Immunological quantitation of chloroplast ferredoxin

An immunoassay for the quantitative determination of ferredoxins in cell-free extracts from plant tissues is described. The method is accurate for the assay of 0.3–1.5 nmol ferredoxin directly from the extracts. The following average values (nmol ferredoxin/mg extractable protein) were obtained: 3.9, 1.8, 5.90, 14.8 and 10.9 for Euglena gracilis, spinach, parsley, lettuce and broccoli, respectively. Specific factors affecting the method are discussed in detail.     

Ferredoxin-dependent methane formation from acetate in cell extracts of Methanosarcina barkeri (strain MS).

Cell extracts of Methanosarcina barkeri grown on acetate catalyzed the conversion of acetyl-CoA to CO2 and CH4 at a specific rate of 50 nmol min-1 mg-1. When ferredoxin was removed from the extracts by DEAE-Sephacel anion exchange chromatography, the extracts were inactive but full activity was restored upon addition of purified ferredoxin from M. barkeri or from Clostridium pasteurianum. The apparent Km for ferredoxin from M. barkeri was determined to be 2.5 M. A ferredoxin dependence was also found for the formation of CO2, H2 and methylcoenzyme M from acetyl-CoA, when methane formation was inhibited by bromoethanesulfonate. Reduction of methyl-coenzyme M with H2 did not require ferredoxin. These and other data indicate that ferredoxin is involved as electron carrier in methanogenesis from acetate. Methanogenesis from acetyl-CoA in cell extracts was not dependent on the membrane fraction, which contains the cytochromes.     

Nondenaturing procedure for rapid preparation of ferredoxin from Clostridium pasteurianum

A procedure is described for the rapid preparation of ferredoxin in high yield from crude extracts of C. pasteurianum. The method involves two successive chromatographic treatments on DEAE-cellulose in concentrated ammonium sulfate. Ferredoxin prepared by this method has an absorption spectrum and iron content similar to ferredoxin prepared by other methods.     

Partial purification of ferredoxin from Ruminococcus albus and its role in pyruvate metabolism and reduction of nicotinamide adenine dinucleotide by H2.

Extracts of Ruminococcus albus were not able to convert pyruvate to acetyl phosphate, CO2, and H2 after passage through a diethylaminoethyl (DEAE)-cellulose column. Activity was restored by a brown protein fraction eluted from the column with 0.4 M Cl-. The protein was partially purified and shown to have the spectral and biological characteristics of ferredoxin. R. albus ferredoxin, Clostridium pasteurianum ferredoxin, and methyl viologen restored activity for pyruvate decomposition by DEAE-cellulose-treated R. albus extracts. R. albus or C. pasteurianum ferredoxin restored the ability of DEAE-cellulose-treated C. pasteurianum extracts to form H2 and acetyl phosphate from pyruvate. Ferredoxin-free extracts of R. albus reduced nicotinamide adenine dinucleotide (NAD) when supplemented with R. albus or C. pasteurianum ferredoxin or with methyl viologen. These extracts reduced NADP with H2 poorly unless both ferredoxin and NAD were added, which indicates the presence of an NADH:NADP transhydrogenase. Flavin mononucleotide and flavin adenine dinucleotide were rapidly reduced by H2 by ferredoxin-free extracts in the absence of ferredoxin.     

Measurement of ferredoxin-dependent sulfite reductase activity in crude extracts from leaves using O-acetyl-L-serine sulfhydrylase in a coupled assay system to measure the sulfide formed

Ferredoxin-dependent sulfite reductase (EC 1.8.7.1) catalyses the reduction of sulfite to sulfide, using reduced ferredoxin as an electron donor. An assay system was developed for measuring this enzyme activity in crude extracts and broken chloroplast preparations from leaves. The assay consists of a coupled system in which the sulfide formed is used for cysteine synthesis by added O-acetyl-L-serine sulfhydrylase (EC 4.2.99.8). Cysteine thus formed is determined with ninhydrin under conditions where O-acetylserine does not react and serves as a measure for ferredoxin-dependent sulfite reductase activity. Cysteine synthesized in the assay can be determined from 10 to 200 nmol. One assay per minute can be performed.     

Characterization of ferredoxins on a nanomole scale

Two method are described for the characterization of ferredoxins. First, mapping of tryptic peptides from 2 to 3 nmol of carboxymethylated ferredoxin by two-dimensional thin-layer electrophoresis and chromatography. Second, gel electrophoresis of tryptic digests of apoferredoxins. The latter method discriminates between ferredoxins of closely related species.     

