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PubMed2000

Stearoyl-acyl carrier protein and unusual acyl-acyl carrier protein desaturase activities are differentially influenced by ferredoxin.

Acyl-acyl carrier protein (ACP) desaturases function to position a single double bond into an acyl-ACP substrate and are best represented by the ubiquitous Delta9 18:0-ACP desaturase. Several variant acyl-ACP desaturases have also been identified from species that produce unusual monoenoic fatty acids. All known acyl-ACP desaturase enzymes use ferredoxin as the electron-donating cofactor, and in almost all previous studies the photosynthetic form of ferredoxin rather than the non-photosynthetic form has been used to assess activity. We have examined the influence of different forms of ferredoxin on acyl-ACP desaturases. Using combinations of in vitro acyl-ACP desaturase assays and [(14)C]malonyl-coenzyme A labeling studies, we have determined that heterotrophic ferredoxin isoforms support up to 20-fold higher unusual acyl-ACP desaturase activity in coriander (Coriandrum sativum), Thunbergia alata, and garden geranium (Pelargonium x hortorum) when compared with photosynthetic ferredoxin isoforms. Heterotrophic ferredoxin also increases activity of the ubiquitous Delta9 18:0-ACP desaturase 1.5- to 3.0-fold in both seed and leaf extracts. These results suggest that ferredoxin isoforms may specifically interact with acyl-ACP desaturases to achieve optimal enzyme activity and that heterotrophic isoforms of ferredoxin may be the in vivo electron donor for this reaction.

Acyl Carrier Protein↗
PubMed1994

Laser flash-induced photoreduction of photosynthetic ferredoxins and flavodoxin by 5-deazariboflavin and by a viologen analogue.

Laser flash photolysis has been used to compare the kinetics of reduction of ferredoxin isoforms from the green alga Monoraphidium braunii, and the ferredoxin and flavodoxin from the cyanobacterium Anabaena PCC 7119, by 5-deazariboflavin semiquinone (dRfH.) and the viologen analogue 1,1'-propylene-2,2'-bipyridyl (PDQ.+). Similar ionic strength-independent second-order rate constants (1.4 x 10(8) M-1 s-1) were obtained for the reduction of both algal ferredoxin isoforms by dRfH.. For the reduction of oxidized flavodoxin by dRfH., a more complex behavior was observed, with a second-order rate constant for dRfH. decay of 1.8 x 10(8) M-1 s-1, and a first-order (i.e. protein concentration independent) rate constant of 450 s-1, that probably corresponds to the protonation of the FMN semiquinone cofactor, which occurs subsequent to electron transfer. A value of 5 x 10(7) M-1 s-1 was obtained for the second-order rate constant of flavodoxin semiquinone reduction by dRfH.. The reduction of ferredoxins and flavodoxin semiquinone by PDQ.+ showed nonlinear protein concentration dependencies, consistent with a minimal two-step mechanism involving complex formation followed by intracomplex electron transfer. A negative ionic strength effect on the kinetic constants was obtained, indicating the existence of attractive electrostatic interactions during electron transfer. With all the ferredoxins the k infinity values (rate constants extrapolated to infinite ionic strength) for the second-order step of the reduction process (complex formation) are smaller than previously reported for spinach ferredoxin, although Anabaena ferredoxin is somewhat more reactive than are the algal ferredoxins with the viologen.(ABSTRACT TRUNCATED AT 250 WORDS)

Anabaena↗
PubMed1989

Construction and characterization of an Azotobacter vinelandii strain with mutations in the genes encoding flavodoxin and ferredoxin I.

