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The acetylation of spinach ferredoxin by acetic anhydride modified about four moles of amino groups. The absorption spectra, CD spectra, the fluorescence of sole tryptophan residue and the biological activity of acetylated ferredoxin were investigated. An equilibrium existed between two different states, D- and N-form, of the acetylated ferredoxin and was dependent on the cation concentration. D-form completely reverted to N-form upon the binding of one mole of cation, Na+ or Mg2+. Although the N-form was indistinguishable from native ferredoxin in every property tested, the D-form was significantly different from the N-form or native ferredoxin and was very unstable, especially at low salt concentrations. It is suggested that the amino groups might be important in maintaining the protein conformation by forming salt linkages, but may not be essential for the activity. Furthermore, since the D-form, unlike the N-form and native ferredoxin, was inactive in the ferredoxin-NADP+ reductase [EC 1.6.7.1] assay system and had no inhibitory effect in this system, it was considered to be incapable of forming a complex with ferredoxin-NADP+ reductase. On the other hand, the N-form of the modified ferredoxin was as active as native ferredoxin. It is suggested that amino groups of spinach ferredoxin are not essential for the redox reaction of ferredoxin or for complex formation with the reductase.
We have used a cell-free rabbit reticulocyte translational system programmed with polyadenylated [poly(A)+] RNA prepared from chick kidney tissue to study the synthesis of nascent ferredoxin, a class of iron-sulphur-containing proteins functional in the renal mitochondrial 1 alpha- and 24-hydroxylases of 25-hydroxyvitamin D3. The synthesis of ferredoxin was monitored by determining [35S]methionine incorporation into ferredoxin and quantified by SDS/PAGE and autoradiography after immunoprecipitation from the total translation products. Compared with normal controls, vitamin D deprivation caused a significant increase in the net synthesis of nascent ferredoxin with an Mr of 12,000-13,000. [3H]Orotate incorporation as uridine into kidney poly(A)+ RNA was stimulated by aminophylline, a potent inducer of 25-hydroxyvitamin D3 24-hydroxylase; however, the amount of nascent ferredoxin synthesis was the same as in normal controls. Also, partially purified chick kidney mitochondrial cyclic AMP-stimulated protein kinase catalysed the phosphorylation of ferredoxin in vitro. The catalytic activity of the ferredoxin in 1 alpha- and 24-hydroxylations of 25-hydroxyvitamin D3 in reconstituted systems consisting of cytochrome P-450 and ferredoxin reductase was altered with ferredoxin phosphorylation. The phosphorylation caused inhibition of the 1 alpha-hydroxylase activity while at the same time it stimulated the 24-hydroxylase. Authentic 1 alpha,25- and 24,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 were used as standards to monitor the separation of the enzymic products by h.p.l.c. using methanol/water (4:1, v/v) as solvent. These results indicate that, in the absence of vitamin D or its metabolites in the deficient state, the synthesis of ferredoxin necessary for the 1 alpha-hydroxylase is accentuated, whereas the stimulation of the 24-hydroxylase requires the phosphorylation of existing ferredoxin without a net gain in its synthesis. This would suggest a post-translational regulation of the 1 alpha- and 24-hydroxylases. A model delineating the various aspects of this study is presented.
Ferredoxin-NADP+ reductase and ferredoxin from the cyanobacterium Anabaena PCC 7119 have been covalently cross-linked by incubation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The covalent adduct, which shows a molecular mass consistent with a 1:1 stoichiometry of the two proteins, maintains nearly 60% of the NADPH-cytochrome c reductase activity of the enzyme saturated with ferredoxin and this value is considerably higher than when equimolar amounts of both proteins are assayed. No ternary complexes with Anabaena flavodoxin or horse heart cytochrome c were formed, suggesting that the binding site on the enzyme is the same for ferredoxin and flavodoxin and that ferredoxin-NADP+ reductase and cytochrome c bind at a common site on ferredoxin. In the noncovalent complex, titrated at pH 7, the oxidation-reduction potential of ferredoxin becomes 15 mV more negative and that of ferredoxin-NADP+ reductase 27 mV more positive compared to the proteins alone. When covalently linked, the midpoint potential of the enzyme has a value similar to that in the noncovalent complex, while the ferredoxin potential is 20 mV more positive compared to ferredoxin alone. The changes in redox potentials have been used to estimate the dissociation constants for the interaction of the different redox forms of the proteins, based on the value of 1.21 microM calculated for the oxidized noncovalent complex.
