A four-iron, four-sulfide ferredoxin with high thermostability from Clostridium thermoaceticum.

A ferredoxin containing only one [Fe4S4] cluster was purified from Clostridium thermoaceticum. It has a molecular weight of about 7,300, a partial specific volume of 0.67, and an isoelectric point of 3.25. Its absorption spectrum has two maxima at 390 nm (epsilon = 16.8 X 10(3)M-1cm-1) and at 280 nm (epsilon = 24.2 X 10(3)M-1cm-1). The absorption at 390 nm is almost half that of other clostridial ferredoxins, which have two [Fe4S4] clusters, and is similar to other ferredoxins with only one [Fe4S4] cluster. The ferredoxin had high thermal stability and retained over 50% of its activity after treatment at 80 degrees C. It functions in the transfer of electrons from pyruvate to nicotinamide adenine dinucleotide phosphate (NADP), which indicates the presence of pyruvate:ferredoxin oxidoreductase and reduced ferredoxin-NADP reductase in C, thermoaceticum. NADPH is used in the synthesis of acetate from CO2 in this organism.     

Isolation and characterization of polyferredoxin from Methanobacterium thermoautotrophicum. The mvhB gene product of the methylviologen-reducing hydrogenase operon.

The methylviologen-reducing hydrogenase operon of Methanobacterium thermoautotrophicum contains an open reading frame, mvhB, the product of which was predicted to have a molecular weight of 44 kDa and to contain as many as 48 iron atoms in 12 [4Fe-4S] clusters, and was therefore suggested to be a polyferredoxin. We have now, for the first time, isolated this polyferredoxin. Its identity with the mvhB gene product was evidenced by a comparison of the N-terminal amino acid sequence. The dark-brown protein of apparent molecular weight 44 kDa was found to contain 53 mol Fe and 43 mol acid-labile sulfur per mol. The UV/visible spectrum showed two maxima at 280 nm and 390 nm, and a shoulder at 308 nm. The A390/A280 ratio was 0.73. The molar extinction coefficient at 390 nm was 170,000 M-1.cm-1. In the dithionite reduced state the protein displayed an EPR spectrum like that of [4Fe-4S] clusters. The results indicate that the mvhB gene product is indeed a polyferredoxin.     

Purification and characterization of a ferredoxin from acetate-grown Methanosarcina thermophila

A ferredoxin, which functions as an electron acceptor for the CO dehydrogenase complex from Methanosarcina thermophila, was purified from acetate-grown cells. It was isolated as a trimer having a native molecular weight of approximately 16,400 and monomer molecular weight of 4,888 calculated from the amino acid composition. The ferredoxin contained 2.80 +/- 0.56 Fe atoms and 1.98 +/- 0.12 acid-labile sulfide. UV-visible absorption maxima were 395 and 295 nm with monomeric extinction coefficients of epsilon 395 = 12,800 M-1 cm-1 and epsilon 295 = 14,460 M-1 cm-1. The A395/A295 ratio ranged from 0.80 to 0.88. There were 5 cysteines per monomer but no methionine, histidine, arginine, or aromatic amino acids. The N-terminal amino acid sequence showed a 4-cysteine cluster with potential to coordinate a Fe:S center. The protein was stable for 30 min at 70 degrees C, but denatured during incubation at 85 degrees C.     

