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PubMed1991

The interaction of ferredoxin and glutamate synthase: cross-linking and immunological studies.

The water-soluble carbodiimide, N-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) serves as an effective reagent for cross-linking spinach leaf ferredoxin and the ferredoxin-dependent spinach leaf enzyme, glutamate synthase. The cross-linked complex was functional in the absence of added ferredoxin, suggesting that ferredoxin is cross-linked to glutamate synthase at the physiological binding site on the enzyme for this iron-sulfur protein electron donor. The ferredoxin:glutamate synthase stoichiometry of the cross-linked complex was estimated to be 2:1. The absorbance spectrum of the oxidized, cross-linked complex was very similar to that of an electrostatically stabilized, noncovalent, 2:1 complex of the two proteins. An antibody raised against spinach NADP+ reductase, which recognizes a ferredoxin-binding site on glutamate synthase, does not recognize the cross-linked ferredoxin-glutamate synthase complex. This implies that the ferredoxin-binding sites on the two enzymes are structurally similar enough so that an antibody raised against one of these ferredoxin-dependent enzymes recognizes an epitope at the ferredoxin-binding site of the second enzyme. Cross-linking of ferredoxin to its binding site on glutamate synthase renders this epitope inaccessible to the antibody.

Antibodies↗
PubMed1981

Characterization of a new type of ferredoxin from Desulfovibrio africanus.

A new ferredoxin designated ferredoxin III has been isolated from Desulfovibrio africanus grown on media high in iron. Native ferredoxin III is a dimer constituted by two identical subunits of approx. 7500. It is distinguished from the two other ferredoxins (I and II) isolated from this microorganism by its amino acids composition, N-terminal sequence, spectral properties and iron-sulfur content. The amino acid composition of D. africanus ferredoxin III is typical of ferredoxins with an excess of acidic over basic residues and the absence of histidine and arginine residues. The absorption spectrum of ferredoxin III exhibits two maxima, at 408 nm (epsilon = 58.5 . 10(3) M-1 . cm-1) and 285 nm (epsilon = 82 . 10(3) M-1 . cm-1), with a shoulder at 305 nm (epsilon = 75 . 10(3) M-1 . cm-1). Its A408/A285 absorbance ratio is 0.78. Ferredoxin III contains approx. 12--13 atoms each of iron and labile sulfur. This is in agreement with the high value of the extinction coefficient at 408 nm, which is slightly higher than 3-fold that of one [4Fe-4S] cluster. However, the number of cysteine residues of the protein (six residues), which is about the half that of iron atoms, is indicative of the presence of a new type of iron-sulfur cluster in ferredoxin III. The protein is unstable in a low ionic strength environment; the addition of neutral salts stabilizes the protein conformation. The data on the biological activity of ferredoxin III as compared to the two other ferredoxins from D. africanus show that the three iron-sulfur proteins function with equal effectiveness as electron carrier in the phosphoroclastic reaction and the H2-sulfite reductase system.

Amino Acid Sequence↗
PubMed1976

Regulation of the NADH and NADPH-ferredoxin oxidoreductases in clostridia of the butyric group.

NADH and NADPH-ferredoxin oxidoreductases have been studied in Clostridium acetobutylicum, Cl. tyrobutyricum and Cl. pasteurianum. The study of the distribution and regulation of these enzymatic activities in well-defined culture conditions, reveals that the essential function of NADPH-ferredoxin oxidoreductase is to produce NADPH, while NADH-ferredoxin oxidoreductase can, depending on cellular conditions, produce or oxidize NADH. When these Clostridia use glycolysis, regulation of the NADH-ferredoxin oxidoreductase by acetyl-CoA (obligatory activator of NADH-ferroxin reductase activity) and by NADH (competitive inhibitor of ferredoxin-NAD+ reductase activity) allow the enzymes to function correlatively with glyceraldehyde-3-phosphate dehydrogenase and thus control the levels of NAD+ and NADH in the cell. In Cl. tyrobutyricum and Cl. pasteurianum, the ferredoxin-NADP+ reductase activities are regulated by NAD+ and NADH in accordance with the intracellular concentrations of these coenzymes. In Cl. tyrobutyricum growing on pyruvate/acetate, NADH and NADPH-ferredoxin reductase activities cannot be detected; only the ferredoxin-NAD+ and ferredoxin-NADP+ reductase activities are found. In this Clostridium, regulation of the ferredoxin-NADP+ reductase activity is the same whether it is grown on glucose or pyruvate. Contrary to this, the ferredoxin-NAD+ reductase activity undergoes a drastic change, since NADH no longer controls the enzymatic activity. In this case regulation is no longer necessary, since glyceraldehyde-3-phosphate dehydrogenase does not function.