1. The ferredoxin from bovine liver mitochondria, so-called hepatoredoxin, was purified and characterized as to its molecular weight, optical absorption spectrum and amino acid composition. 2. These properties were found to be very similar to those of adreno-ferredoxin. 3. To clarify the molecular basis of tissue specificity, the ferredoxin clones were obtained from a bovine liver library and the cDNA sequence of hepato-ferredoxin was determined. 4. The nucleotide sequence of hepato-ferredoxin clone was found to be identical to that of adreno-ferredoxin clone except for a single nucleotide in the 3' non-translated region. 5. Identical amino acid sequence of the two ferredoxins was confirmed by determining the partial amino acid sequence of the purified hepato-ferredoxin. 6. The results indicated that the organ specific activity of purified ferredoxin could not be explained by the different primary structure nor different RNA processing. 7. Other factors may be involved in the tissue specific properties of ferredoxins.
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We report the observation of paramagnetically shifted (hyperfine) proton resonances from vertebrate mitochondrial [2Fe-2S] ferredoxins. The hyperfine signals of human, bovine, and chick [2Fe-2S] ferredoxins are described and compared with those of Anabaena 7120 vegetative ferredoxin, a plant-type [2Fe-2S] ferredoxin studied previously [Skjeldal, L., Westler, W. M., & Markley, J. L. (1990) Arch. Biochem. Biophys. 278, 482-485]. The hyperfine resonances of the three vertebrate ferredoxins were very similar to one another both in the oxidized state and in the reduced state, and slow (on the NMR scale) electron self-exchange was observed in partially reduced samples. For the oxidized vertebrate ferredoxins, hyperfine signals were observed downfield of the diamagnetic envelope from +13 to +50 ppm, and the general pattern of peaks and their anti-Curie temperature dependence are similar to those observed for the oxidized plant-type ferredoxins. For the reduced vertebrate ferredoxins, hyperfine signals were observed both upfield (-2 to -18 ppm) and downfield (+15 to +45 ppm), and all were found to exhibit Curie-type temperature dependence. This pattern and temperature dependence are distinctly different from those found with reduced plant-type ferredoxins which have signal centered around +120 ppm with Curie-type temperature dependence, assigned to cysteines which interact with Fe(III), and signals centered around +20 ppm with anti-Curie temperature dependence, assigned to cysteines which interact with Fe(II) [Dugad, L. B., La Mar, G. N., Banci, L., & Bertini, I. (1990) Biochemistry 29, 2263-2271].(ABSTRACT TRUNCATED AT 250 WORDS)
An immobilized ferredoxin more stable than the conventional immobilized spinach ferrodoxin was prepared by reacting CNBr-Sepharose with ferredoxins isolated from barley and Synechococcus vulcanus, a thermophilic blue-green alga. The dissociation constants of immobilized ferredoxin from spinach, barley and S. vulcanus for spinach ferredoxin-NADP reductase were 0.922, 2.505 and 5.209 microM, respectively, whereas those for barley ferredoxin-NADP reductase were 1.159, 0.579 and 2.851 microM, respectively. The order of stability was S. vulcanus greater than barley greater than spinach. The immobilized ferredoxin was applied to the simultaneous detection of ferredoxin-dependent enzymes in spinach chloroplasts. Over 20 polypeptides were detected. Synechococcus ferredoxin could also be immobilized on a Toyopearl gel and repeatedly used in an automated high-performance liquid chromatographic system.
Antibodies prepared against purified spinach ferredoxin and ferredoxin-NADP+ reductase were used as specific inhibitors of electron-transfer reactions dependent on either ferredoxin or ferredoxin-NADP+ reductase; The possible role of both electron carriers in cyclic electron transport was checked using cytochrome b6 photoreactions as indicator. It could be demonstrated that the ferredoxin antibody inhibits cytochrome b6 photoreduction. Ferredoxin-NADP+ reductase, however, appears not to be involved in this pathway: reductase antibody in concentrations sufficient to completely inhibit electron transport to NADP+ had no effect on cytochrome b6 photoreduction. Quantitative treatment of the immunoassay data showed that osmotically shocked chloroplasts contain both bound ferredoxin and ferredoxin-NADP+ reductase in concentration approximately equal to that of cytochrome b6.
