and sodium chloride (t(1/2), approximately 25 min at 25 degrees C).

Amino Acids↗
PubMed1996

2-oxoacid:ferredoxin oxidoreductase from the thermoacidophilic archaeon, Sulfolobus sp. strain 7.

The purified 2-oxoacid:ferredoxin oxidoreductase of a thermoacidophilic and aerobic crenarchaeote, Sulfolobus sp. strain 7, consists of 70-kDa alpha and 37-kDa beta subunits, and contains one thiamine pyrophosphate (TPP), one [4Fe-4S]2+.1+ cluster, and two magnesium atoms per alpha beta structure. It exhibits a broad substrate specificity toward 2-oxoacids such as 2-oxoglutarate, 2-oxobutyrate, and pyruvate. The gene encoding the archaeal oxidoreductase was cloned, and the two open reading frames encoding the alpha (632 amino acids) and beta subunits (305 amino acids), respectively, were sequenced. Careful sequence alignment revealed several consensus motifs of this enzyme family, as well as possible cofactor binding residues of the Sulfolobus enzyme. This new structural information also indicates that (i) several genetic fusions and reorganization of the early, possibly alpha beta gamma delta-type enzyme similar to those from hyperthermophiles have taken place during evolution of the 2-oxoacid:ferredoxin (flavodoxin) oxidoreductase superfamily, which might have occurred in different ways in early aerobic archaea and early anaerobic bacteria, and that (ii) enzymes with different subunit compositions should have an essentially similar catalytic mechanism with one TPP and at least one [4Fe-4S] cluster as the minimal set of redox centers.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1995

In Situ Association of Calvin Cycle Enzymes, Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activase, Ferredoxin-NADP+ Reductase, and Nitrite Reductase with Thylakoid and Pyrenoid Membranes of Chlamydomonas reinhardtii Chloroplasts as Revealed by Immunoelectron Microscopy.

The in situ localization of the chloroplast enzymes ribulose-1,5-bisphosphate carboxylase (Rubisco), Rubisco activase, ribose-5-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, aldolase, nitrite reductase, ferredoxin-NADP+ reductase, and H+-ATP synthase was studied by immunoelectron microscopy in Chlamydomonas reinhardtii. Immunogold labeling revealed that, despite Rubisco in the pyrenoid matrix, Calvin cycle enzymes, Rubisco activase, nitrite reductase, ferredoxin-NADP+ reductase, and H+-ATP synthase are associated predominantly with chloroplast thylakoid membranes and the inner surface of the pyrenoid membrane. This is in accord with previous enzyme localization studies in higher plants (K.H. Suss, C. Arkona, R. Manteuffel, K. Adler [1993] Proc Natl Acad Sci USA 90: 5514-5518). Pyrenoid tubules do not contain these enzymes. The pyrenoid matrix consists of Rubisco but is devoid of the other photosynthetic enzymes investigated. Evidence for the occurrence of two Rubisco forms differing in their spatial localization has also been obtained: Rubisco form I appears to be membrane associated like other Calvin cycle components, whereas Rubisco form II is confined to the pyrenoid matrix. It is proposed that enzyme form I represents an active Rubisco when assembled into Calvin cycle enzyme complexes, whereas Rubisco form II may be part of a CO2-concentrating mechanism. Pyrenoidal Calvin cycle complexes are thought to be highly active in CO2 fixation and important for the synthesis of starch around the pyrenoid.

Journal Article↗
PubMed1980

Effect of Light on Chemical Modification of Chloroplast Ferredoxin-NADP Reductase.

Chemical modification of spinach chloroplasts by phenylglyoxal and dansyl chloride resulted in inhibition of NADP photoreduction. The rate of inactivation was higher with both reagents when modification was carried out in the light with methylviologen or phenazine methosulfate present. Uncouplers prevent the effect of light. Electron transport from water to methylviologen was not affected by the modifiers.The presence of 10 millimolar NADP completely protected the membrane-bound reductase against inactivation by phenylglyoxal. With lower concentrations, protection was higher in the light than in the dark. The apparent dissociation constants of the enzyme-substrate complex for NADP were 0.9 and 0.1 millimolar for the dark and light inactivation, respectively. Inactivation of NADP photoreduction by dansyl chloride was completely prevented by ferredoxin, but only partially by nucleotides.The diaphorase activity was inhibited in chloroplasts modified by phenylglyoxal, but not when modified by dansyl chloride.The results suggest that energizing thylakoid membranes by light induces a conformational change in membrane-bound ferredoxin-NADP reductase, and that the reductase is an allotopic enzyme.

