Cloning, sequencing, and overexpression of a [2Fe-2S] ferredoxin gene from Escherichia coli.

Escherichia coli contains a soluble, [2Fe-2S] ferredoxin of unknown function (Knoell, H.-E., and Knappe, J. (1974) Eur. J. Biochem. 50, 245-252). Using antiserum to the purified protein to screen E. coli genomic expression libraries, we have cloned a gene (designated fdx) encoding this protein. The DNA sequence of the gene predicts a polypeptide of 110 residues after removal of the initiator methionine (polypeptide M(r) = 12,186, holoprotein M(r) = 12,358). The deduced amino acid sequence is strikingly similar to those of the ferredoxins found in animal mitochondria which function with cytochrome P450 enzymes and to the ferredoxin from Pseudomonas putida which functions with P450cam. The overall sequence identity is approximately 36% when compared with human mitochondrial and P. putida ferredoxins, and the identities include 4 cysteine residues proposed to coordinate the iron cluster. The protein was overproduced approximately 500-fold using an expression plasmid, and the holoprotein was assembled and accumulated in amounts exceeding 30% of the total cell protein. The overexpressed ferredoxin exhibits absorption, circular dichroism, and electron paramagnetic resonance spectra closely resembling those of the animal ferredoxins and P. putida ferredoxin.     

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.     

An investigation of the iron-sulphur proteins of benzene dioxygenase from Pseudomonas putida by electron-spin-resonance spectroscopy.

Benzene dioxygenase from Pseudomonas putida comprises three components, namely a flavoprotein (NADH:ferredoxin oxidoreductase; Mr 81000), an intermediate electron-transfer protein, or ferredoxin (Mr 12000) with a [2Fe-2S] cluster, and a terminal dioxygenase containing two [2Fe-2S] iron-sulphur clusters (Mr 215000), which requires two additional Fe2+ atoms/molecule for oxygenase activity. The ferredoxin and the dioxygenase give e.s.r. signals in the reduced state with rhombic symmetry and average g values of 1.92 and 1.896 respectively. The mid-point redox potentials were determined by e.s.r. titration at pH 7.0 to be -155 mV and -112 mV respectively. The signal from the dioxygenase shows pronounced g anisotropy and most closely resembles those of 4-methoxybenzoate mono-oxygenase from Pseudomonas putida and the [2Fe-2S] 'Rieske' proteins of the quinone-cytochrome c region of electron-transport chains of respiration and photosynthesis.     

Purification, characterization, and crystallization of the components of the nitrobenzene and 2-nitrotoluene dioxygenase enzyme systems

The protein components of the 2-nitrotoluene (2NT) and nitrobenzene dioxygenase enzyme systems from Acidovorax sp. strain JS42 and Comamonas sp. strain JS765, respectively, were purified and characterized. These enzymes catalyze the initial step in the degradation of 2-nitrotoluene and nitrobenzene. The identical shared reductase and ferredoxin components were monomers of 35 and 11.5 kDa, respectively. The reductase component contained 1.86 g-atoms iron, 2.01 g-atoms sulfur, and one molecule of flavin adenine dinucleotide per monomer. Spectral properties of the reductase indicated the presence of a plant-type [2Fe-2S] center and a flavin. The reductase catalyzed the reduction of cytochrome c, ferricyanide, and 2,6-dichlorophenol indophenol. The ferredoxin contained 2.20 g-atoms iron and 1.99 g-atoms sulfur per monomer and had spectral properties indicative of a Rieske [2Fe-2S] center. The ferredoxin component could be effectively replaced by the ferredoxin from the Pseudomonas sp. strain NCIB 9816-4 naphthalene dioxygenase system but not by that from the Burkholderia sp. strain LB400 biphenyl or Pseudomonas putida F1 toluene dioxygenase system. The oxygenases from the 2-nitrotoluene and nitrobenzene dioxygenase systems each had spectral properties indicating the presence of a Rieske [2Fe-2S] center, and the subunit composition of each oxygenase was an alpha(3)beta(3) hexamer. The apparent K(m) of 2-nitrotoluene dioxygenase for 2NT was 20 muM, and that for naphthalene was 121 muM. The specificity constants were 7.0 muM(-1) min(-1) for 2NT and 1.2 muM(-1) min(-1) for naphthalene, indicating that the enzyme is more efficient with 2NT as a substrate. Diffraction-quality crystals of the two oxygenases were obtained.     