Flavodoxin and ferredoxin I have both been implicated as components of the electron transport chain to nitrogenase in the aerobic bacterium Azotobacter vinelandii. Recently, the genes encoding flavodoxin (nifF) and ferredoxin I (fdxA) were cloned and sequenced and mutants were constructed which are unable to synthesize either flavodoxin (DJ130) or ferredoxin I (LM100). Both single mutants grow at wild-type rates under N2-fixing conditions. Here we report the construction of a double mutant (DJ138) which does not synthesize either flavodoxin or ferredoxin I. When plated on ammonium-containing medium, this mutant had a very small colony size when compared with the wild type, and in liquid culture with ammonium, this double mutant grew three times slower than the wild type or single mutant strains. This demonstrated that there is an important metabolic function unrelated to nitrogen fixation that is normally carried out by either flavodoxin or ferredoxin. If either one of these proteins is missing, the other can substitute for it. The double mutant phenotype can now be used to screen site-directed mutant versions of ferredoxin I for functionality in vivo even though the specific function of ferredoxin I is still unknown. The double mutant grew at the same slow rate under N2-fixing conditions. Thus, A. vinelandii continues to fix N2 even when both flavodoxin and ferredoxin I are missing, which suggests that a third as yet unidentified protein also serves as an electron donor to nitrogenase.

Azotobacter↗
PubMed1992

Biosynthesis of phycobilins. Ferredoxin-supported nadph-independent heme oxygenase and phycobilin-forming activities from Cyanidium caldarium.

The unicellular red alga, Cyanidium caldarium, synthesizes phycocyanobilin from protoheme via biliverdin IX alpha. In vitro transformation of protoheme to biliverdin IX alpha and biliverdin IX alpha to phycobilins were previously shown to require NADPH, ferredoxin, and ferredoxin-NADP+ reductase, as well as specific heme oxygenase and phycobilin formation enzymes. The role of NADPH in these reactions was investigated in this study. The C. caldarium enzymatic activities that catalyze biliverdin IX alpha formation from protoheme, and phycobilin formation from biliverdin IX alpha, were partially purified by differential (NH4)2SO4 precipitation. The enzyme fractions, when supplemented with a light-driven ferredoxin-reducing photosystem I fraction derived from spinach leaves, catalyzed light-dependent transformation of protoheme to biliverdin IX alpha and biliverdin IX alpha to phycobilins, with or without the addition of NADPH and ferredoxin-NADP+ reductase. In the dark, neither reaction occurred unless NADPH and ferredoxin-NADP+ reductase were supplied. These results indicate that the only role of NADPH in both reactions of phycobilin biosynthesis, in vitro, is to reduce ferredoxin via ferredoxin-NADP+ reductase and that reduced ferredoxin can directly supply the electrons needed to drive both steps in the transformation of protoheme to phycocyanobilin.

Cell Fractionation↗
PubMed1996

Characterization of a redox active cross-linked complex between cyanobacterial photosystem I and soluble ferredoxin.

A covalent stoichiometric complex between photosystem I (PSI) and ferredoxin from the cyanobacterium Synechocystis sp. PCC 6803 was generated by chemical cross-linking. The photoreduction of ferredoxin, studied by laser flash absorption spectroscopy between 460 and 600 nm, is a fast process in 60% of the covalent complexes, which exhibit spectral and kinetic properties very similar to those observed with the free partners. Two major phases with t(1/2) <1 micros and approximately 10-14 micros are observed at two different pH values (5.8 and 8.0). The remaining complexes do not undergo fast ferredoxin reduction and 20-25% of the complexes are still able to reduce free ferredoxin or flavodoxin efficiently, thus indicating that ferredoxin is not bound properly in this proportion of covalent complexes. The docking site of ferredoxin on PSI was determined by electron microscopy in combination with image analysis. Ferredoxin binds to the cytoplasmic side of PSI, with its mass center 77 angstroms distant from the center of the trimer and in close contact with a ridge formed by the subunits PsaC, PsaD and PsaE. This docking site corresponds to a close proximity between the [2Fe- 2S] center of ferredoxin and the terminal [4Fe-4S] acceptor FII of PSI and is very similar in position to the docking site of flavodoxin, an alternative electron acceptor of PSI.

Cyanobacteria↗
PubMed2005

A conserved histidine-aspartate pair is required for exovinyl reduction of biliverdin by a cyanobacterial phycocyanobilin:ferredoxin oxidoreductase.