Butyrates↗
PubMed1994

Laser flash absorption spectroscopy study of ferredoxin reduction by photosystem I in Synechocystis sp. PCC 6803: evidence for submicrosecond and microsecond kinetics.

The kinetics of reduction of soluble ferredoxin by photosystem I (PSI), both purified from the cyanobacterium Synechocystis sp. PCC 6803, were investigated by flash-absorption spectroscopy between 460 and 600 nm. Most experiments were made with isolated monomeric PSI reaction centers prepared with the detergent beta-dodecyl maltoside. Analysis of absorption transients, in parallel at 480 and 580 nm and under several conditions, shows the existence of three different first-order components in the presence of ferredoxin (t1/2 approximately 500 ns, 20 microseconds, and 100 microseconds). A second-order phase of ferredoxin reduction is also present [k = (2-5) x 10(8) s-1 at pH 8 and at moderate ionic strength]. Similar first-order kinetic components were found with membranes from Synechocystis, with dissolved crystals of trimeric PSI reaction centers from Synechococcus, and also when ferredoxin from Synechocystis is replaced by ferredoxin from Chlamydomonas reinhardtii. The three first-order phases exhibit similar, though not identical, spectra which are consistent with electron transfer from the [4Fe-4S] centers of PSI to the [2Fe-2S] center of ferredoxin and are all attributed to reduction of ferredoxin bound to PSI. At pH 8 and at moderate ionic strength, the dissociation constants associated with each of these components are also similar, with a global value varying between 0.2 and 0.8 microM in different cyanobacterial preparations. The presence of three exponential components is discussed assuming homogeneity of the two partners and using the estimated values for the shortest possible distance of approach of soluble ferredoxin from the different iron-sulfur centers of PSI. It is concluded that the 500-ns phase corresponds to electron transfer from either FA- or FB-, the terminal iron-sulfur acceptors of PSI, to ferredoxin and that the immediate electron donor to ferredoxin is reduced within less than 500 ns. The presence of at least two different types of PSI-ferredoxin complex, all competent in electron transfer, is also deduced from the kinetic behavior.

Cyanobacteria↗
PubMed1997

Specific interaction of the [2Fe-2S] ferredoxin from Clostridium pasteurianum with the nitrogenase MoFe protein.

Putative physiological partners of the [2Fe-2S] ferredoxin from Clostridium pasteurianum have been searched by running crude soluble extracts of this bacterium through an affinity column to which the [2Fe-2S] ferredoxin had been covalently bound. Subsequent washing of the column with buffers of increasing ionic strength revealed a strong and specific interaction of the ferredoxin with the MoFe protein of nitrogenase. This interaction was further investigated by performing cross-linking experiments with mixtures of the two purified proteins in solution. Analysis of the reactions by SDS-polyacrylamide gel electrophoresis evidenced only two covalently linked products. These were identified by N-terminal sequencing as the alpha and beta subunits of the MoFe protein, each cross-linked to a single polypeptide chain of the ferredoxin. This result, taking into account the dimeric structure of the ferredoxin in solution, strongly suggests an interaction of the ferredoxin with the MoFe protein at a site contributed to by both subunits of the MoFe protein. The ionic strength dependence of the interaction evidenced by affinity chromatography was confirmed in the cross-linking reactions, and its specificity was assessed by showing that no cross-linking occurred when the [2Fe-2S] C. pasteurianum ferredoxin was denatured or replaced by spinach ferredoxin or by clostridial rubredoxin, or when the MoFe protein from C. pasteurianum was either inactivated or replaced by its counterpart from Azotobacter vinelandii. It has also been observed that the ferredoxin inhibits cross-linking between the nitrogenase Fe protein and the MoFe protein, which suggests overlapping binding sites of the ferredoxin and of the Fe protein on the MoFe protein. Cross-linking experiments implementing a number of molecular variants of the [2Fe-2S] C. pasteurianum ferredoxin demonstrated that glutamate residues 31, 34, and 38 are important contributors to the interaction with the MoFe protein.