Crystal structure of NADH-dependent ferredoxin reductase component in biphenyl dioxygenase

Oxidative biodegradation of aromatic compounds by bacteria usually begins with hydroxylation of the aromatic ring by multi-component dioxygenases like benzene dioxygenase, biphenyl dioxygenase, and others. These enzymes are composed of ferredoxin reductase, ferredoxin, and terminal oxygenase. Reducing equivalents that originate from NADH are transferred from ferredoxin reductase to ferredoxin and, in turn, to the terminal oxygenase, thus resulting in the activation of a dioxygen. BphA4 is the ferredoxin reductase component of biphenyl dioxygenase from Pseudomonas sp. strain KKS102. The amino acid sequence of BphA4 exhibits significant homology with the putidaredoxin reductase of the cytochrome P450cam system in Pseudomonas putida, as well as with various other oxygenase-coupled NADH-dependent ferredoxin reductases (ONFRs) of bacteria. To date, no structural information has been provided for the ferredoxin reductase component of the dioxygenase systems. In order to provide a structural basis for discussing the mechanism of electron transport between ferredoxin reductase and ferredoxin, crystal structures of BphA4 and its NADH complex were solved. The three-dimensional structure of BphA4 is different from those of ferredoxin reductases whose structures have already been determined, but adopts essentially the same fold as the enzymes of the glutathione reductase (GR) family. Also the three-dimensional structure of the first two domains of BphA4 adopts a fold similar to that of adrenodoxin reductase (AdR) in the mitochondrial cytochrome P450 system. Comparing the amino acid sequence with what is known of the three-dimensional structure of BphA4 strongly suggests that the other ONFRs have secondary structural features that are similar to that of BphA4. This analysis of the crystal structures of BphA4 suggests that Lys53 and Glu159 seem to be involved in the hydride transfer from NADH to FAD. Since the amino acid residues around the active site, some of which seem to be important to electron transport, are highly conserved among ONFRs, it is likely that the mechanism of electron transport of BphA4 is quite applicable to other ONFRs.     

NMR studies of Azotobacter vinelandii and Pseudomonas putida seven-iron ferredoxins. Direct assignment of beta-cysteinyl carbon NMR resonances and further proton NMR assignments of cysteinyl and aromatic resonances.

Ferredoxins are proteins which contain iron and inorganic sulfide and are capable of electron transport. They are found in a wide range of organisms, from anaerobic bacteria, to plants and mammals. Although NMR spectroscopy has been used to study ferredoxins since the 1970s, little important structural or biochemical information has resulted from these investigations. The major difficulty has been the effect of the paramagnetic iron-sulfur clusters on the peptide resonances, hindering nuclear Overhauser effect (NOE) studies and causing broad line widths. These effects are most pronounced on resonances arising from the nuclei closest to the iron-sulfur center. Unfortunately, these are likely to be the most interesting nuclei, as they report the events and geometry in the vicinity of the active sites. In this paper, the first direct assignment of beta-cysteinyl 13C resonances for any iron-sulfur protein is reported for the spectrum of Pseudomonas putida ferredoxin. These resonances are of special significance, as they arise from the atoms on the protein closest to the iron centers, with the exception of the directly bound cysteinyl sulfur atoms. In addition, cysteinyl and ring system 1H NMR resonance assignments are made for the spectra of P. putida ferredoxin and Azotobacter vinelandii ferredoxin I.     

