Isolation and characterization of an Fe,-S8 ferredoxin (ferredoxin II) from Clostridium thermoaceticum.

A second ferredoxin protein was isolated from the thermophilic anaerobic bacterium Clostridium thermoaceticum and termed ferredoxin II. This ferredoxin was found to contain 7.9 +/- 0.3 iron atoms and 7.4 +/- 0.4 acid-labile sulfur atoms per mol of protein. Extrusion studies of the iron-sulfur centers showed the presence of two [Fe4-S4] centers per mol of protein and accounted for all of the iron present. The absorption spectrum was characterized by maxima at 390 nm (epsilon 390 = 30,400 M-1cm-1) and 280 nm (epsilon 280 = 41.400 M-1 cm-1) and by a shoulder at 300 nm. The ration of the absorbance of the pure protein at 390 nm to the absorbance at 280 nm was 0.74. Electron paramagnetic resonance data showed a weak signal in the oxidized state, and the reduced ferredoxin exhibited a spectrum typical of [Fe4-S4] clusters. Double integration of the reduced spectra showed that two electrons were necessary for the complete reduction of ferredoxin II. Amino histidine, and 1 arginine, and a molecular weight of 6,748 for the native protein. The ferredoxin is stable under anaerobic conditions for 60 min at 70 degrees C. The average oxidation-reduction potential for the two [Fe4-S4] centers was measured as -365 mV.     

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.     

Characterization of indo-1 and quin-2 as spectroscopic probes for Zn2(+)-protein interactions

1-[2-Amino-5-(6-carboxyindol-2-yl)phenoxyl]-2-(2'- amino-5'-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid (indo-1) and 2-[2-(bis(carboxymethyl)amino-5-methylphenoxy) methyl]-6- methyl-8-[bis-(carboxymethyl)amino]quinoline (quin-2) are sensitive, spectral indicators for Zn2+. Additions of subsaturating Zn2+ to 10-80 microM indo-1 or quin-2 at pH 7.0 produce uv difference spectra with isosbestic wavelengths at 342 and 282 nm or at 342, 317, and 252 nm, respectively. Formation of 1:1 Zn2+:indicator complexes at pH 7.0 and 20 degrees C in the absence (presence) of 100 mM KCl gives delta epsilon max = -2.4 +/- 0.2 X 10(4) M-1 cm-1 at 367 nm (-2.1 +/- 0.2 X 10(4) M-1 cm-1 at 365 nm) for indo-1 and delta epsilon max = -2.7 +/- 0.1 X 10(4) M-1 cm-1 at 266 nm (-2.6 +/- 0.1 X 10(4) M-1 cm-1 at 265 nm) for quin-2. Competition experiments at pH 7.0 and 20 degrees C with indo-1 and quin-2 and also 4-(2-pyridylazo)resorcinol (PAR) as the second chelator in the absence (presence) of 100 mM KCl yield apparent affinity constants: K'A = 2.5 +/- 1.0 X 10(10) M-1 (6.2 +/- 0.5 X 10(9) M-1) for indo-1 binding Zn2+ and K'A = 9.4 +/- 3.3 X 10(11) M-1 (2.7 +/- 0.1 X 10(11) M-1) for quin-2 binding Zn2+. The above constants provide the basis for rapid steady-state spectrophotometric determinations of the affinity of a protein for Zn2+ with K'A approximately 10(10) - 10(13) M-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and biochemical characterization of a putative [6Fe-6S] prismane-cluster-containing protein from Desulfovibrio vulgaris (Hildenborough).

A novel iron-sulfur protein has been isolated from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough). It is a stable monomeric protein, which has a molecular mass of 52 kDa, as determined by sedimentation-equilibrium centrifugation. Analysis of the metal and acid-labile sulfur content of the protein revealed the presence of 6.3 +/- 0.4 Fe/polypeptide and 6.2 +/- 0.7 S2-/polypeptide. Non-iron transition metals, heme, flavin and selenium were absent. Combining these data with the observation of a very anisotropic S = 1/2 [6Fe-6S]3+ prismane-like EPR signal in the dithionite-reduced protein, we believe that we have encountered the first example of a prismane-cluster-containing protein. The prismane protein has a slightly acidic amino acid composition and isoelectric point (pI = 4.9). The ultraviolet/visible spectrum is relatively featureless (epsilon 280 = 81 mM-1.cm-1, epsilon 400 = 25 mM-1.cm-1, epsilon 400,red = 14 mM-1.cm-1). The shape of the protein is approximately globular (S20.w = 4.18 S). The N-terminal amino acid sequence is MFS/CFQS/C QETAKNTG. Polyclonal antibodies against the protein were raised. Cytoplasmic localization was inferred from subcellular fractionation studies. Cross-reactivity of antibodies against this protein indicated the occurrence of a similar protein in D. vulgaris (Monticello) and Desulfovibrio desulfuricans (ATCC 27774). We have not yet identified a physiological function for the prismane protein despite trials for some relevant enzyme activities.     