Chromatography, Affinity↗
PubMed1998

Identification and characterization of the natural electron donor ferredoxin and of FAD as a possible prosthetic group of benzoyl-CoA reductase (dearomatizing), a key enzyme of anaerobic aromatic metabolism.

Under anoxic conditions most aromatic compounds are metabolized via benzoyl-CoA which becomes reduced by benzoyl-CoA reductase (dearomatizing); this enzyme was recently described in the bacterium Thauera aromatica [Boll, M. & Fuchs, G. (1995) Eur. J. Biochem. 234, 921-933]. It catalyzes the reaction benzoyl-CoA + 2 e- + 2 H+ + 2 MgATP + 2 H2O --> cyclohexa-1,5-diene-1-carboxyl-CoA + 2 MgADP + 2 Pi. The iron-sulfur protein has a native molecular mass of 160-170 kDa and consists of four different subunits. In addition a flavin may be present. The nature of the potential prosthetic group and the natural electron donor were determined. Purified benzoyl-CoA reductase preparations contained 0.25-0.3 mol FAD/mol enzyme. Cells grown anaerobically with aromatic substrates contained a ferredoxin which represented the main, if not the only ferredoxin present. It was purified from 200 g cells with a yield of 60 mg and its N-terminal amino acid sequence was determined. The native molecular mass was 9659 +/- 2 Da as determined by electrospray mass spectrometry. The protein contained 7.6 +/- 0.6 mol iron and 7.6 +/- 1 mol acid-labile sulfur/mol. The ultraviolet-visible spectrum of the protein was typical for ferredoxins with maxima at 280 nm and 390 nm (in the oxidized state). The estimated molar absorption coefficients were 63500 M(-1) cm(-1) at 280 nm and 40500 M(-1) cm(-1) at 390 nm. The difference spectrum between the oxidized and the reduced form had a maximum at 415 nm with delta epsilon415 = 8200 M(-1) cm(-1). 1 mol ferredoxin became reduced/mol dithionite added, suggesting the presence of two [4Fe-4S] clusters. The average midpoint potential of the iron-sulfur clusters was -450 mV. The ferredoxin gene was cloned and sequenced. It was located in a gene cluster coding for enzymes involved in anaerobic aromatic metabolism. The amino acid sequence of the T. aromatica ferredoxin showed high similarities to several other ferredoxins containing 2[4Fe-4S] clusters, e.g. from Clostridia and phototrophic bacteria. The reduced ferredoxin served as electron donor for benzoyl-CoA reduction at a three times higher rate compared with the rate obtained with the artificial electron donor reduced methyl viologen. The turnover number with the natural electron donor of 5 s(-1) can explain the bacterial growth rate with benzoate as substrate. Half-maximal enzyme activity was obtained with 6 microM reduced ferredoxin, at an estimated cellular concentration of 70 microM ferredoxin. Both the low apparent Km value and the turnover number are consistent with the proposed role of ferredoxin in aromatic-ring reduction.

Amino Acid Sequence↗
PubMed1974

Synthesis and properties of Clostridium acidi-urici (Leu2)-ferredoxin: a function of the peptide chain and evidence against the direct role of the aromatic residues in electron transfer.