Phycocyanobilin:ferredoxin oxidoreductase is a member of the ferredoxin-dependent bilin reductase family and catalyzes two vinyl reductions of biliverdin IXalpha to produce phycocyanobilin, the pigment precursor of both phytochrome and phycobiliprotein chromophores in cyanobacteria. Atypically for ferredoxin-dependent enzymes, phycocyanobilin:ferredoxin oxidoreductase mediates direct electron transfers from reduced ferredoxin to its tetrapyrrole substrate without metal ion or organic cofactors. We previously showed that bound bilin radical intermediates could be detected by low temperature electron paramagnetic resonance and absorption spectroscopies (Tu, S., Gunn, A., Toney, M. D., Britt, R. D., and Lagarias, J. C. (2004) J. Am. Chem. Soc. 126, 8682-8693). On the basis of these studies, a mechanism involving sequential electron-coupled proton transfers to protonated bilin substrates buried within the phycocyanobilin:ferredoxin oxidoreductase protein scaffold was proposed. The present investigation was undertaken to identify catalytic residues in phycocyanobilin:ferredoxin oxidoreductase from the cyanobacterium Nostoc sp. PCC7120 through site-specific chemical modification and mutagenesis of candidate proton-donating residues. These studies identified conserved histidine and aspartate residues essential for the catalytic activity of phycocyanobilin:ferredoxin oxidoreductase. Spectroscopic evidence for the formation of stable enzyme-bound biliverdin radicals for the H85Q and D102N mutants support their role as a "coupled" proton-donating pair during the reduction of the biliverdin exovinyl group.

Amino Acid Sequence↗
PubMed1976

Events Surrounding the Early Development of Euglena Chloroplasts: VII. Photocontrol of the Source Of Reducing Power for Chloramphenicol Reduction by the Ferredoxin-NADP Reductase System.

d(-)threo-Chloramphenicol blocks chlorophyll and plastid protein synthesis in Euglena. During chloroplast development in white light, but not in red, the cells escape from chloramphenicol inhibition and chlorophyll formation is restored. Concomitantly, chloramphenicol is reduced. Reduction of chloramphenicol in an enzyme extract from Euglena requires NADPH and ferredoxin for maximal activity. Methyl viologen replaces ferredoxin, and when chemically reduced, ferredoxin or methyl viologen reduces chloramphenicol directly. This suggests that the enzyme involved is ferredoxin-NADP reductase. In agreement, crude extracts from wild type and W(3)BUL, a mutant lacking detectable plastids and plastid DNA, when separated on acrylamide gels, show a single band which reduces methyl viologen with NADPH, and its mobility is similar in wild type and in mutant W(3)BUL. The reductase is inducible by light and increases 3-fold in wild type in white or red light and 1.5-fold in W(3)BUL in white light. DCMU does not block chloramphenicol reduction in vivo indicating that electrons originate from sources other than photosynthetic electron transport. We infer that chloramphenicol is reduced by ferredoxin which receives electrons via ferredoxin-NADP reductase. The limiting step is not the enzyme but the source of reducing power which can be supplied from the cytoplasm, probably under control of the blue light receptor. Ferredoxin and ferredoxin NADP reductase appear to be coded in the nuclear genome, synthesized on cytoplasmic ribosomes, and join a group of enzymes which cannot be precisely localized, since they may be active anywhere from their site of synthesis in the cytoplasm to their place of deposition in the chloroplast.

Journal Article↗
PubMed1987

Protein modulase appears to be a complex of ferredoxin, ferredoxin/thioredoxin reductase, and thioredoxin.