The reaction between ferrous polyaminocarboxylate complexes and hydrogen peroxide: an investigation of the reaction intermediates by stopped flow spectrophotometry

The reactions of Fe(II)EDTA, Fe(II)DTPA, and Fe(II)HEDTA with hydrogen peroxide near neutral pH have been investigated. All these reactions have been assumed to proceed through an active intermediate, I1, (Formula: see text) where pac is one of the three polyaminocarboxylates mentioned above. I1, whether .OH radical or an iron complex, reacts with ethanol, formate, and other scavengers at rates relative to k2 that, with the exception of t-butanol and benzoate, are similar, but not identical, to those expected for the.OH radical. In contrast, at pH 3, in the absence of ligands the reaction of I1 with Fe2+ was inhibited by ethanol and t-butanol and the reactivity of I1 towards these two scavengers relative to ferrous ion is identical to that exhibited by the hydroxyl radical. When pac = HEDTA, the intermediate of the first reaction reacts with formate ion to form the ferrous HEDTA ligand radical complex, which is characterized by absorption maxima at 295 nm (epsilon = 2,640 M-1 cm-1) and 420 nm (epsilon = 620 M-1 cm-1). For the reaction of Fe(II)HEDTA with H2O2, the following mechanism is proposed: (Formula: see text) where k17 = 4.2 X 10(4) M-1 sec-1 and k19 = 5 +/- 0.2 sec-1.     

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.     

6-Nitro-L-tryptophan: a novel spectroscopic probe of trp aporepressor and human serum albumin

The binding of 6-nitro-L-tryptophan to trp aporepressor and human serum albumin has been examined by visible difference spectroscopy and circular dichroism. 6-Nitro-L-tryptophan, prepared by nitration of L-tryptophan with nitric acid in glacial acetic acid, exhibits a visible and near-uv absorption spectrum with lambda max at about 330 nm (epsilon = 7 X 10(3) M-1 cm-1) and a shoulder near 380 nm in H2O. In the presence of trp aporepressor, the visible absorption intensity is sharply diminished. Visible difference spectral titration data give KD = 1.27 X 10(-4) M and n = 0.95 per subunit at 25 degrees C. While 6-nitro-L-tryptophan exhibits no significant circular dichroism between 300 and 500 nm, the complex with trp aporepressor exhibits strong circular dichroism signals, with a negative maximum at 386 nm (delta epsilon = -7.5 M-1 cm-1) and a positive maximum at 310 nm (delta epsilon = +6 M-1 cm-1). Circular dichroism titration data give KD = 1.69 X 10(-4) M and n = 0.90 per subunit at 25 degrees C. The KD values determined spectroscopically are in excellent agreement with that determined by equilibrium dialysis, KD = 1.5 X 10(-4) M at 25 degrees C. In the presence of human serum albumin, the spectrum of 6-nitro-L-tryptophan exhibits a blue shift and an increase in absorption intensity; similar changes are observed in solvents of low dielectric contrast such as 80% aqueous dioxane. Visible difference spectral titration data give KD = 8.0 X 10(-5) M and n = 0.95 for human serum albumin. The complex of 6-nitro-L-tryptophan with human serum albumin exhibits a strong positive circular dichroism maximum at 380 nm (delta epsilon = +9.8 M-1 cm-1) with a shoulder at 310-320 nm. Circular dichroism titration data give KD = 6.4 X 10(-5) M and n = 0.83, in good agreement with the visible difference spectral results. Taken together, our results demonstrate the utility of 6-nitro-L-tryptophan as a spectroscopic probe for tryptophan-binding proteins.     

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.     

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.     

Determination of the enzymatic activity of lysine amidase

A technique is proposed for determining lysinamidase and aminolactamase activities of lysinamidase (EC 3.5.1.n.). It is based on spectrophotometric measurement of the optical density decrease of the substrate solution at 227 nm. For cyclic lysinamide L-alpha-amino-epsilon-caprolactam epsilon 227 M = 151 M-1.cm-1, for linear lysinamide epsilon 227 M = 73 M-1.cm-1, and for lysine epsilon 227 M = 5 M-1.cm-1. The technique is simple and requires no additional reagents.     

Serratia marcescens endonuclease. Properties of the enzyme

Some physico-chemical properties of endonuclease (EC 3.1.4.9) from Serratia marcescens were studied and the amino acid composition of the enzyme was determined. The protein molecule was shown to contain one SH-group and one S-S-bond, which renders it different from the well studied nuclease (EC 3.1.4.7) from Staph. pyogenes. The conditions for reconstitution of the S-S-bond by dithioerythritol for quantitative estimation of cysteine residues of the endonuclease molecule were selected. The N-terminal amino acid was found to be threonine. The UV spectra for the enzyme are typical for proteins; A 0,1% 1cm,280nm is 1.46, epsilon 25 degrees 280nm,pH7,4 is 47292 M-1 cm-1. The sedimentation coefficient in phosphate buffer sW, 20 degrees is 3.4 S, pI is 6.5 and 7.5.     