Tyrosyl or other aromatic residues generally occur in two conserved positions in the peptide chain of clostridial-type ferredoxins and have been implicated in the electron transfer function of these iron-sulfur proteins. We have prepared and determined some of the properties of a derivative of Clostridium acidi-urici ferredoxin, [Leu(2)]-ferredoxin, in which a leucyl residue has been substituted for the tyrosyl residue in position 2 from the amino terminus. [Leu(2)]-ferredoxin is fully active as an electron carrier in two biological assays, the phosphoroclastic enzyme system and the ferredoxin-dependent reduction of cytochrome c in the presence of ferredoxin-TPN reductase and TPNH. Quantitative electron paramagnetic resonance experiments indicate that [Leu(2)]-ferredoxin accepts nearly two electrons upon enzymatic reduction by pyruvate-ferredoxin oxidoreductase and an excess of pyruvate. If electron transfer to an iron-sulfur cluster is the rate-limiting step in the assays used, and if the rate of electron transfer through Tyr(30) is not much faster than through Tyr(2), these results indicate that the primary pathway of electron transfer in clostridial-type ferredoxins is not via Tyr or other aromatic amino-acid residues. The syntheses of other ferredoxin derivatives with amino-acid substitutions or deletions in positions 1 and 2 indicate that a large bulky residue, but not necessarily an aromatic residue, is needed in position 2 for the stability of this ferredoxin. The residue in position 2, therefore, appears to act as a hydrophobic shield for an iron-sulfur cluster.

Amino Acid Sequence↗
PubMed1977

Horsetail (Equisetum arvense) ferredoxins I and II Amino acid sequences and gene duplication.

Amino acid sequences of two ferredoxins isolated from Equisetum arvense were determined by conventional procedures. Ferredoxins I and II of E. arvense had 95 and 93 residues, respectively, and nearly identical sequences each with only one amino acid difference from ferredoxins. I and II of E. telmateia (1). The overall structural characteristics of these two ferredoxins were therefore very similar to those of E. telmateia ferredoxins. Ferredoxins I and II from E. arvense differ in 31 sites and those from E. telmateia in 29 sites from each other. These facts suggested that duplication of the ferredoxin gene in one organism occurred at an early evolutionary stage long before the divergence of the two horsetail species. The number of differences in amino acids between horsetail ferredoxins and other chloroplast-type ferredoxins indicated that the duplication occurred after divergence of horsetails from other plants. Comparing green plant ferredoxins, it was estimated that this gene duplication occurred about 250 million years ago. Some comments on the unique amino acid substitutions in horsetail ferredoxins are also presented.

Amino Acid Sequence↗
PubMed1980

Complex-forming properties of spinach NADP+ reductase with ferredoxin, ferrocyanide and NADP+.

The flavoprotein NADP+ reductase from spinach chloroplasts may form a ternary complex with one molecule of NADP+ and one molecule of ferredoxin. Spectroscopic titration studies show that the NADP+ binding site and the ferredoxin binding site are totally independent, that is previous binding of ferredoxin does not modify binding of NADP+, and conversely. Since NADP+ reductase conditions the diaphorase reaction, that is an electron transfer between NADPH and various acceptors such as ferricyanide, the binding of ferrocyanide and its possible interaction with NADP+ and ferredoxin has been studied. Ferrocyanide behaves as a competitive inhibitor with respect to both NADP+ and ferredoxin. This seems paradoxical since NADP+ and ferredoxin are independently bound at two different non-overlapping sites of the flavoprotein. This apparent paradox may be resolved by a theoretical analysis of the interactions between either ferrocyanide and NADP+, or ferrocyanide and ferredoxin. Theory shows that if ferrocyanide is non-specifically bound at two independent sites, namely the NADP+ and the ferredoxin binding sites, it appears competitive with respect to both NADP+ and ferredoxin, although ternary flavoprotein-ferredoxin-ferrocyanide and flavoprotein-NADP+-ferrocyanide complexes are formed. The binding constants of NADP+, ferredoxin and ferrocyanide for the enzyme have been determined. These results are discussed in connection with the possible mechanism of the diaphorase reaction.