Journal Article↗
PubMed1981

Uncouplers stimulate photosynthesis in intact chloroplasts by enhancing light-activation of enzymes regulated by the ferredoxin-thioredoxin system.

Some uncouplers stimulate CO(2)-dependent O(2) evolution by intact spinach chloroplasts at pH 8.6. This effect is not due to alkalinization of the stroma. The stimulation is observed only when photosynthesis has been partly inhibited by the presence of H(2)O(2), generated in a Mehler-type reaction by the broken chloroplasts which always contaminate the intact chloroplast preparations. The addition of methyl viologen increases the Mehler-type reaction and results in greater inhibition of photosynthesis. The addition of excess catalase stimulates photosynthesis by preventing accumulation of H(2)O(2). The uncouplers stimulate photosynthesis primarily by enhancing the light-activation of enzymes that are regulated by the ferredoxin-thioredoxin system, and this effect results from the influence of the uncouplers on the redox poising of the ferredoxin in the intact chloroplasts.

Journal Article↗
PubMed1982

Characterization of a Photosynthesizing Reconstituted Spinach Chloroplast Preparation : REGULATION BY PRIMER, ADENYLATES, FERREDOXIN, AND PYRIDINE NUCLEOTIDES.

A particulate preparation (MgP) capable of photosynthetic CO(2) assimilation without the addition of stromal protein was obtained by rupturing whole spinach (Spinacia oleracea var. America) chloroplasts in 15 millimolar MgCl(2) buffered with Tricine at pH 8.5. This CO(2) assimilation was dependent upon light, inorganic phosphate, ferredoxin, ADP, NAD or NADP, and primer. Excepting glycolate, the products of CO(2) fixation by MgP were similar to those found with whole chloroplasts.Glycerate-3-phosphate (PGA), fructose-1, 6-bisphosphate (FBP), and ribose-5-phosphate (R5P) but not fructose-6-P (F6P) functioned as primers. Concentrations of PGA and FBP but not of R5P higher than 2 millimolar were inhibitory to CO(2) fixation. A lag of CO(2) fixation was observed with PGA and FBP but not with R5P. This lag as well as inhibition by NADP, ADP, and ATP in the FBP-primed preparation was eliminated by an equimolar mixture of FBP plus F6P indicating FBPase as the sensitive site. NADP, ADP, and ATP also blocked CO(2) fixation by the PGA-fortified preparation but inhibition was even more sensitive than that observed when FBP was added. Inhibition by AMP in the PGA and FBP-primed preparations was not affected by the addition of F6P. When R5P was the starting primer, inhibition of CO(2) fixation was relatively insensitive to the adenylates and NADP. In contrast to the parent whole chloroplast, CO(2) fixation by MgP was insensitive to high (5 millimolar) inorganic phosphate. Depending upon the ferredoxin concentration, NAD was as effective as NADP in supporting CO(2) fixation.

Journal Article↗
PubMed1982

Interaction of Ferredoxin-NADP Oxidoreductase with the Thylakoid Membrane.

The binding of ferredoxin-NADP reductase to spinach chloroplast membranes was studied by washing the membranes with different media. Release of the enzyme from the thylakoids was greater in 0.75 millimolar EDTA but was not complete inasmuch as 20% the activity remained membrane-bound after three washes.A Scatchard plot of binding experiments suggests the presence of one type of binding site and a stoichiometry of 3 to 4 nanomoles of reductase per micromole of chlorophyll was calculated. Rebinding has a nonspecific requirement for cations. Their effectiveness increased with their valency. Rebinding of purified enzyme to depleted membranes resulted in a stimulation of its diaphorase activity.It is suggested that binding of ferredoxin-NADP reductase to thylakoid membranes is dependent upon neutralization of negative charges.

Journal Article↗
PubMed1986

Detection of the messenger RNA encoding for the ferredoxin-dependent glutamate synthase in maize leaf.