Ferredoxins found in animal mitochondria function in electron transfer from NADPH-dependent ferredoxin reductase (Fd-reductase) to cytochrome P450 enzymes. To identify residues involved in binding of human ferredoxin to its electron transfer partners, neutral amino acids were introduced in a highly conserved acidic region (positions 68-86) by site-directed mutagenesis of the cDNA. Mutant ferredoxins were produced in Escherichia coli, and separate assays were used to determine the effect of substitutions on the capacity of each mutant to bind to Fd-reductase and cytochrome P450scc and to participate in the cholesterol side chain cleavage reaction. Replacements at several positions (mutants D68A, E74Q, and D86A) did not significantly affect activity, suggesting that acidic residues at these positions are not required for binding or electron transfer interactions. In contrast, substitutions at positions 76 and 79 (D76N and D79A) caused dramatic decreases in activity and in the affinity of ferredoxin for both Fd-reductase and P450scc; this suggests that the binding sites on ferredoxin for its redox partners overlap. Other substitutions (mutants D72A, D72N, E73A, E73Q, and D79N), however, caused differential effects on binding to Fd-reductase and P450scc, suggesting that the interaction sites are not identical. We propose a model in which Fd-reductase and P450scc share a requirement for ferredoxin residues Asp-76 and Asp-79 but have other determinants that differ and play an important role in binding. This model is consistent with the hypothesis that ferredoxin functions as a mobile shuttle in steroidogenic electron transfer, and it is considered unlikely that a functional ternary complex is formed.
Ferredoxin immobilized on Sepharose 4B was prepared by reaction of CNBr-Sepharose 4B with spinach ferredoxin. The ferredoxin-Sepharose 4B conjugated ferredoxin-NADP+ reductase (NADPH: ferredoxin oxidoreductase, [EC 1.6.7.1]) in dilute buffer solution and released it in high salt concentrations. A novel method of preparation for the reductase was established by a combination of affinity adsorption on the ferredoxin-Sepharose 4B column with usual purification procedures. It was found using the new method, that there are two forms of ferredoxin-NADP+ reductase, FNR I and FNR II, in spinach. Comparative studies of the two components suggest that FNR I may be a dimer of FNR II.
Purified antisera against ferredoxin, ferredoxin-NADP+ reductase and plastocyanin agglutinated osmotically shocked and washed spinach chloroplasts, prepared according to standard procedures. The monomeric antibody (immunoglobulin G fraction) of the reductase antiserum agglutinated chloroplasts specifically and directly, indicating that protruding structures (for example, the coupling factor) do not act as steric hindrances as has been suggested. With ferredoxin antiserum, the presence of a pentameric antibody (immunoglobulin M fraction) was obligatory to observe a positive agglutination reaction. Immunoglobulin G only inhibited ferredoxin-dependent reactions, like NADP+-photoreduction, but did not cause agglutination. Ferredoxin seems to be located in depressions of the membrane, possibly caused by a partial release of this protein in shocked chloroplasts. Similar results were obtained with purified immunoglobulins from a plastocyanin antiserum. Again the immunoglobulin G fraction inhibited electron transport reactions catalyzed by plastocyanin, whereas immunoglobulin M showed a positive agglutination, but had no influence on electron transport. It is concluded that ferredoxin, ferredoxin-NADP+ reductase and plastocyanin are peripheral electron transport components, located at the outer thylakoid membrane.
Wheat ferredoxin was purified from the leaves of common wheat (Triticum aestivum). The absorption spectrum showed maxima at 465, 425, 332, and 278 nm. The absorbance ratio, A425 nm/A278 nm was 0.49, and the millimolar extinction coefficient at 425 nm was 10.8 mM-1. cm-1. The amino acid composition was determined to be Lys5, His2, Arg1, Asp11, Thr5, Ser7, Glu18, Pro5, Gly6, Ala7, Cys5, Val7, Met1, Ile4, Leu7, Tyr4, Phe1, and Trp1. The total number of amino acid residues was 97. The molecular weight was calculated from the amino acid composition to be 10,829, including iron and sulfur atoms. This value was confirmed by other methods, which were based on the contents of non-heme iron and of terminal amino acid. The N-terminal amino acid was alanine, and the C-terminal amino acid sequence was -Glu-Leu-Thr-AlaCOOH. Comparative studies were performed between T. aestivum ferredoxin and ferredoxins isolated from closely related species; these were T. aegilopoides, T. durum, Ae. squarrosa, and Ae. ovata. No significant differences in the properties of these ferredoxins were detected. It was also shown that these ferredoxins are immunologically homologous. It is, therefore, likely that one molecular species of ferredoxin is distributed through two genera of Triticum and Aegilops.