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

The [2Fe-2S] ferredoxin from Clostridium pasteurianum had previously been shown to interact specifically with the nitrogenase MoFe protein, and electrostatic forces were found to be important contributors to the interaction. This phenomenon has now been analyzed in detail by using ferredoxin variants in which charge inversions or cancellations were introduced on all charged residues. The mutated forms of the ferredoxin were covalently cross-linked to the MoFe protein. The reaction products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and their nitrogenase activity was measured. The latter displayed a consistent inverse correlation with the amount of cross-linked MoFe protein. The data allowed an unambiguous identification of the ferredoxin residues (glutamates 31, 34, 38, 39, 84, 85) that are involved in the interaction with the MoFe protein. Furthermore, whereas the wild-type ferredoxin yielded approximately equal amounts of cross-linked products with the alpha and beta subunits of the MoFe protein, some of its molecular variants displayed a differential decrease of reactivity towards one or the other of these subunits. The positions on the ferredoxin molecule of the residues interacting with the MoFe protein were determined using the recently elucidated crystal structure of the homologous [2Fe-2S] ferredoxin from Aquifex aeolicus.     

Comparison of the spin-lattice relaxation properties of the two classes of [2Fe-2S] clusters in proteins

Two classes of [2Fe-2S] proteins have been defined according to the mean value gav of their g tensor components (Bertrand, P., Guigliarelli, B., Gayda, J.P., Beardwood, P. and Gibson, J.F. (1985) Biochim. Biophys. Acta 831, 261-266). To characterize their magnetic properties better, we have compared the spin-lattice relaxation behavior of typical proteins which belong to these two classes, namely Spirulina maxima and adrenal ferredoxin for the gav approximately 1.96 class, Thermus thermophilus Rieske protein and Pseudomonas putida benzene dioxygenase for the gav approximately 1.91 class. For all these proteins, the data support the existence of an efficient Orbach process in the highest temperature range, which allows the determination of the exchange coupling parameter, J. From the comparison of the J values obtained in each class, it is concluded that the structural factors which determine the value of the g tensor and the strength of the antiferromagnetic exchange interactions are different.     

The downfield resonances in the 1H NMR spectra of Azotobacter vinelandii and Pseudomonas putida seven-iron ferredoxins

Pseudomonas putida and Azotobacter vinelandii ferredoxins each contain one [4Fe-4S] cluster and one [3Fe-4S] cluster. Their polypeptide chains are nearly identical, differing by only 15 residues out of a total of 106. T1 measurements and temperature dependence studies of the 1H NMR spectrum of each ferredoxin demonstrate that all six resolved downfield resonances are near an iron-sulfur center. The five most downfield resonances are shown to arise from protons on cysteinyl beta-carbons by incorporation of cysteine deuterated at the beta-carbon into cell protein. The sixth peak (10.5 ppm) is shown to be a non-cysteinyl proton. This peak resolves into two resonances of approximately equal intensity at temperatures below 15 degrees or above 25 degrees C. A nuclear Overhauser effect observed between the two downfield-most resonances of A. vinelandii ferredoxin indicates that they originate from a geminal pair of beta-cysteinyl protons. An Overhauser effect observed between the resonances at 22.3 and 15.7 ppm, in conjunction with other results, implies that the resonance at 22.3 ppm arises from a beta-proton on the 3Fe-center-bound Cys16, while the resonance at 15.7 ppm arises from Cys45 beta-proton, which is bound to the 4Fe center. The five most downfield resonances are pH-dependent. The sixth peak (10.5 ppm in P. putida ferredoxin) is pH-independent. Possible origins for the observed pH dependencies are discussed.     

NMR spectroscopic studies of the hydrogenosomal [2Fe-2S] ferredoxin from Trichomonas vaginalis: hyperfine-shifted 1H resonances

The hyperfine-shifted 1H NMR resonances of oxidized and reduced Trichomonas vaginalis ferredoxin, a functionally unique [2Fe-2S] ferredoxin, have been studied. The oxidized protein spectrum displayed a pattern of six broad upfield-shifted resonances between 13 and 40 ppm with chemical shifts distinct from those of other [2Fe-2S] ferredoxins. All hyperfine 1H resonances of the oxidized ferredoxin displayed anti-Curie temperature dependences. Reduced T. vaginalis ferredoxin displayed hyperfine resonances both upfield and downfield of the diamagnetic region. These resonances showed Curie temperature dependences. Overall the hyperfine-shifted NMR spectrum of T. vaginalis ferredoxin, along with other spectroscopic properties, suggested different structural properties for the active center of oxidized hydrogenosomal ferredoxins from those of other [2Fe-2S] ferredoxins.     