Interference of Nucleases in Cyanobacterium Ferredoxin Purification

Abstract

Isolation of cyanobacterial ferredoxin is normally carried out using nucleases in order to degrade the nucleic acids that accompany this protein during the purification procedure. However, this practice presents the inconvenience that these proteins remain in trace amounts in the purified ferredoxin preparations, although they are not visible by electrophoretical techniques. Evidence of that fact is shown in this report and an alternative procedure is described for the rapid preparation of ferredoxin from crude extracts of Anabaena PCC 7119. The method involves a treatment of the crude extract with streptomycin sulphate, a high molecular weight polication that precipitates the nucleic acids in the beginning of the purification.     

The effect of ferredoxin(BED) overexpression on benzene dioxygenase activity in Pseudomonas putida ML2.

The benzene dioxygenase from Pseudomonas putida ML2 is a multicomponent complex comprising a flavoprotein reductase, a ferredoxin, and a terminal iron-sulfur protein (ISP). The catalytic activity of the isolated complex shows a nonlinear relationship with protein concentration in cell extracts, with the limiting factor for activity in vitro being ferredoxin(BED). The relative levels of the three components were analyzed by using 125I-labelled antibodies, and the functional molar ratio of ISP(BED), ferredoxin(BED), and reductase(BED) was shown to be 1:0.9:0.8, respectively. The concentration of ferredoxin(BED) was confirmed by quantitative electron paramagnetic resonance spectroscopy of the 2Fe-2S centers in ferredoxin(BED) and ISP(BED) of whole cells. These results demonstrate that the ferredoxin(BED) component is a limiting factor in dioxygenase activity in vitro. To determine if it is a limiting factor in vivo, a plasmid (pJRM606) overproducing ferredoxin(BED) was introduced into P. putida ML2. The benzene dioxygenase activity of this strain, measured in cell extracts, was fivefold greater than in the wild type, and the activity was linear with protein concentration in cell extracts above 2 mg/ml. Western blotting (immunoblotting) and electron paramagnetic resonance spectroscopic analysis confirmed an elevated level of ferredoxin(BED) protein and active redox centers in the recombinant strain. However, in these cells, the increased level of ferredoxin(BED) had no effect on the overall rate of benzene oxidation by whole cells. Thus, we conclude that ferredoxin(BED) is not limiting at the high intracellular concentration (0.48 mM) found in cells.     

Ferredoxin requirement for electron transport from the carbon monoxide dehydrogenase complex to a membrane-bound hydrogenase in acetate-grown Methanosarcina thermophila

Cell extracts from acetate-grown Methanosarcina thermophila contained CO-oxidizing:H2-evolving activity 16-fold greater than extracts from methanol-grown cells. Following fractionation of cell extracts into soluble and membrane components, CO-dependent H2 evolution and CO-dependent methyl-coenzyme M methylreductase activities were only present in the soluble fraction, but addition of the membrane fraction enhanced both activities. A b-type cytochrome(s), present in the membrane fraction, was linked to a membrane-bound hydrogenase. CO-oxidizing:H2-evolving activity was reconstituted with: (i) CO dehydrogenase complex, (ii) a ferredoxin, and (iii) purified membranes with associated hydrogenase. The ferredoxin was a direct electron acceptor for the CO dehydrogenase complex. The ferredoxin also coupled CO oxidation by CO dehydrogenase complex to metronidazole reduction.     

Purification and characterization of a soybean flour-inducible ferredoxin reductase of Streptomyces griseus.

We have purified an NADH-dependent ferredoxin reductase from crude extracts of Streptomyces griseus cells grown in soybean flour-enriched medium. The purified protein has a molecular weight of 60,000 as determined by sodium dodecyl sulfate gel electrophoresis. The enzyme requires Mg2+ ion for catalytic activity in reconstituted assays, and its spectral properties resemble those of many other flavin adenine dinucleotide-containing flavoproteins. A relatively large number of hydrophobic amino acid residues are found by amino acid analysis, and beginning with residue 7, a consensus flavin adenine dinucleotide binding sequence, GXGXXGXXXA, is revealed in this protein. In the presence of NADH, the ferredoxin reductase reduces various electron acceptors such as cytochrome c, potassium ferricyanide, dichlorophenolindophenol, and nitroblue tetrazolium. However, only cytochrome c reduction by the ferredoxin reductase is enhanced by the addition of ferredoxin. In the presence of NADH, S. griseus ferredoxin and cytochrome P-450soy, the ferredoxin reductase mediates O dealkylation of 7-ethoxycoumarin.     