Hydrogenosomal ferredoxin of the anaerobic protozoon, Tritrichomonas foetus

A low molecular weight iron-sulfur protein has been purified from Tritrichomonas foetus by deoxycholate extraction of whole cells, ion exchange chromatography, and gel filtration. The purified protein was essentially homogeneous as judged by isoelectric focusing, polyacrylamide gel electrophoresis, and gel filtration. A pI of 4.3 was observed. The molecular weight of the protein was estimated to be 12,000. Chemical and spectral analysis showed the protein to have a [2Fe-2S] cluster. The absorbance spectrum of the oxidized protein showed maxima at 280, 340, 458 and shoulders at 410 and 550 nm. The maximum observed A458/A280 ratio was 0.82 and the absorbance of the oxidized protein at 458 nm was 8,000 M-1 X cm-1. The low temperature EPR spectrum of the protein reduced with dithionite revealed axial symmetry with features at g values of g = 1.94 and g = 2.02. The oxidized protein gave no EPR signal in the g = 1.8 to 2.2 range. Cell fractionation studies indicated the localization of this protein in the hydrogenosome. The protein was able to function as an electron transport component in the reduction of metronidazole (a 5-nitroimidazole derivative) by pyruvate:ferredoxin oxidoreductase and hydrogenase from T. foetus and also from Trichomonas vaginalis and Clostridium pasteurianum as well as in the reduction of cytochrome c by plant NADPH:ferredoxin oxidoreductase. This protein has the characteristics of a ferredoxin and is likely to be a physiological electron carrier in hydrogenosomal pyruvate oxidation.     

Purification and characterization of a heat stable ferredoxin isolated fromClostridium thermocellum

A thermostable ferredoxin was purified from Clostridium thermocellum. The final preparation was homogeneous as judged by electrophoresis in sodium dodecyl sulfate polyacrylamide gel and sedimentation equilibrium. It contains eight atoms of iron and eight acid-labile sulfur groups per molecule, the molecular weight is estimated to be 6 400 and the isoelectric point 3.35. Its amino-acid composition is characterized by the absence of histidine residues and the presence of eight cysteine residues. The absorption spectrum has a maximum at 390 nm with a molar absorption coefficient of 39 x 10(3) M1 cm-1, similar to that of other bacterial eight iron ferredoxins. The purified ferredoxin has high thermal stability, since the spectrophotometric absorption of the protein at 390 nm did not change after one hour at 70 degrees C and only thirty five per cent of absorbance were lost after one hour at 80 degrees C. With regard to the electron carrier activity, the stability is slightly higher, only twenty five per cent of the activity were lost after one hour at 80 degrees C. During pyruvate oxidation, ferredoxin functions in the transfer of electrons to hydrogenase and also in the back reaction during pyridine nucleotide reduction by a ferredoxin -NAD oxidoreductase using hydrogen as electron donor.     

Isolation, characterization, and biological activity of the Methanococcus thermolithotrophicus ferredoxin.

A ferredoxin has been isolated from the thermophilic methanogen Methanococcus thermolithotrophicus. The native protein was a monomer exhibiting a molecular weight of 7,262, calculated from the amino acid composition. Its absorption spectrum had two maxima at 390 and 283 nm, with an absorbance ratio A390/A283 of 0.79. The absorption at 390 nm (E = 29 mM-1 cm-1) and the content of iron of the protein are in agreement with the presence of two 4Fe-4S clusters in M. thermolithotrophicus ferredoxin. Its amino acid composition showed the presence of eight cysteine residues, which is the required number of cysteines for the binding of two 4Fe-4S clusters. The protein was characterized by the lack of histidine, arginine, and leucine and a high content of valine. It was unusually stable to high temperatures but not to oxygen. The ESR spectrum of the protein in the oxidized state showed a minor signal at g = 2.01, corresponding to an oxidized 3Fe-4S cluster. The protein, which was difficult to reduce with dithionite or reduced mediators, exhibited in its reduced state a spectrum typical of two interacting reduced 4Fe-4S clusters. M. thermolithotrophicus ferredoxin functioned as an electron acceptor for the CO dehydrogenase complex with an extract free of ferredoxin. No reaction was detected with F420 or hydrogenase.     