Protein modulase and ferredoxin/thioredoxin reductase are soluble proteins that have been suggested to catalyze the light-dependent modulation of enzyme activity in the stromal compartment of the chloroplast. Protein modulase is active in vitro without additional ferredoxin and thioredoxin, whereas ferredoxin/thioredoxin reductase requires additional ferredoxin and thioredoxin. We hypothesize that protein modulase is a complex protein composed of ferredoxin/thioredoxin reductase, ferredoxin, and thioredoxin. In reconstituted chloroplast systems, antiserum directed against ferredoxin, at concentrations sufficient to inhibit the photoreduction of NADP, had no effect on light modulation. Antiserum directed against thioredoxin gave variable results: one batch of polyclonal antibodies inhibited light modulation, another was stimulatory, and another was without effect. These results suggest that the ferredoxin and thioredoxin active in light modulation are not free in solution. Furthermore, molecular sieve chromatography of stromal proteins results in the elution of four species that catalyze light modulation. Based on whether or not ferredoxin and/or thioredoxin must be added for activity, these four species have been tentatively identified as protein modulase, a complex of ferredoxin/thioredoxin reductase and ferredoxin, a complex of ferredoxin/thioredoxin reductase and thioredoxin, and ferredoxin/thioredoxin reductase. That is, the four correspond to all the possible combinations of ferredoxin, ferredoxin/thioredoxin reductase, and thioredoxin. We suggest that buffer ionic strength affects the interactions among these proteins and in part determines the fate of the protein modulase complex in vitro.

Journal Article↗
PubMed2002

Properties of 2-oxoglutarate:ferredoxin oxidoreductase from Thauera aromatica and its role in enzymatic reduction of the aromatic ring.

Benzoyl coenzyme A (benzoyl-CoA) reductase is a key enzyme in the anaerobic metabolism of aromatic compounds catalyzing the ATP-driven reductive dearomatization of benzoyl-CoA. The enzyme from Thauera aromatica uses a reduced 2[4Fe-4S] ferredoxin as electron donor. In this work, we identified 2-oxoglutarate:ferredoxin oxidoreductase (KGOR) as the ferredoxin reducing enzyme. KGOR activity was increased 10- to 50-fold in T. aromatica cells grown under denitrifying conditions on an aromatic substrate compared to that of cells grown on nonaromatic substrates. The enzyme was purified from soluble extracts by a 60-fold enrichment with a specific activity of 4.8 micromol min(-1) mg(-1). The native enzyme had a molecular mass of 200 +/- 20 kDa (mean +/- standard deviation) and consisted of two subunits with molecular masses of 66 and 34 kDa, suggesting an (alphabeta)(2) composition. The UV/visible spectrum was characteristic for an iron-sulfur protein; the enzyme contained 8.3 +/- 0.5 mol of Fe, 7.2 +/- 0.5 mol of acid-labile sulfur, and 1.6 +/- 0.2 mol of thiamine diphosphate (TPP) per mol of protein. The high specificity for 2-oxoglutarate and the low K(m) for ferredoxin ( approximately 10 microM) indicated that both are the in vivo substrates of the enzyme. KGOR catalyzed the isotope exchange between (14)CO(2) and C(1) of 2-oxoglutarate, representing a typical reversible partial reaction of 2-oxoacid oxidoreductases. The two genes coding for the two subunits of KGOR were found adjacent to the gene cluster coding for enzymes and ferredoxin of the catabolic benzoyl-CoA pathway. Sequence comparisons with other 2-oxoacid oxidoreductases indicated that KGOR from T. aromatica belongs to the Halobacterium type of 2-oxoacid oxidoreductases, which lack a ferredoxin-like module which contains two additional [4Fe-4S](1+/2+) clusters/monomer. Using purified KGOR, ferredoxin, and benzoyl-CoA reductase, the 2-oxoglutarate-driven reduction of benzoyl-CoA was shown in vitro. This demonstrates that ferredoxin acts as an electron shuttle between the citric acid cycle and benzoyl-CoA reductase by coupling the oxidation of the end product of the benzoyl-CoA pathway, acetyl-CoA, to the reduction of the aromatic ring.

Amino Acid Sequence↗
PubMed1988

Antigenic similarities between ferredoxin-dependent nitrite reductase and glutamate synthase from Chlamydomonas reinhardtii.