The cross reactivity of sera from rabbits sensitized to performic acid oxidized ferredoxin (O-Fd) and of spleen cells from mice sensitized to O-Fd was analysed using several chemically modified forms of ferredoxin in the complement fixation test and the in vitro lymphocyte stimulation assay. Only O-Fd and native ferredoxin (native-Fd) gave positive responses in both assays. Dinitrophenylated-O-Fd (DNP-O-Fd) and acid precipitated ferredoxin (TCA-Fd) were able to fix complement (C') but did not simulate DNA synthesis in vitro. Ferredoxin alkylated with N-ethylmaleimioxin (CM-Fd) was unable to stimulate DNA synthesis and was marginally able to fix C'. Methylated-O-Fd (meth-O-Fd) was not recognized in either assay. The various ferredoxin preparations were tested for their ability to sensitize mice for use in the in vitro lymphocyte stimulation assay. Only O-Fd, NEM-Fd and native-Fd were capable of sensitizing lymphocytes for a proliferative response in vitro to the test antigens. This correlates with the observation that only these antigens were able to induce DNA synthesis in O-Fd-sensitized lymphocytes. The nature of the cells responding in vitro was examined by treating the cells with rabbit anti-mouse immunoglobulin and C' or rabbit anti-mouse brain associated theta and C'. The 24-hr response was found to be sensitive to both sera while the 120-hr response was sensitive only to the anti-theta sera.
The molecular weight of spinach ferredoxin-NADP reductase [EC 1.6.99.4] was estimated to be 33,100 by the sedimentation equilibrium method. On the basis of this molecular weight, the amino acid composition of the reductase was determined. The reactivity of ferredoxin toward p-chloromercuribenzoate was investigated. By measuring the time course of the reaction, 1 mol of ferredoxin was found to react with about 8 mol of p-chloromercuribenzoate in 10 min. Under low ionic strength conditions (1 mM NaCl), the second-order rate constants of this reaction determined spectrophotometrically at 420 and 250 nm were 3,640 and 3,690 M-1.S-1, respectively; under high ionic strength conditions (100 mM NaCl), these rate constants were 1,360 and 1,270 M-1.S-1, respectively. In the presence of the reductase, the rate constants under low and high ionic strength conditions were 54 and 1,040 M-1.S-1, respectively. By investigation of the solvent perturbation effects on the aromatic amino acid residues with 20% ethylene glycol, it was found that ferredoxin, ferredoxin-NADP reductase, and the complex between these proteins had 2.8, 6.3, and 3.8 mol of exposed tyrosyl residues per mol of protein, respectively. It therefore seems likely that about 5 tyrosyl residues may exist in the neighborhood of the binding site of the complex of these proteins.
Methods are described for the simultaneous isolation of ferredoxin-NADP+ reductase, ferredoxin and flavodoxin from large quantities of the cyanobacterium Anabaena PCC 7119 allowing the use of a single batch of cells. The ultraviolet-visible spectra and the extinction coefficients of ferredoxin-NADP+ reductase and ferredoxin were determined. The purification procedure also yields enriched fractions of phycobiliproteins and cytochrome c553.
Chemical modification of ferredoxin--NADP+ reductase from the cyanobacteria Anabaena has been performed using the alpha-dicarbonyl reagent phenylglyoxal. Inactivation of both the diaphorase and cytochrome-c reductase activities, characteristic of the enzyme, indicates the involvement of one or more arginyl residues in the catalytic process of the enzyme. The determination of the rate constants for the inactivation process under different conditions, including those in which substrates, NADP+ and ferredoxin, as well as other NADP+ analogs were present, indicates the involvement of two different groups in the inactivation process, one that reacts very rapidly with the reagent (kobs = 8.3 M-1 min-1) and is responsible for the binding of NADP+, and a second less reactive group (kobs = 0.9 M-1 min-1), that is involved in the binding of ferredoxin. Radioactive labeling of the enzyme with [14C]phenylglyoxal confirms that two groups are modified while amino acid analysis of the modified protein indicates that the modified groups are arginine residues. The identification of the amino acid residues involved in binding and catalysis of the substrates of ferredoxin--NADP+ reductase will help to elucidate the mechanism of the reaction catalyzed by this important enzyme.
A 285-base pair open reading frame was found immediately upstream of the fdxN gene (encoding ferredoxin I) of Rhodobacter capsulatus and coded for a 95-amino acid protein with a predicted molecular weight of 10,156. The deduced amino acid sequence contained 5 cysteines, 4 of which exhibited spacing characteristic of [2Fe-2S] plant and cyanobacterial ferredoxins. The amino acid sequence was found to share approximately 25% amino acid similarity with plant-type ferredoxins. The gene was named fdxC. Expression of the fdxC and fdxN genes together in Escherichia coli was accomplished by subcloning the genes in the vector pUC18 downstream of the lac promoter. Cells containing this plasmid produced a red and a brown protein corresponding to the fdxC and fdxN gene products, respectively. EPR and UV-visible absorption spectroscopy confirmed that the FdxC protein contained a [2Fe-2S] cluster and the FdxN protein contained two [4Fe-4S] clusters and that the centers were correctly assembled and inserted in the ferredoxins expressed in E. coli. Transcription (Northern blot) analysis showed that the genes were transcribed only under nitrogen-limiting (nif-derepressing) growth conditions.