A new,fast, and sensitive assay for NADH-ferredoxin oxidoreductase detection in clostridia

A new, simple and very sensitive assay for NADH-ferredoxin or flavodoxin reductase activity is described. The assay is based on the nonenzymatic reduction of the metronidazole by ferredoxin or flavodoxin. In the presence of NADH, ferredoxin or flavodoxin and cell-free extract of clostridia, no metronidazole reduction is observed; the reaction occurs only if acetyl-CoA is added to the reaction mixture. Metronidazole reduction is quantitated by the spectrophotometric measurement at 320 nm. In this assay the change in absorbance is linearly related to the amount of clostridial extract for concentration of 0.1 to 0.8 mg/ml and to the flavodoxin or ferredoxin for concentrations of 0.5 to 8 nmol/ml.     

Isolation and characterization of pig kidney mitochondrial ferredoxin:NADP+ oxidoreductase

A two-step affinity chromatography procedure, using 2',5'-ADP-agarose and adrenodoxin-Sepharose 4B affinity supports, was used to purify mitochondrial ferredoxin:NADP+ oxidoreductase (EC 1.18.1.2, formerly EC 1.6.7.1) from pig kidney. The 450:270 nm absorbance ratio of the enzyme was 0.128, and it had a specific activity of 16,305 nmol/min/mg for the reduction of cytochrome c. The mitochondrial enzyme was a monomer which contained one molecule of FAD and had calculated molecular masses of 51,500 and 48,000 daltons when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high performance liquid chromatography gel exclusion chromatography, respectively. The porcine enzyme had a Km for NADPH of 0.94 microM and it expressed maximal activity when coupled with its homologous ferredoxin, although it was also active with the heterologous ferredoxin from bovine adrenal. The purified ferredoxin:NADP+ oxidoreductase supported the in vitro reduction of membrane-bound adrenal mitochondrial P-450, and it was demonstrated from immunologic studies that the enzyme shares some common epitopes with bovine adrenodoxin:NADP+ oxidoreductase.     

Hydroxylations in biosynthesis of bile acids. Isolation of a cytochrome P-450 from rabbit liver mitochondria catalyzing 26-hydroxylation of C27-steroids

A cytochrome P-450 catalyzing 26-hydroxylation of C27-steroids was purified from liver mitochondria of untreated rabbits. The enzyme fraction contained 10 nmol of cytochrome P-450/mg of protein and showed only one protein band with a minimum Mr = 53,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified mitochondrial cytochrome P-450 showed apparent molecular weight similar to microsomal cytochromes P-450LM4 but differed in spectral and catalytic properties from these microsomal isozymes. The purified cytochrome P-450 catalyzed 26-hydroxylation of cholesterol, 5-cholestene-3 beta,7 alpha-diol, 7 alpha-hydroxy-4-cholesten-3-one, 5 beta-cholestane-3 alpha,7 alpha-diol, and 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol up to 1000 times more efficiently than the mitochondria. The cytochrome P-450 required both ferredoxin and ferredoxin reductase for catalytic activity. Microsomal NADPH-cytochrome P-450 reductase could not replace ferredoxin and ferredoxin reductase. The cytochrome P-450 was inactive in 7 alpha-, 12 alpha- and 25-hydroxylations of C27-steroids. The results suggest that mitochondrial 26-hydroxylation of various C27-steroids is catalyzed by the same species of cytochrome P-450.     

Coupled ferredoxin and crotonyl coenzyme A (CoA) reduction with NADH catalyzed by the butyryl-CoA dehydrogenase/Etf complex from Clostridium kluyveri