Polyclonal antisera were prepared against ferredoxin-nitrite reductase (EC 1.7.7.1) and ferredoxin-glutamate synthase (glutamate synthase (ferredoxin); EC 1.4.7.1) from the green alga Chlamydomonas reinhardtii. The anti-glutamate synthase antibodies recognized both glutamate synthase and nitrite reductase, but inhibited only the ferredoxin-linked activity of the latter enzyme and not the activity dependent on methyl viologen. Analogously, the anti-nitrite reductase antibodies recognized glutamate synthase and nitrite reductase but the first enzyme was only poorly inhibited. Free ferredoxin protected the nitrite reductase against its inactivation by anti-glutamate synthase antibodies. These results indicate that the ferredoxin-dependent glutamate synthase and nitrite reductase from this alga share common antigenic determinants, and that these are located at the ferredoxin-binding domains.

Chlamydomonas↗
PubMed1997

On the role of the acidic cluster Glu 92-94 of spinach ferredoxin I.

The role of the acidic cluster Glu 92-94 of spinach ferredoxin I in the interaction both with the photosystem I multisubunit complex and the ferredoxin-NADP+ reductase, either membrane-bound or purified, was studied by kinetic characterization of site-directed mutants. Three mutants of ferredoxin have been produced to evaluate the effects of elimination of one or two negative charges in the three specific positions of the acidic cluster. Kinetic characterization of the ferredoxin mutants E92A/E93A, E93A and E93A/E94A as electron carriers in the photosynthetic electron transport chain, allowed to establish that the two latter mutants were nearly indistinguishable from the wild-type protein in their ability to be photoreduced by photosystem I and as electron donor to the reductase in the NADP+ photoreduction with thylakoid membranes. The E92A/E93A ferredoxin mutant behaved very similarly to E92 mutants previously characterized. Thus, the elimination of the carboxyl groups adjacent to residue 92 did not further impaired ferredoxin I main function, i.e., as an electron carrier in NADP+ photoreduction. The two double mutants showed a reduced rate in the cross-linking of ferredoxin to the reductase promoted by a soluble carbodiimide, indicating an involvement of the acidic cluster in the formation of the active covalent complex between the two proteins.

Amino Acid Sequence↗
PubMed2003

Insights into the design of a hybrid system between Anabaena ferredoxin-NADP+ reductase and bovine adrenodoxin.

The opportunity to design enzymatic systems is becoming more feasible due to detailed knowledge of the structure of many proteins. As a first step, investigations have aimed to redesign already existing systems, so that they can perform a function different from the one for which they were synthesized. We have investigated the interaction of electron transfer proteins from different systems in order to check the possibility of heterologous reconstitution among members of different chains. Here, it is shown that ferredoxin-NADP+ reductase from Anabaena and adrenodoxin from bovine adrenal glands are able to form optimal complexes for thermodynamically favoured electron transfer reactions. Thus, electron transfer from ferredoxin-NADP+ reductase to adrenodoxin seems to proceed through the formation of at least two different complexes, whereas electron transfer from adrenodoxin to ferredoxin-NADP+ reductase does not take place due because it is a thermodynamically nonfavoured process. Moreover, by using a truncated adrenodoxin form (with decreased reduction potential as compared with the wild-type) ferredoxin-NADP+ reductase is reduced. Finally, these reactions have also been studied using several ferredoxin-NADP+ reductase mutants at positions crucial for interaction with its physiological partner, ferredoxin. The effects observed in their reactions with adrenodoxin do not correlate with those reported for their reactions with ferredoxin. In summary, our data indicate that although electron transfer can be achieved in this hybrid system, the electron transfer processes observed are much slower than within the physiological partners, pointing to a low specificity in the interaction surfaces of the proteins in the hybrid complexes.

Adrenodoxin↗
PubMed1976

Electrochemistry of drug action I: electrooreduction of ferredoxins.