Ferredoxin-dependent glutamate synthase (EC 1.4.7.1), glutamate oxoglutarate aminotransferase (glutamate synthase) (GOGAT) messenger RNA was extracted from maize (Zea mays L.) leaves and partially purified through oligo(dT)-cellulose chromatography and ultracentrifugation in a sucrose gradient. mRNA were translated in vitro using a reticulocyte system. The glutamate synthase subunit was characterized by immunoprecipitation with antibodies raised against the rice (Oryza sativa L.) ferredoxin-glutamate synthase. The in vitro synthesized protein and the 145 kilodaltons genuine maize leaf subunit of GOGAT were found to comigrate in sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments.

Journal Article↗
PubMed1986

Localization and Quantitative Determination of Ferredoxin-NADP Oxidoreductase, a Thylakoid-Bound Enzyme in the Cyanobacterium Anabaena sp. Strain 7119.

Thylakoid membrane preparations obtained from mechanically disrupted (sonicated) cells of the cyanobacterium Anabaena sp. strain 7119 show a membrane-bound ferredoxin-NADP(+) oxidoreductase (EC 1.18.1.2) as determined either by specific antibodies or by using the ferredoxin-dependent NADPH-cytochrome c reductase activity, which is a specific test for this enzyme. However, in contrast with higher plant thylakoids, a low yield of the cyanobacterial reductase-only about 20% of the total amount of this protein estimated in whole cell homogenates-was obtained as a membrane-bound form when Mg(2+) was present during the disruption treatment. It is noteworthy that the addition of water-soluble nonionic polymers, namely polyethylene glycol and polyyinylpyrrolidone, dramatically increased the yield of the thylakoid-bound reductase, reaching values up to 80 to 85% of the total enzyme. Using these thylakoid membrane preparations, a quantitative determination of the reductase has been performed for the first time for cyanobacterial thylakoids. The value determined by immunoelectrophoresis-from 8 to 10 nanomoles per micromole of chlorophyll-is clearly higher than those reported for chloroplast thylakoids.

Journal Article↗
PubMed1987

Regulation of Cyclic Photophosphorylation during Ferredoxin-Mediated Electron Transport : Effect of DCMU and the NADPH/NADP Ratio.

Addition of ferredoxin to isolated thylakoid membranes reconstitutes electron transport from water to NADP and to O(2) (the Mehler reaction). This electron flow is coupled to ATP synthesis, and both cyclic and noncyclic electron transport drive photophosphorylation. Under conditions where the NADPH/NADP(+) ratio is varied, the amount of ATP synthesis due to cyclic activity is also varied, as is the amount of cyclic activity which is sensitive to antimycin A. Partial inhibition of photosystem II activity with DCMU (which affects reduction of electron carriers of the interphotosystem chain) also affects the level of cyclic activity. The results of these experiments indicate that two modes of cyclic electron transfer activity, which differ in their antimycin A sensitivity, can operate in the thylakoid membrane. Regulation of these activities can occur at the level of ferredoxin and is governed by the NADPH/NADP ratio.

Journal Article↗
PubMed1989

Localization of ferredoxin isoproteins in mesophyll and bundle sheath cells in maize leaf.

Four ferredoxin isoproteins were identified in the C(4) plant Zea mays L. by analysis of extracts from leaves, mesocotyls, and roots of the young seedlings. The relative amounts of the isoproteins isolated from the photosynthetic and nonphotosynthetic organs were different. All the isoproteins were present in the leaves of green and etiolated plants, whereas two out of the four isoproteins were not detected in the roots or in the mesocotyls. During the greening of etiolated seedlings, the level of the two isoproteins unique to the leaf increased markedly. Analysis of the cellular and subcellular distribution of the two major leaf isoproteins showed that one isoprotein was present in the chloroplasts of both mesophyll and bundle sheath cells, whereas the other was only found in the chloroplasts of bundle sheath cells. This is the first report of the cell-specific expression of ferredoxin isoproteins in the leaves of a C(4) plant.

Journal Article↗
PubMed1993

Structure at pH 6.5 of ferredoxin I from Azotobacter vinelandii at 2.3 A resolution.