Purification, amino-acid sequence and some properties of the ferredoxin isolated from Bacillus acidocaldarius

Ferredoxin was isolated from the aerobic, thermophilic and acidophilic bacterium Bacillus acidocaldarius and its sequence of 78 amino acids completely determined by automated Edman degradation of the protein and of peptides derived from chemical cleavage between aspartic acid and proline and from enzymatic digestions. The optical spectrum of the oxidized protein has a broad maximum around 400 nm. The ferredoxin is thermostable: its absorbance begins to decrease only at incubation over 71 degrees C. The number of iron and inorganic sulphur atoms per molecule was determined to be 5.3 and 5.0, respectively. The calculated molar extinction coefficient was 23 000 M-1 X cm-1, the molecular mass of the apoferredoxin 8 872 Da. Contrary to all expectations, the sequence of B. acidocaldarius ferredoxin shows very little homology to that of B. stearothermophilus but closely resembles that of Thermus thermophilus.     

Purification and characterization of a 7Fe-ferredoxin from Rhodobacter capsulatus

A ferredoxin was purified anaerobically from Rhodobacter capsulatus grown photoheterotrophically with excess ammonia. This ferredoxin, called ferredoxin II (FdII), had a molecular weight of approximatively 15,000 by gel filtration and 14,000 by SDS polyacrylamide gel electrophoresis indicating that it is monomeric. Its absorption spectrum (oxidized form) exhibited maxima at 280 nm and 400 nm; the A400/A280 ratio had a calculated value of 0.55. Chemical determination of its iron and sulfur atom content, the value of the extinction coefficient at 400 nm (epsilon 400 = 26.8 mM-1 cm-1) and EPR spectra indicated that ferredoxin II contained one [3Fe-4S] and one [4Fe-4S] cluster. Upon reduction with excess dithionite only the [3Fe-4S] cluster became reduced. The reduction of both clusters was achieved by using 5-deazaflavin as photocatalyst. Ferredoxin II was also purified from bacteria grown under nitrogen limiting (nif derepressing) conditions. In in vitro assays, ferredoxin II catalyzed electron transport between illuminated chloroplasts and nitrogenase.     

Amino acid sequence of [2Fe-2S] ferredoxin from Clostridium pasteurianum

The complete amino acid sequence of the [2Fe-2S] ferredoxin from the saccharolytic anaerobe Clostridium pasteurianum has been determined by automated Edman degradation of the whole protein and of peptides obtained by tryptic and by staphylococcal protease digestion. The polypeptide chain consists of 102 amino acids, including 5 cysteine residues in positions 11, 14, 24, 56, and 60. The sequence has been analyzed for hydrophilicity and for secondary structure predictions. In its native state the protein is a dimer, each subunit containing one [2Fe-2S] cluster, and it has a molecular weight of 23,174, including the four iron and inorganic sulfur atoms. The extinction coefficient of the native protein is 19,400 M-1 cm-1 at 463 nm. The positions of the cysteine residues, four of which are most probably the ligands of the [2Fe-2S] cluster, on the polypeptide chain of this protein are very different from those found in other [2Fe-2S] proteins, and in other ferredoxins in general. In addition, whole sequence comparisons of the [2Fe-2S] ferredoxin from C. pasteurianum with a number of other ferredoxins did not reveal any significant homologies. The likely occurrence of several phylogenetically unrelated ferredoxin families is discussed in the light of these observations.     

Purification and characterization of the ferredoxin component of 25-hydroxycholecalciferol 1 alpha-hydroxylase.

The chick kidney mitochondrial iron--sulphur protein (ferredoxin), a component of the NADPH--cytochrome P-450 reductase functional in the 1 alpha-hydroxylation of 25-hydroxycholecalciferol, was purified to homogeneity by chromatography on DEAE-cellulose, gel filtration on Sephadex G-100 and preparative electrophoresis on polyacrylamide gel. A novel NADPH--cytochrome c reductase assay utilizing crude renal NADPH--ferredoxin reductase was used for the detection of the ferredoxin. A mol. wt. of 53 000 was determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by Sephadex G-100 gel filtration of the 125I-labelled ferredoxin. The ferredoxin has a sedimentation constant (S 20, w) of 2.66S, an A411/A280 of 0.4, and a molar absorptivity of 7300 cm-1 . M-1. The electron-paramagnetic-resonance spectrum after reduction with Methyl Viologen and dithionite was characteristic of ferredoxins with signals at g = 1.956 and 2.025. Two iron and two labile sulphur atoms per molecule of ferredoxin were released by acid. Ouchterlony immunodiffusion tests by using goat anti-(bovine adrenal ferredoxin) antiserum showed precipitin reactions with the bovine adrenal ferredoxin and the chick renal ferredoxin as antigens, suggesting that the renal ferredoxin shares antigenic determinants(s) with the natural adrenal antigen. Amino acid analysis showed that of the total number of residues per molecule of ferredoxin, glutamic acid and aspartic acid are the most abundant residues, comprising 17 and 15% respectively.     