Cell extracts of butyrate-forming clostridia have been shown to catalyze acetyl-coenzyme A (acetyl-CoA)- and ferredoxin-dependent formation of H₂ from NADH. It has been proposed that these bacteria contain an NADH:ferredoxin oxidoreductase which is allosterically regulated by acetyl-CoA. We report here that ferredoxin reduction with NADH in cell extracts from Clostridium kluyveri is catalyzed by the butyryl-CoA dehydrogenase/Etf complex and that the acetyl-CoA dependence previously observed is due to the fact that the cell extracts catalyze the reduction of acetyl-CoA with NADH via crotonyl-CoA to butyryl-CoA. The cytoplasmic butyryl-CoA dehydrogenase complex was purified and is shown to couple the endergonic reduction of ferredoxin (E₀′ = −410 mV) with NADH (E₀′ = −320 mV) to the exergonic reduction of crotonyl-CoA to butyryl-CoA (E₀′ = −10 mV) with NADH. The stoichiometry of the fully coupled reaction is extrapolated to be as follows: 2 NADH + 1 oxidized ferredoxin + 1 crotonyl-CoA = 2 NAD⁺ + 1 ferredoxin reduced by two electrons + 1 butyryl-CoA. The implications of this finding for the energy metabolism of butyrate-forming anaerobes are discussed in the accompanying paper.     

Pyruvate: Ferredoxin oxidoreductase and its activation by ATP in the blue-green alga Anabaena variabilis

1. The breakdown of pyruvate was examined in whole cells and cell-free extracts of the blue-green alga Anabaena variabilis. Decarboxylation of specifically labelled pyruvate indicated a similar metabolic route to that of acetate, although no pyruvate oxidase was present. Pyruvate: ferredoxin oxidoreductase was detected in cell-free extracts and after DEAE-cellulose treatment, addition of ferredoxin was necessary for pyruvate decarboxylation; acetyl-CoA was the first product of the reaction.     

Lipid biosynthesis in developing and germinating soybean cotyledons: The formation of oleate by a soluble stearyl acyl carrier protein desaturase

Extracts of developing soybean cotyledons contain a highly specific stearyl acyl carrier protein (ACP) desaturase which in the presence of NADPH, O₂, ferredoxin and ferredoxin: NADP⁺ reductase, rapidly converts stearyl ACP to oleyl ACP. The enzyme system has a high affinity for O₂, near-maximal activity being obtained at only 10 μm O₂. The pH optimum for the desaturase is 6.0. Stearic acid and stearyl-CoA, alone or in the presence of acyl carrier protein, are totally inactive. Although the enzyme is found in extracts prepared from developing soybean seeds (15–50 days after flowering), activity was not detected in extracts of germinated seeds.     

The pyruvate: ferredoxin oxidoreductase in heterocysts of the cyanobacterium Anabaena cylindrica

Heterocyst preparations have been obtained which actively perform nitrogen fixation (C2H2 reduction) and contain the enzymes of glycolysis and some of the tricarboxylic acid cycle. Pyruvate: ferredoxin oxidoreductase has been unambiguously demonstrated in extracts from heterocysts by the formation of acetylcoenzyme A, CO2 and reduced methyl viologen (ferredoxin) from pyruvate, coenzyme A and oxidized methyl viologen (ferredoxin) as well as by the synthesis of pyruvate from CO2, acetylcoenzyme A and reduced methyl viologen. Pyruvate supports C2H2 reduction by isolated heterocysts, however, with lower activity than Na2S2O4 and H2. alpha-Ketoglutarate: ferredoxin oxidoreductase is absent in Anabaena cylindrica, confirming that the organism has an incomplete tricarboxylic acid cycle.     

Isolation, characterization, and biological activity of ferredoxin-NAD+ reductase from the methane oxidizer Methylosinus trichosporium OB3b.

A ferredoxin-NAD+ oxidoreductase (EC 1.18.1.3) has been isolated from extracts of the obligate methanotroph Methylosinus trichosporium OB3b. This enzyme was shown to couple electron flow from formate dehydrogenase (NAD+ requiring) to ferredoxin. Ferredoxin-NAD+ reductase was purified to homogeneity by conventional chromatography techniques and was shown to be a flavoprotein with a molecular weight of 36,000 +/- 1,000. This ferredoxin reductase was specific for NADH (Km, 125 microM) and coupled electron flow to the native ferredoxin and to ferredoxins from spinach, Clostridium pasteurianum, and Rhodospirillum rubrum (ferredoxin II). M. trichosporium ferredoxin saturated the ferredoxin-NAD+ reductase at a concentration 2 orders of magnitude lower (3 nM) than did spinach ferredoxin (0.4 microM). Ferredoxin-NAD+ reductase also had transhydrogenase activity which transferred electrons and protons from NADH to thionicotinamide adenine dinucleotide phosphate (Km, 9 microM) and from NADPH to 3-acetylpyridine adenine dinucleotide (Km, 16 microM). Reconstitution of a soluble electron transport pathway that coupled formate oxidation to ferredoxin reduction required formate dehydrogenase, NAD+, and ferredoxin-NAD+ reductase.     