Allosteric Regulation↗
PubMed1977

Direct assignment of the cysteinyl, the slowly exchangeable, and the aromatic ring 1H nuclear magnetic resonances in clostridial-type ferredoxins.

We have directly assigned the 1H NMR corresponding to the cysteinyl protons, the slowly exchangeable protons, and the aromatic ring protons in the 1H NMR spectrum of Clostridium acidi-urici ferredoxin by isotopic labeling and 13C NMR decoupling techniques. We also show that the resonance pattern in the 8- to 20-ppm (from 2,2-dimethyl-2-sialapentanesulfonic acid) region of the 1H NMR spectra of oxidized Clostridium acidi-urici, Clostridium pasteurianum, Clostridium perfringens, and Peptococcus aerogenes ferredoxins are very similar, and we assign the resonances in this region by analogy with the spectrum of C. acidi-urici ferredoxin. The 1H NMR spectra of the beta protons of the cysteinyl residues of these ferredoxins differ, however, from the 1H NMR spectra of equivalent beta protons of the methylene carbon atoms bonded via a sulfur atom to [4Fe-4S] clusters in synthetic inorganic analogues. In the spectra of the synthetic compounds, the beta protons appear as a single resonance shifted 10 ppm from its unbonded reference position. In the spectra of oxidized clostridial ferredoxins, the cysteinyl beta protons appear as a series of at least eight resolved resonances with shifts that range from 6 to 14 ppm, relative to the free amino acid resonance position. This difference in the spectra of the protein and the synthetic compounds probably results from the fact that the equivalent beta protons of the synthetic compounds are not constrained and are free to rotate and thus assume the same average orientation with respect to the [4Fe-4S] cluster. The shift pattern in the 9- to 14-ppm region is identical in three different clostridial ferredoxins. This suggests that the molecular environments of the corresponding cysteinyl residues are identical. Significant differences in the resonance positions occur, however, in the 14- to 18-ppm region, suggesting that the physical environments of these cysteinyl residues differ. This may reflect differences in the orientation of the corresponding cysteinyl residues relative to the [4Fe-4S] clusters or differences in charge density at the cysteinyl beta protons or both. The slowly exchangeable protons were identified by comparing the 1H NMR spectra of ferredoxins reconstituted in H2O and 2H2O. The remaining resonances in the 8- to 20-ppm region were assigned to each of the 2 tyrosyl residues in C. acidi-urici ferredoxin. This was done by comparing the 1H NMR spectra of C. acidi-urici [(3',5'-2H2)Tyr]ferredoxin and C. acidi-urici [PHE2]ferredoxin with that of C. acidi-urici native ferredoxin.

Amino Acids↗
PubMed1994

Sequence, assembly and evolution of a primordial ferredoxin from Thermotoga maritima.

A gene coding for the ferredoxin of the primordial, strictly anaerobic and hyperthermophilic bacterium Thermotoga maritima was cloned, sequenced and expressed in Escherichia coli. The ferredoxin gene encodes a polypeptide of 60 amino acids that incorporates a single 4Fe-4S cluster. T. maritima ferredoxin expressed in E. coli is a heat-stable, monomeric protein, the spectroscopic properties of which show that its 4Fe-4S cluster is correctly assembled within the mesophilic host, and that it remains stable during purification under aerobic conditions. Removal of the iron-sulfur cluster results in an apo-ferredoxin that has no detectable secondary structure. This observation indicates that in vivo formation of the ferredoxin structure is coupled to the insertion of the iron-sulfur cluster into the polypeptide chain. Sequence comparison of T. maritima ferredoxin with other 4Fe-4S ferredoxins revealed high sequence identities (75% and 50% respectively) to the ferredoxins from the hyperthermophilic members of the Archaea, Thermococcus litoralis and Pyrococcus furiosus. The high sequence similarity supports a close relationship between these extreme thermophilic organisms from different phylogenetic domains and suggests that ferredoxins with a single 4Fe-4S cluster are the primordial representatives of the whole protein family. This observation suggests a new model for the evolution of ferredoxins.