Purification and characterization of a 7Fe ferredoxin from a thermophilic hydrogen-oxidizing bacterium, Bacillus schlegelii.

A ferredoxin (Fd) was purified from a thermophilic hydrogen-oxidizing bacterium, Bacillus schlegelii. This ferredoxin was a monomer with apparent molecular weight of 13,000 and contained 7 mol Fe/mol ferredoxin. The oxidized ferredoxin showed the characteristic EPR spectrum for [3Fe-4S]1+ (1.2 spin/mol Fd). This signal disappeared upon reduction with dithionite and new signals due to [3Fe-4S]0 and [4Fe-4S]1+ (0.7 spin/mol Fd) appeared. The quantitation of EPR signals and the iron content reveal that B. schlegelii ferredoxin contains one [3Fe-4S]1+/0 and one [4Fe-4S]2+/1+ cluster. The ferredoxin has the characteristic distribution of cysteines (-Cys8-X7-Cys16-X3-Cys20-Pro-) for 7Fe ferredoxins in the N-terminus.     

Physicochemical properties of cloned nucleocapsid protein from HIV. Interactions with metal ions

The nucleocapsid (NC) protein (p15) of the human immunodeficiency virus (HIV) has been cloned and overproduced (under the control of a phage T7 promoter) in soluble form in an Escherichia coli host. The soluble NC protein is a fusion protein containing 15 amino acids from the T7 gene 10 and 7 amino acids from the HIV p24 protein at the N-terminus to make a protein of 171 amino acids. The plasmid containing the fusion gene is designated p15DF. A homogeneous product has been isolated from the induced cells and, when isolated under aerobic conditions, contains 0.3-0.5 mol of Zn/mol of protein and has only 2 titratable SH groups. Reduction and refolding in the presence of Zn(II) yields a protein containing 2.0 mol of Zn/mol of protein and 6 titratable SH groups. On the other hand, if the cells are sonicated in 2 mM CdCl2 and purified at pH 5.0, an unoxidized protein containing 2 mol of Cd/mol of protein is obtained. The Cd(II) ions can be exchanged with Zn(II), Co(II), or 113Cd(II). The Co(II)2 NC protein shows d-d electronic transitions at 695 nm [epsilon = 675 M-1 cm-1 per Co(II)] and 640 nm [epsilon = 825 M-1 cm-1 per Co(II)] compatible with regular tetrahedral geometry around both Co(II) ions. The Co(II)2 and Cd(II)2 NC proteins show intense charge-transfer bands in the near-UV, at 355 nm (epsilon = approximately 4000 M-1 cm-1) and 310 nm (epsilon = approximately 8000 M-1 cm-1) for the Co(II) protein and 255 nm (epsilon = approximately 10(4) M-1 cm-1) for the Cd(II)2 NC protein, compatible with -S- coordination. 113Cd NMR of the 113Cd(II)2 NC protein shows two 113Cd NMR signals at 659 and 640 ppm, respectively, each integrating to approximately 1 Cd(II) ion. The downfield chemical shifts suggest coordination of each 113Cd(II) ion to 3 sulfur donor atoms. The spectroscopic data fully support the prediction that the NC protein binds metal ions to each of the tandem repeats of the -Cys-X2-Cys-X4-His-X4-Cys- sequence contained in the N-terminal half of the molecule. 113Cd NMR shows, however, that the sites are not identical. Isolation of the NC protein under standard aerobic conditions results in oxidation of the sulfhydryl groups and loss of the coordinated Zn(II) ions, while preparation of the NC protein as the Cd(II) derivative at low pH protects the sulfhydryl groups from oxidation.     