Ferredoxin serves as an electron carrier in the oxidation-reduction system in anaerobic microorganisms, transferring electrons from a low potential donor to electron-accepting biochemicals. The anaerobicidal activity of some drugs may be due to their interference with the electron transport function of ferredoxin. Two types of ferredoxins (isolated from Clostridium pasteurianum and spinach) were studied, and their electrochemical reduction and biochemical properties were analyzed using a sensitive ac polarographic technique. The reduction potential of both ferredoxins was linearly related to pH. The mechanisms of electron transport in ferredoxin molecules were found to be related to their sulfur-iron bonds. The dissociation of the sulfur-iron bonds resulted in the formation of a free sulfhydryl group and the interruption of the electroactivity of ferredoxin. This sulfur-iron dissociation process was found to be pH dependent. The electroreduction of ferredoxins was an energy-requiring, pH-dependent process.

Clostridium↗
PubMed1995

Protein expression, selective isotopic labeling, and analysis of hyperfine-shifted NMR signals of Anabaena 7120 vegetative [2Fe-2S]ferredoxin.

Two alternative T7 RNA promoter/polymerase systems have been employed for the heterologous expression of a plant-type [2Fe-2S]ferredoxin, Anabaena 7120 vegetative ferredoxin, in Escherichia coli at high levels (approximately 20 mg/liter of culture). One system was used when 15N-labeling the ferredoxin uniformly by growing E. coli with 15NH4Cl as the nitrogen source; the other was used in conjunction with auxotrophic host strains to enrich the protein selectively by incorporating 2H-, 13C-, and 15N-labeled amino acids. The labeled ferredoxin samples were studied by 1H, 2H, 13C, and 15N NMR spectroscopy. Results from 1H and 2H NMR studies of samples containing [2H alpha]Cys, [2H beta 2, beta 3]Cys, [13 C beta]-Cys, and [15N]Cys have confirmed previous cysteinyl proton resonance assignments (L. Skjeldal, W. M. Westler, B.-H. Oh, A. M. Krezel, H. M. Holden, B. L. Jacobson, I. Rayment, and J. L. Markley (1991) Biochemistry 30, 7363-7368). All four 13C NMR peaks arising from the four cysteinyl beta-carbons and all four 15N NMR peaks from the four cysteinyl nitrogens were resolved in spectra of both the oxidized and reduced ferredoxins. The nitrogen resonance of Cys46, which is located in a unique (Ala-Cys) dipeptide, was assigned by detection of 13Ci-15Ni+1 coupling in a ferredoxin sample with incorporated [13C']Ala and [15N]Cys. The nitrogen signal of Cys 41 was assigned tentatively on the basis of its chemical shift and T1 relaxation time. The cysteinyl beta-carbon resonances in the reduced state have been assigned to individual residues on the basis of correlations with their (previously assigned) beta-protons. The beta-carbons resonance from Cys46 in the oxidized state has been assigned by its correlation with the corresponding resonance in the reduced state; this was accomplished by following the progressive air oxidation of a protein sample reduced by dithionite in the presence of methyl viologen. The spin-lattice relaxation times of the beta-carbons of the two cysteines coordinated to Fe)III) were similar in the oxidized and reduced states. This suggests that the antiferromagnetic coupling present in the reduced cluster has little influence on the electronic relaxation time of the Fe(III). Studies of the temperature dependence of the 1H, 13C, and 15N signals of the cysteinyl ligands to the [2Fe-2S] cluster show that the slope of the temperature dependence (delta delta/delta T-1) can be different for different atom types within a given residue. For example, in the reduced ferredoxin, although delta delta/delta T-1 is positive for Cys49 1H beta 2 and 1H beta 3, it is negative for Cys49 13C beta. Although delta delta/delta T-1 is negative for protons of cysteines ligated to Fe(II) and positive for protons of cysteines ligated to Fe(III), it is positive for all the cysteinyl nitrogens.(ABSTRACT TRUNCATED AT 400 WORDS)

Anabaena↗
PubMed2000

Importance of the region including aspartates 57 and 60 of ferredoxin on the electron transfer complex with photosystem I in the cyanobacterium Synechocystis sp. PCC 6803.