Amino Acid Sequence↗
PubMed1995

Spinach ferredoxin i: overproduction in Escherichia coli and purification.

Ferredoxin I is the most abundant form of photosynthetic-type ferredoxin present in spinach chloroplasts. A cDNA clone encoding the precursor of spinach ferredoxin I has been engineered to synthesize the mature form of the plant protein in Escherichia coli. Among several different plasmid constructions, the expression system based on phage T7 promoter (vector pET-11d) was found to be the most efficient for spinach ferredoxin overproduction. Upon induction, ferredoxin I accounted for about 2.5% of soluble E. coli protein. A rapid procedure for the purification of the recombinant protein, which yielded at least 1 mg of homogeneous ferredoxin I per gram of cells (fresh wt), was developed. The recombinant protein was found to be identical to ferredoxin I isolated from spinach, both by mass spectrometry analysis and by N-terminal protein sequencing, indicating in vivo removal of the N-terminal methionine. Ferredoxin I was synthesized as the holoprotein, correctly assembled with the [2Fe-2S] cluster as judged by its absorption spectrum, and was fully active in the assay with its physiological partner (ferredoxin-NADP+ reductase). The expression system described here is amenable to the structure-function relationship study of spinach ferredoxin I through site-directed mutagenesis and NMR spectroscopy.

Amino Acid Sequence↗
PubMed2005

Chemical modification studies of tryptophan, arginine and lysine residues in maize chloroplast ferredoxin:sulfite oxidoreductase.

The ferredoxin-dependent sulfite reductase from maize was treated, in separate experiments, with three different covalent modifiers of specific amino acid side chains. Treatment with the tryptophan-modifying reagent, N-bromosuccinimide (NBS), resulted in a loss of enzymatic activity with both the physiological donor for the enzyme, reduced ferredoxin, and with reduced methyl viologen, a non-physiological electron donor. Formation of the 1:1 ferredoxin/sulfite reductase complex prior to treating the enzyme with NBS completely protected the enzyme against the loss of both activities. Neither the secondary structure, nor the oxidation-reduction midpoint potential (Em) values of the siroheme and [4Fe-4S] cluster prosthetic groups of sulfite reductase, nor the binding affinity of the enzyme for ferredoxin were affected by NBS treatment. Treatment of sulfite reductase with the lysine-modifying reagent, N-acetylsuccinimide, inhibited the ferredoxin-linked activity of the enzyme without inhibiting the methyl viologen-linked activity. Complex formation with ferredoxin protects the enzyme against the inhibition of ferredoxin-linked activity produced by treatment with N-acetylsuccinimide. Treatment of sulfite reductase with N-acetylsuccinimide also decreased the binding affinity of the enzyme for ferredoxin. Treatment of sulfite reductase with the arginine-modifying reagent, phenylglyoxal, inhibited both the ferredoxin-linked and methyl viologen-linked activities of the enzyme but had a significantly greater effect on the ferredoxin-dependent activity than on the reduced methyl viologen-linked activity. The effects of these three inhibitory treatments are consistent with a possible role for a tryptophan residue the catalytic mechanism of sulfite reductase and for lysine and arginine residues at the ferredoxin-binding site of the enzyme.

Acetylation↗
PubMed1993

Characterization of the ferredoxin gene transcripts in bovine liver and brain.

1. To study the expression of a ferredoxin gene in extra-adrenocortical tissues, the amounts and structures of the ferredoxin gene transcripts in bovine liver and brain were studied and compared to those of that in adrenocortex. 2. The sizes and amounts of the ferredoxin mRNAs were analyzed by means of Northern blotting, RNA slot blotting and RT-PCR. 3. The nucleotide sequences were determined for 39 ferredoxin cDNA clones from bovine liver. 4. The results indicated that the sizes of the ferredoxin mRNAs in liver and brain were the same as that in adrenocortex, however, their amounts were approx 1/30th and less than the latter, respectively. 5. Although the multiple forms of ferredoxin cDNA, differing in the poly(A) addition sites, were also found in liver, a minor form of ferredoxin cDNA, produced through alternative promoter usage and splicing, could not be detected in liver. 6. The nucleotide sequences of all hepato-ferredoxin cDNA clones obtained were identical to that of a major type of adreno-ferredoxin. 7. These results showed that the expression level of the ferredoxin gene in different tissues was controlled by the amount of mRNA.