FA/FB Protein from the Spinach Photosystem I Complex: Isolation in a Native State and Some Properties of the Iron-Sulfur Clusters

The F_A/F_B protein of the photosystem I complex was isolatedfrom spinach leaves in a native state by use of anaerobic systems.The protein contained 8.5 non-heme iron atoms and 8.0 acid-labilesulfur atoms per molecule, consistent with the current conceptthat it has two [4Fe-4S] clusters. Its absorption spectrum wasvery similar to those of bacterial-type ferredoxins. The ratioof the absorbance at 390 nm to that at 280 nm was 0.6, and themolar extinction coefficient at 390 nm was 32,000 M^–.cm^–.Theoxidation-reduction properties of the iron-sulfur clusters wereexamined by redox potentiometry and EPR spectroscopy. The twoclusters were distinguishable in terms of their oxidation-reductionmidpoint potentials; their E_m values were determined to be about-470mV and-560 mV, respectively. (Received July 16, 1990; Accepted October 8, 1990)     

How to measure and predict the molar absorption coefficient of a protein.

The molar absorption coefficient, epsilon, of a protein is usually based on concentrations measured by dry weight, nitrogen, or amino acid analysis. The studies reported here suggest that the Edelhoch method is the best method for measuring epsilon for a protein. (This method is described by Gill and von Hippel [1989, Anal Biochem 182:319-326] and is based on data from Edelhoch [1967, Biochemistry 6:1948-1954]). The absorbance of a protein at 280 nm depends on the content of Trp, Tyr, and cystine (disulfide bonds). The average epsilon values for these chromophores in a sample of 18 well-characterized proteins have been estimated, and the epsilon values in water, propanol, 6 M guanidine hydrochloride (GdnHCl), and 8 M urea have been measured. For Trp, the average epsilon values for the proteins are less than the epsilon values measured in any of the solvents. For Tyr, the average epsilon values for the proteins are intermediate between those measured in 6 M GdnHCl and those measured in propanol. Based on a sample of 116 measured epsilon values for 80 proteins, the epsilon at 280 nm of a folded protein in water, epsilon (280), can best be predicted with this equation: epsilon (280) (M-1 cm-1) = (#Trp)(5,500) + (#Tyr)(1,490) + (#cystine)(125) These epsilon (280) values are quite reliable for proteins containing Trp residues, and less reliable for proteins that do not. However, the Edelhoch method is convenient and accurate, and the best approach is to measure rather than predict epsilon.     

Studies on nitrate reductase of Clostridium perfringens. II. Purification and some properties of ferredoxin.

A ferredoxin was purified from Clostridium perfringens by DEAE-cellulose chromatography and Sephadex G-50 gel filtration. It had absorption maxima at 390 and 280 nm. The molecular weight was estimated to be 6,000 by Sephadex gel filtration and from the results of amino acid analysis. The isoelectric point was 3.0. It contained four atoms of iron, four atoms of labile sulfur, and six cysteine residues. This ferredoxin as well as ferredoxin from C. pasteurianum acted as an electron donor for nitrate reductase from C. perfringens. The ferredoxin could also act as an electron donor for the hydrogenase from C. pasteurianum in hydrogen evolution.     

Purification and properties of ferredoxin and rubredoxin from Butyribacterium methylotrophicum.