Oxidative release of nitrite from 2-nitrotoluene by a three-component enzyme system from Pseudomonas sp. strain JS42.

Pseudomonas sp. strain JS42 utilizes 2-nitrotoluene (2NT) as the sole source of carbon and energy for growth. Intact cells catalyze the oxidation of 2NT to 3-methylcatechol and nitrite in a reaction that requires molecular oxygen. Cell extracts oxidized 2NT to 3-methylcatechol and nitrite in the presence of NAD(P)H and ferrous iron. Ion-exchange chromatography yielded three protein fractions (A, B, and C) which were all required for the oxidation of 2NT to 3-methylcatechol and nitrite. Component B (reductase2NT) catalyzed a NAD(P)H-dependent reduction of cytochrome c. Solutions of component A (ISP2NT) were brown and showed absorption maxima at 458 and 324 nm. Two major bands with M(r)s 52,500 and 28,000 were observed when ISP2NT was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Component C could be replaced by ferredoxin NAP from the Pseudomonas putida NCIB 9816-4 naphthalene dioxygenase system and was given the designation ferredoxin2NT. Experiments with 18O2 showed that both oxygen atoms were added to the aromatic ring of 2NT to yield 3-methylcatechol. The enzyme is a new multicomponent enzyme system which we have designated 2NT 2,3-dioxygenase.     

Gene components responsible for discrete substrate specificity in the metabolism of biphenyl (bph operon) and toluene (tod operon).

bph operons coding for biphenyl-polychlorinated biphenyl degradation in Pseudomonas pseudoalcaligenes KF707 and Pseudomonas putida KF715 and tod operons coding for toluene-benzene metabolism in P. putida F1 are very similar in gene organization as well as size and homology of the corresponding enzymes (G. J. Zylstra and D. T. Gibson, J. Biol. Chem. 264:14940-14946, 1989; K. Taira, J. Hirose, S. Hayashida, and K. Furukawa, J. Biol. Chem. 267:4844-4853, 1992), despite their discrete substrate ranges for metabolism. The gene components responsible for substrate specificity between the bph and tod operons were investigated. The large subunit of the terminal dioxygenase (encoded by bphA1 and todC1) and the ring meta-cleavage compound hydrolase (bphD and todF) were critical for their discrete metabolic specificities, as shown by the following results. (i) Introduction of todC1C2 (coding for the large and small subunits of the terminal dioxygenase in toluene metabolism) or even only todC1 into biphenyl-utilizing P. pseudoalcaligenes KF707 and P. putida KF715 allowed them to grow on toluene-benzene by coupling with the lower benzoate meta-cleavage pathway. Introduction of the bphD gene (coding for 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase) into toluene-utilizing P. putida F1 permitted growth on biphenyl. (ii) With various bph and tod mutant strains, it was shown that enzyme components of ferredoxin (encoded by bphA3 and todB), ferredoxin reductase (bphA4 and todA), and dihydrodiol dehydrogenase (bphB and todD) were complementary with one another. (iii) Escherichia coli cells carrying a hybrid gene cluster of todClbphA2A3A4BC (constructed by replacing bphA1 with todC1) converted toluene to a ring meta-cleavage 2-hydroxy-6-oxo-hepta-2,4-dienoic acid, indicating that TodC1 formed a functional multicomponent dioxygenase associated with BphA2 (a small subunit of the terminal dioxygenase in biphenyl metabolism), BphA3, and BphA4.     

Cytochrome P450 alkane hydroxylases of the CYP153 family are common in alkane-degrading eubacteria lacking integral membrane alkane hydroxylases

Several strains that grow on medium-chain-length alkanes and catalyze interesting hydroxylation and epoxidation reactions do not possess integral membrane nonheme iron alkane hydroxylases. Using PCR, we show that most of these strains possess enzymes related to CYP153A1 and CYP153A6, cytochrome P450 enzymes that were characterized as alkane hydroxylases. A vector for the polycistronic coexpression of individual CYP153 genes with a ferredoxin gene and a ferredoxin reductase gene was constructed. Seven of the 11 CYP153 genes tested allowed Pseudomonas putida GPo12 recombinants to grow well on alkanes, providing evidence that the newly cloned P450s are indeed alkane hydroxylases.     