Purification and characterization of a ferredoxin from Rhizobium japonicum bacteroids

An eight-iron, eight-sulfur ferredoxin from Rhizobium japonicum bacteroids of soybean root nodules has been purified to apparent homogeneity as judged by disc gel electrophoresis. The purification procedure included chromatography on DEAE-cellulose, Bio-Gel P-60, and hydroxylapatite. Specific activities of several purified preparations of bacteroid ferredoxin ranged from 1700 to 1900 nmol of C2H4 produced . min-1 . mg-1 in the reaction mediating electron transfer between illuminated chloroplasts and bacteroid nitrogenase. A molecular weight of 6740 for the protein was determined by low speed sedimentation equilibrium and a molecular weight of 6500 was estimated from the mobility of bacteroid ferredoxin relative to the mobility of standard proteins during sodium dodecyl sulfate disc gel electrophoresis. All of the common amino acids were present except arginine, methionine, and tryptophan. The absorbance spectrum of the oxidized protein exhibited maxima at 285 nm and 380 nm with a shoulder near 305 nm. The A380/A285 ratio was 0.76 and the extinction coefficient at 380 nm for the oxidized protein was found to be 30,800 M-1. Equilibration of bacteroid ferredoxin with methyl viologen at various potentials revealed a midpoint oxidation-reduction potential of -484 mV. Spectrophotometric examination of iron-sulfur clusters extruded from bacteroid ferredoxin with benzenethiol and the transfer of its iron-sulfur clusters to other ferredoxins established the presence of two [4Fe-4S] clusters in a molecule of bacteroid ferredoxin. The EPR spectrum of oxidized ferredoxin consisted of a small signal at g = 2.02 integrating to 0.19 spin/molecule. The EPR spectrum of ferredoxin reduced with 5-deazaflavin exhibited a signal with features at g values of 1.88, 1.94, 2.01, and 2.07, and integrated to 1.7 spins/molecule. The EPR properties of bacteroid ferredoxin are characteristic of a ferredoxin operating between the 1+ and 2+ oxidation levels. Bacteroid ferredoxin mediated electron transfer to clostridial hydrogenase, but was not reduced by the clostridial phosphoroclastic system in the presence of pyruvate. Bacteroid ferredoxin reduced by illuminated 5-deazariboflavin also supported a high rate of C2H2 reduction by bacteroid nitrogenase which was free of Na2S2O4. It was concluded, on this basis, that bacteroid ferredoxin has the capability of functioning as the electron donor for nitrogenase in R. japonicum.     

Characterization of a female-specific hepatic mitochondrial cytochrome P-450 whose steady-state level is modulated by testosterone

Polyclonal antibody to mitochondrial P-450c27/25 reacted with two proteins of apparent molecular masses of 52 kilodaltons (kDa) and 50 kDa from the female rat liver mitochondrial proteins bound to an omega-octylaminoagarose column. The two proteins were purified to greater than 85% homogeneity by DEAE-Sephacel and hydroxylapatite column chromatography, and both were found to be P-450 as judged by dithionite-reduced CO difference spectra. Both of the P-450 forms required mitochondrial-specific ferredoxin and ferredoxin reductase for in vitro reconstitution of enzyme activities, suggesting that they are mitochondrial forms. The 52-kDa P-450 exhibited the properties of mitochondrial 27/25-hydroxylase with respect to high vitamin D3 25-hydroxylase activity [1.4 nmol (nmol of P-450)-1 min-1] and N-terminal amino acid sequence. The 50-kDa P-450, on the other hand, lacked significant vitamin D3 25-hydroxylase activity, but showed 17 beta-reductase [0.380-0.400 nmol (nmol of P-450)-1 min-1] and 17 beta-oxidase [0.1-0.16 nmol (nmol of P-450)-1 min-1] activities with both androgens and estrogens as substrates. Immunoblot analysis of proteins using a monoclonal antibody specific for P-450c27/25 showed a 2-3-fold higher level of this enzyme in the female liver mitochondria than in the males. Similarly, use of a polyclonal antibody in the immunoblot analysis showed that the 50-kDa P-450 is female-specific. The relative level of P-450c27/25 was reduced significantly in castrated females, while the level of the female-specific 50-kDa P-450 was increased. However, the levels of both enzymes were increased in castrated males.(ABSTRACT TRUNCATED AT 250 WORDS)