Adrenal Cortex↗
PubMed2001

Metabolic activation of mitomycin C by NADPH-ferredoxin reductase in vitro.

Mammalian NADPH-ferredoxin reductase (EC 1.18.1.2) functions in the mitochondrial electron transport chain for cytochrome P-450-dependent steroid hydroxylation. Significant homology of three-dimensional structure exists in the surroundings of FAD between NADPH-ferredoxin reductase and NADH-cytochrome b5 reductase. The latter is involved in the bioreduction of mitomycin C (MC), a prototype antitumor agent. In this study, we assessed the capacity of NADPH-ferredoxin reductase to activate MC. Mitomycin C increased the NADPH oxidase activity of NADPH-ferredoxin reductase. In the absence of ferredoxin, the Km value of NADPH-ferredoxin reductase for MC was 73.5 +/- 2.3 microM. While in the presence of 500 nM ferredoxin, a Lineweaver-Burk plot exhibited a biphasic curve. NADPH-ferredoxin reductase-mediated reduction of MC resulted in the formation of an alkylated complex of 4-(p-nitrobenzyl) pyridine and an increase in plasmide DNA single-strand breaks under hypoxic conditions. With the addition of 500 nM ferredoxin, the amount of the alkylated complex of 4-(p-nitrobenzyl) pyridine and the plasmide DNA single-strand breaks increased by 40% and 37%, respectively. However, neither alkylated complex of 4-(p-nitrobenzyl) pyridine nor DNA strand breaks was observed in the presence of SOD and catalase under aerobic conditions. These findings demonstrate that NADPH-ferredoxin reductase is capable of catalyzing the bioactivation of mitomycin C under hypoxic conditions in vitro.

Alkylation↗
PubMed1989

Laser flash photolysis studies of the kinetics of reduction of spinach and Clostridium ferredoxins by a viologen analogue: electrostatically controlled nonproductive complex formation and differential reactivity among the iron-sulfur clusters.

We have studied the transient kinetics of electron transfer from a positively charged viologen analogue (propylene diquat), reduced by pulsed laser excitation of the deazariboflavin/EDTA system, to the net negatively charged ferredoxins from spinach and Clostridium pasteurianum. Spinach ferredoxin showed monophasic kinetics over the ionic strength range studied, consistent with the presence of only a single iron-sulfur center. Clostridium ferredoxin at low ionic strength showed biphasic kinetics, which indicates a differential reactivity of the two iron-sulfur centers of this molecule toward the electron donor. The kobsd values for the initial fast phase observed with Clostridium ferredoxin were ionic strength dependent, whereas the slow-phase kinetics were ionic strength independent. This correlates with the highly asymmetric charge distribution on the surface of the bacterial protein relative to the two iron-sulfur clusters. The kinetics corresponding to spinach ferredoxin reduction were also ionic strength dependent, and the results obtained with these kinetics and with the fast phase of the bacterial ferredoxin reduction were consistent with a mechanism involving electrostatically stabilized complex formation. For spinach ferredoxin, the second-order rate constant extrapolated to infinite ionic strength was 2-fold smaller, and the extrapolated limiting first-order rate constant was 10-fold smaller, than for Clostridium ferredoxin, indicating a smaller intrinsic reactivity of the spinach protein toward the electron donor. Differences in the rate constant values and the ionic strength dependencies with both ferredoxins are consistent with differences in cluster structure and environment and protein size and charge distribution. For both proteins, the total amount of ferredoxin reduced increased with the ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)

Clostridium↗
PubMed2002

Properties of the recombinant ferredoxin-dependent glutamate synthase of Synechocystis PCC6803. Comparison with the Azospirillum brasilense NADPH-dependent enzyme and its isolated alpha subunit.