A ferredoxin and a rubredoxin from Butyribacterium methylotrophicum, which displays a carbonyl-dependent acetyl-coenzyme A synthesis, were purified to electrophoretic homogeneity. The two electron carriers showed absorption spectra similar to those in Clostridium species. The ferredoxin displayed absorption peaks at 280 and 391 nm, while rubredoxin displayed absorption peaks at 279, 382, and 482 nm. Minimum molecular weights calculated from the respective amino acid compositions were 5,727 for ferredoxin and 5,488 for rubredoxin, excluding iron and inorganic sulfur atoms. Both electron carriers were isolated as monomers, according to gel-filtration data. Electron spin resonance analysis revealed that the ferredoxin was a 2[4Fe-4S]-type and that both clusters had a midpoint redox potential value of -410 mV, whereas rubredoxin contained one acid-stable iron and had a redox value of -40 mV. The coupling of these electron carriers to hydrogenase and carbon monoxide dehydrogenase activities was investigated. Rubredoxin showed higher activity towards carbon monoxide dehydrogenase, whereas ferredoxin showed higher activity towards hydrogenase.     

Spectroscopic characterization of the novel iron-sulfur cluster in Pyrococcus furiosus ferredoxin

Pyrococcus furiosus ferredoxin is the only known example of a ferredoxin containing a single [4Fe-4S] cluster that has non-cysteinyl ligation of one iron atom, as evidenced by the replacement of a ligating cysteine residue by an aspartic acid residue in the amino acid sequence. The properties of the iron-sulfur cluster in both the aerobically and anaerobically isolated ferredoxin have been characterized by EPR, magnetic circular dichroism, and resonance Raman spectroscopies. The anaerobically isolated ferrodoxin contains a [4Fe-4S]+,2+ cluster with anomalous properties in both the oxidized and reduced states which are attributed to aspartate and/or hydroxide coordination of a specific iron atom. In the reduced form, the cluster exists with a spin mixture of S = 1/2 (20%) and S = 3/2 (80%) ground states. The dominant S = 3/2 form has a unique EPR spectrum that can be rationalized by an S = 3/2 spin Hamiltonian with E/D = 0.22 and D = +3.3 +/- 0.2 cm-1. The oxidized cluster has an S = 0 ground state, and the resonance Raman spectrum is characteristic of a [4Fe-4S]2+ cluster except for the unusually high frequency for the totally symmetric breathing mode of the [4Fe-4S] core, 342 cm-1. Comparison with Raman spectra of other [4Fe-4S]2+ centers suggests that this behavior is diagnostic of anomalous coordination of a specific iron atom. The iron-sulfur cluster is shown to undergo facile and quantitative [4Fe-4S] in equilibrium [3Fe-4S] interconversion, and the oxidized and reduced forms of the [3Fe-4S] cluster have S = 1/2 and S = 2 ground states, respectively. In both redox states the [3Fe-4S]0,+ cluster exhibits spectroscopic properties analogous to those of similar clusters in other bacterial ferredoxins, suggesting non-cysteinyl coordination for the iron atom that is removed by ferricyanide oxidation. Aerobic isolation induces partial degradation of the [4Fe-4S] cluster to yield [3Fe-4S] and possibly [2Fe-2S] centers. Evidence is presented to show that only the [4Fe-4S] form of this ferredoxin exists in vivo.     

Amino acid sequence of ferredoxin II from the phototroph Rhodospirillum rubrum: Characteristics of a 7Fe ferredoxin

The complete sequence of amino acids of ferredoxin II (FdII) from Rhodospirillum rubrum was determined by repetitive Edman degradation using pyridylethylated-ferredoxin and oxidized, denatured ferredoxin. Peptides derived from trypsin, pepsin, Glu-C endoproteinase, Arg-C endoproteinase, tryptophan specific cleavage and partial acid hydrolysis and C-terminal sequence from carboxypeptidase digestion were used to construct the total sequence. RrFdII is a polypeptide of 104 amino acids having a calculated molecular weight of 11556 excluding the iron and sulfur atoms. The complete amino acid sequence was: PYVVTENCIKCKYQDCVEVCPVDCFYEGENFLVINPDECIDCGVCNPECPAEAIAGKWLEINRKFADLWPNITRKGPAL ADADDWKDKPDKTGLLSENPGKGTV. Sequence comparisons, EPR characteristics and iron analyses indicate that RrFdII has structural features in common with ferredoxins containing [3Fe-4S], [4Fe-4S] centers. Of 104 amino acids, 60 (58%) including all 9 cysteines, are found in identical locations in the 7Fe ferredoxin prototype, Azotobacter vinelandii FdI.The protein sequence data reported in this paper will appear in the SWISS-PROT database and EMBL Data Library under the accession number P80448.     