Ferredoxin reduction by photosystem I has been studied by flash-absorption spectroscopy. Aspartate residues 20, 57, and 60 of ferredoxin were changed to alanine, cysteine, arginine, or lysine. On the one hand, electron transfer from photosystem I to all mutated ferredoxins still occurs on a microsecond time scale, with half-times of ferredoxin reduction mostly conserved compared to wild-type ferredoxin. On the other hand, the total amplitude of the fast first-order reduction varies largely when residues 57 or 60 are modified, in apparent relation to the charge modification (neutralized or inverted). Substituting these two residues for lysine or arginine induce strong effects on ferredoxin binding (up to sixfold increase in K(D)), whereas the same substitution on aspartate 20, a spatially related residue, results in moderate effects (maximum twofold increase in K(D)). In addition, double mutations to arginine or lysine were performed on both aspartates 57 and 60. The mutated proteins have a 15- to 20-fold increased K(D) and show strong modifications in the amplitudes of the fast reduction kinetics. These results indicate that the acidic area of ferredoxin including aspartates 57 and 60, located opposite to the C-terminus, is crucial for high affinity interactions with photosystem I.

Aspartic Acid↗
PubMed1999

Vertebrate-type and plant-type ferredoxins: crystal structure comparison and electron transfer pathway modelling.

Crystallographic analysis of a fully functional, truncated bovine adrenodoxin, Adx(4-108), has revealed the structure of a vertebrate-type [2Fe-2S] ferredoxin at high resolution. Adrenodoxin is involved in steroid hormone biosythesis in adrenal gland mitochondria by transferring electrons from adrenodoxin reductase to different cytochromes P450. Plant-type [2Fe-2S] ferredoxins interact with photosystem I and a diverse set of reductases.A systematic structural comparison of Adx(4-108) with plant-type ferredoxins which share about 20 % sequence identity yields these results. (1) The ferredoxins of both types are partitioned into a large, strictly conserved core domain bearing the [2Fe-2S] cluster and a smaller interaction domain which is structurally different for both subfamilies. (2) In both types, residues involved in interactions with reductase are located at similar positions on the molecular surface and coupled to the [2Fe-2S] cluster via structurally equivalent hydrogen bonds. (3) The accessibility of the [2Fe-2S] cluster differs between Adx(4-108) and the plant-type ferredoxins where a solvent funnel leads from the surface to the cluster. (4) All ferredoxins are negative monopoles with a clear charge separation into two compartments, and all resulting dipoles but one point into a narrow cone located in between the interaction domain and the [2Fe-2S] cluster, possibly controlling predocking movements during interactions with redox partners. (5) Model calculations suggest that FE1 is the origin of electron transfer pathways to the surface in all analyzed [2Fe-2S] ferredoxins and that additional transfer probability for electrons tunneling from the more buried FE2 to the cysteine residue in position 92 of Adx is present in some.

Adrenodoxin↗
PubMed1995

Overexpression in Escherichia coli of the fdxA gene encoding Rhodobacter capsulatus ferredoxin II.

Different gene expression systems were tested with the aim of overproducing the 7Fe ferredoxin (FdII) from Rhodobacter capsulatus in Escherichia coli. Plasmids bearing the Ptac, ParaB, fdxA gene, encoding FdII, under the control of the PlacUV5, P phi 10 promoters, were compared for their efficiency to promote the synthesis of recombinant ferredoxin. Using a P phi 10-based expression system, recombinant ferredoxin was obtained in a soluble apoform and accumulated up to 20% of the total cell protein. The ferredoxin polypeptide purified from such cells exhibited the correct molecular mass and no detectable heterogeneity when analyzed by mass spectrometry. When other expression systems were used, the ferredoxin was synthesized in holoform but in relatively smaller amounts (0.2 mg/liter of culture). Factors such as promoter strength, efficient translation signals, and stability of the recombinant mRNA were shown to have little effect on the amount of 7Fe ferredoxin produced in E. coli. It is inferred that iron-sulfur clusters insertion may be a rate-limiting factor for synthesis of the 7Fe ferredoxin in the enterobacterium.

Bacterial Proteins↗
PubMed1993

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-1 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.

Amino Acid Sequence↗