The properties of the recombinant ferredoxin-dependent glutamate synthase of Synechocystis PCC6803 were determined by means of kinetic and spectroscopic approaches in comparison to those exhibited by the bacterial NADPH-dependent enzyme form. The ferredoxin-dependent enzyme was found to be similar to the bacterial glutamate synthase alpha subunit with respect to cofactor content (one FMN cofactor and one [3Fe-4S] cluster per enzyme subunit), overall absorbance properties, and reactivity of the FMN N(5) position with sulfite, as expected from the similar primary structure of ferredoxin-dependent glutamate synthase and of the bacterial NADPH-dependent glutamate synthase alpha subunit. The ferredoxin- and NADPH-dependent enzymes were found to differ with respect to the apparent midpoint potential values of the FMN cofactor and of the [3Fe-4S] cluster, which are less negative in the ferredoxin-dependent enzyme form. This feature is, at least in part, responsible for the efficient oxidation of L-glutamate catalyzed by this enzyme form, but not by the bacterial NADPH-dependent counterpart. At variance with earlier reports on ferredoxin-dependent glutamate synthase, in the Synechocystis enzyme the [3Fe-4S] cluster is not equipotential with the flavin cofactor. The present studies also demonstrated that binding of reduced ferredoxin to ferredoxin-dependent glutamate synthase is essential in order to activate reaction steps such as glutamine binding, hydrolysis, or ammonia transfer from the glutamine amidotransferase site to the glutamate synthase site of the enzyme. Thus, ferredoxin-dependent glutamate synthase seems to control and coordinate catalytic activities taking place at its subsites by regulating the reactions of the glutamine amidotransferase site. Association with reduced ferredoxin appears to be necessary, but not sufficient, to trigger the required activating conformational changes.

Amino Acid Oxidoreductases↗
PubMed1996

Human ferredoxin: overproduction in Escherichia coli, reconstitution in vitro, and spectroscopic studies of iron-sulfur cluster ligand cysteine-to-serine mutants.

Human ferredoxin, the human equivalent of bovine adrenodoxin, is a small iron-sulfur protein with one [2Fe-2S] cluster. It functions, as do other vertebrate ferredoxins, to transfer electrons during the processes of steroid hormone synthesis. A DNA fragment encoding the mature form of human ferredoxin was cloned into an expression vector under control of the T7 RNA polymerase/promoter system. The protein was overproduced in Escherichia coli, and the [2Fe-2S] cluster was incorporated into the protein by in vitro reconstitution. The overall yield was approximately 30 mg of purified, reconstituted ferredoxin per liter of culture. Four of the five cysteines in human ferredoxin are coordinated to the iron-sulfur cluster. First, the non-ligand cysteine (cysteine-95) was mutated to alanine, and then double mutants were created in which each of the other four cysteines (at positions 46, 52, 55, and 92) were mutated individually to serine. The wild-type ferredoxin and each of the five mutant proteins were studied by UV-visible spectroscopy and electron paramagnetic resonance spectroscopy. The EPR gav values of all five mutants were very similar to that of wild-type human ferredoxin. In the reduced state, three of the cysteine-to-serine mutants exhibited axial EPR spectra similar to that of wild-type, but one of the double mutants (C52S/C95A) exhibited a rhombic EPR spectrum. The UV-visible spectroscopic properties of the wild-type and the C95A mutant ferredoxins were identical, but those of the other cysteine-to-serine mutant proteins of human ferredoxin were quite different from those of the wild-type protein and each other. These results, along with those from cysteine-to-serine mutations in other ferredoxins, provide the basis for a more comprehensive theoretical and practical understanding of the features important to the ligation of [2Fe-2S] clusters, although they do not yet permit determination of which two cysteines ligate Fe(II) and which ligate Fe(III) in the reduced protein.

Amino Acid Sequence↗