Expression of human ferredoxin in Saccharomyces cerevisiae: mitochondrial import of the protein and assembly of the [2Fe-2S] center.

Vertebrate ferredoxins function in the transfer of reducing equivalents from NADPH:ferredoxin oxidoreductase to cytochrome P450 enzymes involved in steroid metabolism. We report here the expression of human mitochondrial ferredoxin in the yeast Saccharomyces cerevisiae. The full-length ferredoxin protein containing the ferredoxin mitochondrial leader sequence could not be stably expressed in S. cerevisiae, but a fusion protein consisting of the mature portion of ferredoxin linked to the mitochondrial leader sequence of the S. cerevisiae cytochrome c oxidase subunit Va protein (COX5a) could be stably expressed. The COX5a:ferredoxin fusion protein was targeted to the mitochondria as a preprotein and was cleaved at the normal processing site of the COX5a presequence during import into the matrix. Absorption spectra and electron transfer activity of the isolated fusion protein established that the [2Fe-2S] center was correctly assembled and incorporated into the recombinant ferredoxin in this heterologous system.     

Crystallization and preliminary analysis of oxidized, recombinant, heterocyst [2Fe-2S] ferredoxin from Anabaena 7120.

The [2Fe-2S] ferredoxin produced in the heterocyst cells of Anabaena 7120 plays a key role in nitrogen fixation, where it serves as an electron acceptor from various sources and an electron donor to nitrogenase. Crystals of recombinant heterocyst ferredoxin, coded for by the fdx H gene from Anabaena 7120 and overproduced in Escherichia coli, have been grown from ammonium sulfate solutions and are suitable for high resolution X-ray crystallographic analysis. They belong to the hexagonal space group P6(1) or P6(5) with unit cell dimensions of a = b = 44.2 A and c = 80.6 A. The crystals contain one molecule per asymmetric unit and diffract to a nominal resolution of 1.6 A. The molecular structure of this heterocyst ferredoxin is of special interest in that 4 of the 22 amino acid positions thought to be absolutely conserved in nonhalophilic ferredoxins are different and, based on amino acid sequence alignments, three of these positions are located in the metal-cluster binding loop. Consequently, a high-resolution X-ray analysis of this [2Fe-2S] ferredoxin, and subsequent three-dimensional comparisons with other known ferredoxin models, will provide new insight into structure/function relationships for this class of redox proteins.     

Evidence for the presence of a [2Fe-2S] ferredoxin in bean sprouts

An iron-sulfur protein with properties similar to those of ferredoxins found in the leaves of higher plants has been isolated from bean sprouts--a non-photosynthetic plant tissue. The bean sprout protein has a molecular mass of 12.5 kDa and appears to contain a single [2Fe-2S] cluster. The absorbance and circular dichroism spectra of the bean sprout protein resemble those of spinach leaf ferredoxin and the bean sprout protein can replace spinach ferredoxin as an electron donor for NADP+ reduction, nitrite reduction and thioredoxin reduction by spinach leaf enzymes. Although the reduced bean sprout protein (Em = -440 mV) is a slightly stronger reductant than spinach ferredoxin and appears to be less acidic than spinach ferredoxin, the two proteins are similar enough so that the bean sprout protein is recognized by an antibody raised against spinach ferredoxin.     

Efficient degradation of trichloroethylene by a hybrid aromatic ring dioxygenase.

Engineering of hybrid gene clusters between the toluene metabolic tod operon and the biphenyl metabolic bph operon greatly enhanced the rate of biodegradation of trichloroethylene. Escherichia coli cells carrying a hybrid gene cluster composed of todC1 (the gene encoding the large subunit of toluene terminal dioxygenase in Pseudomonas putida F1), bphA2 (the gene encoding the small subunit of biphenyl terminal dioxygenase in Pseudomonas pseudoalcaligenes KF707), bphA3 (the gene encoding ferredoxin in KF707), and bphA4 (the gene encoding ferredoxin reductase in KF707) degraded trichloroethylene much faster than E. coli cells carrying the original toluene dioxygenase genes (todC1C2BA) or the original biphenyl dioxygenase genes (bphA1A2A3A4).     

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.