A novel and remarkably thermostable ferredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus.

The archaebacterium Pyrococcus furiosus is a strict anaerobe that grows optimally at 100 degrees C by a fermentative-type metabolism in which H2 and CO2 are the only detectable products. A ferredoxin, which functions as the electron donor to the hydrogenase of this organism was purified under anaerobic reducing conditions. It had a molecular weight of approximately 12,000 and contained 8 iron atoms and 8 cysteine residues/mol but lacked histidine or arginine residues. Reduction and oxidation of the ferredoxin each required 2 electrons/mol, which is consistent with the presence of two [4Fe-4S] clusters. The reduced protein gave rise to a broad rhombic electronic paramagnetic resonance spectrum, with gz = 2.10, gy = 1.86, gx = 1.80, and a midpoint potential of -345 mV (at pH 8). However, this spectrum represented a minor species, since it quantitated to only approximately 0.3 spins/mol. P. furiosus ferredoxin is therefore distinct from other ferredoxins in that the bulk of its iron is not present as iron-sulfur clusters with an S = 1/2 ground state. The apoferredoxin was reconstituted with iron and sulfide to give a protein that was indistinguishable from the native ferredoxin by its iron content and electron paramagnetic resonance properties, which showed that the novel iron-sulfur clusters were not artifacts of purification. The reduced ferredoxin also functioned as an electron donor for H2 evolution catalyzed by the hydrogenase of the mesophilic eubacterium Clostridium pasteurianum. P. furiosus ferredoxin was resistant to denaturation by sodium dodecyl sulfate (20%, wt/vol) and was remarkably thermostable. Its UV-visible absorption spectrum and electron carrier activity to P. furiosus hydrogenase were unaffected by a 12-h incubation of 95 degrees C.     

Overproduction, purification, and characterization of chlorocatechol dioxygenase, a non-heme iron dioxygenase with broad substrate tolerance.

We show here that purified chlorocatechol dioxygenase from Pseudomonas putida is able to oxygenate a wide range of substituted catechols with turnover numbers ranging from 2 to 29 s-1. This enzyme efficiently cleaves substituted catechols bearing electron-donating or multiple electron-withdrawing groups in an intradiol manner with kcat/KM values between 0.2 x 10(7) and 1.4 x 10(7) M-1 s-1. These unique catalytic properties prompted a comparison with the related but highly specific enzymes catechol 1,2-dioxygenase and protocatechuate 3,4-dioxygenase. The chlorocatechol dioxygenase gene (clcA) from the Pseudomonas plasmid pAC27 was subcloned into the expression vector pKK223-3, allowing production of chlorocatechol dioxygenase to approximately 7-8% of total cellular protein. An average of 4 mg of purified enzyme has been obtained per gram of wet cells. Protein and iron analyses indicate an iron stoichiometry of 1 iron/57.5-kDa homodimer, alpha 2Fe. The electronic absorption spectrum contains a broad tyrosinate to iron charge transfer transition centered at 430 nm (epsilon = 3095 M-1 cm-1 based on iron concentration) which shifts to 490 nm (epsilon = 3380 M-1 cm-1) upon catechol binding. The resonance Raman spectrum of the native enzyme exhibits characteristic tyrosine ring vibrations. Electron paramagnetic resonance data for the resting enzyme (g = 4.25, 9.83) is consistent with high-spin iron (III) in a rhombic environment. This similarity between the spectroscopic properties of the Fe(III) centers in chlorocatechol dioxygenase and the more specific dioxygenases suggests a highly conserved catalytic site. We infer that the unique catalytic properties of chlorocatechol dioxygenase are due to other characteristics of its substrate binding pocket.