Ferredoxin I from Azotobacter vinelandii (AvFdI) is an iron-sulfur protein composed of 106 amino acids, seven Fe atoms and eight inorganic S* atoms. A crystallographic redetermination of its structure showed the originally reported structure to be incorrect. We report here the crystal structure of AvFdI at pH 6.5. Extensive refinement has led to a final R value of 0.170 for all 6986 non-extinct reflections in the range 10-2.3 A using a solvent model which includes 98 discrete solvent atoms with occupancies between 0.3 and 1.0 and an average B value of 22.5 A(2). The first half of the peptide chain closely resembles that of the 55-residue ferredoxin from Peptococcus aerogenes (PaFd), while the remainder consists of three turns of helix and a series of loops which form a cap over part of the molecular core. Despite the similarities in structure and surroundings, the corresponding 4Fe4S* clusters in PaFd and AvFdI have strikingly different redox potentials; a possible explanation has been sought in the differing hydration models for the two molecules.

Journal Article↗
PubMed1995

Structure of the ferredoxin from Clostridium acidurici: model at 1.8 A resolution.

Ferredoxins (Fd) are electron-carrier proteins, the active sites of which are organized around clusters made of iron and inorganic sulfur. The Fd from Clostridium acidurici is 55 amino acids long and contains two [4Fe-4S] clusters. Crystals have been obtained in the space group P4(3)2(1)2, a = b = 34.441 (5), c = 74.778 (9) A. The structure was solved by molecular replacement using the Fd from Peptostreptcoccus asaccharolyticus as a search model, these two ferredoxins having 37 residues in common. Refinement using molecular-dynamics techniques was then initiated. Successive rounds of model building and refinement gave a structure that includes 45 water molecules with R = 15%. At this stage, the electron-density map clearly revealed discrepancies in the position of two amino acids in the published primary sequence. Refinement based on these modifications led to R = 14.3% for 3921 reflections up to 1.8 A, resolution. The geometry of the two clusters has been found to be in good agreement with that previously obtained at a lower resolution. Interactions of polypeptide chain with the [4Fe-4S] clusters, the cluster geometry as well as the hydrogen bonds involving S, Sgamma, N and water molecules are reported.

Journal Article↗
PubMed1992

Improved purification, crystallization and primary structure of pyruvate:ferredoxin oxidoreductase from Halobacterium halobium.

An improved purification procedure, including nickel chelate affinity chromatography, is reported which resulted in a crystallizable pyruvate:ferredoxin oxidoreductase preparation from Halobacterium halobium. Crystals of the enzyme were obtained using potassium citrate as the precipitant. The genes coding for pyruvate:ferredoxin oxidoreductase were cloned and their nucleotide sequences determined. The genes of both subunits were adjacent to one another on the halobacterial genome. The derived amino acid sequences were confirmed by partial primary structure analysis of the purified protein. The structural motif of thiamin-diphosphate-binding enzymes was unequivocally located in the deduced amino acid sequence of the small subunit.

Amino Acid Sequence↗
PubMed1994

Amino acid sequence of spinach ferredoxin:thioredoxin reductase variable subunit.

Ferredoxin:thioredoxin reductase (FTR) is an iron-sulfur protein, which, in the presence of ferredoxin and thioredoxin, catalyses the light-dependent activation of several photosynthetic enzymes. Spinach FTR consists of two dissimilar polypeptide chains, A and B, present in equal amounts. Whereas subunit B seems to be responsible for the catalytic activity, subunit A has no known catalytic function. We found earlier that the N-terminus of subunit A, also called the variable subunit, shows terminal redundancy and that 2-3 of its serine residues are phosphorylated [Tsugita, A. Yano, K., Gardet-Salvi, L. & Schürmann, P. (1991) Protein Sequence Data Anal. 4, 9-13]. We now report the complete amino acid sequence of subunit A, determined by conventional protein sequencing methods. The polypeptide chain with a calculated molecular mass of 12,669 Da consists of 112 amino acids and has a calculated isoelectric point of 5.4. The analysis of the sequence supports the idea that this subunit has no catalytic function. The comparison with a known cyanobacterial FTR reveals about 58% similarity and the striking presence of a N-terminal extension in the spinach protein. This extension may be responsible for the reported size variability of this subunit.

Amino Acid Sequence↗