The reaction of superoxide radical with iron complexes of EDTA studied by pulse radiolysis.

The reactions of Fe3+-EDTA and Fe2+-EDTA with O2- and CO2- were investigated in the pH range 3.8--11.8. Around neutral pH O2- reduces Fe3+-EDTA with a rate constant which is pH dependent kpH 5.8--8.1 = 2 - 10(6)--5 - 10(5) M-1 - s-1. At higher pH values this reaction becomes much slower. The CO2- radical reduces Fe3+-EDTA with kpH 3.8--1- = 5 +/- 1 - 10(7) M-1 - s-1 independent of pH. At pH 9--11.8, Fe2+-EDTA forms a complex with O2- with kFe2+-EDTA + O2 = 2 - 10(6)--4 - 10(6) M-1 - s-1 which is pH dependent. We measured the spectrum of Fe2+-EDTA-O2- and calculated epsilon 290 over max = 6400 +/- 800 M-1 - cm-1 in air-saturated solutions. In O2-saturated solutions another species is formed with a rate constant of 7 +/- 2 s-1. This intermediate absorbs around 300 nm but we were not able to identify it.     

Co(II) derivatives of Cu,Zn-superoxide dismutase with the cobalt bound in the place of copper. A new spectroscopic tool for the study of the active site

Co(II) derivatives of Cu,Zn-superoxide dismutase having cobalt substituted for the copper (Co,Zn-superoxide dismutase and Co,Co-superoxide dismutase) were studied by optical and EPR spectroscopy. EPR and electronic absorption spectra of Co,Zn-superoxide dismutase are sensitive to solvent perturbation, and in particular to the presence of phosphate. This behaviour suggests that cobalt in Co,Zn-superoxide dismutase is open to solvent access, at variance with the Co(II) of the Cu,Co-superoxide dismutase, which is substituted for the Zn. Phosphate binding as monitored by optical titration is dependent on pH with an apparent pKa = 8.2. The absorption spectrum of Co,Zn-superoxide dismutase in water has three weak bands in the visible region (epsilon = 75 M-1 X cm-1 at 456 nm; epsilon = 90 M-1 X cm-1 at 520 nm; epsilon = 70 M-1 X cm-1 at 600 nm) and three bands in the near infrared region, at 790 nm (epsilon = 18 M-1 X cm-1), 916 nm (epsilon = 27 M-1 X cm-1) and 1045 nm (epsilon = 25 M-1 X cm-1). This spectrum is indicative of five-coordinate geometry. In the presence of phosphate, three bands are still present in the visible region but they have higher intensity (epsilon = 225 M-1 X cm-1 at 544 nm; epsilon = 315 M-1 X cm-1 at 575 nm; epsilon = 330 M-1 X cm-1 at 603 nm), whilst the lowest wavelength band in the near infrared region is at much lower energy, 1060 nm (epsilon = 44 M-1 X cm-1). The latter property suggests a tetrahedral coordination around the Co(II) centre. Addition of 1 equivalent of CN- gives rise to a stable Co(II) low-spin intermediate, which is characterized by an EPR spectrum with a highly rhombic line shape. Formation of this CN- complex was found to require more cyanide equivalents in the case of the phosphate adduct, suggesting that binding of phosphate may inhibit binding of other anions. Titration of the Co,Co-derivative with CN- provided evidence for magnetic interaction between the two metal centres. These results substantiate the contention that Co(II) can replace the copper of Cu,Zn-superoxide dismutase in a way that reproduces the properties of the native copper-binding site.     

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 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.     

Turbidimetric method for quantitative determination of triton X-100 with silicotungstic acid

The Formation of Triton X-100-silicotungstic acid complex was studied. Quantitative turbidimetric determination of the detergent based on this process was suggested. This method allows to determining the complex formation at any wavelength in the range from 350 (epsilon 350 = 15,600 cm-1 M-1) to 600 nm (epsilon 600 = = 9090 cm-1 M-1). The calibration curve for Triton X-100 recorded at 350 nm is linear in the concentration range of 0 to 30 micrograms/ml. A sigmoid calibration curve was observed at longer wavelengths. A linear fragment of the calibration curve recorded at 600 nm was found at a concentration of Triton X-100 of about 5 micrograms/ml. The complex nature of calibration curves can be explained by heterogeneity of the complex dispersion.     

Isolation and characterization of a rubredoxin and an (8Fe-8S) ferredoxin from Desulfuromonas acetoxidans

A two cluster (4Fe-4S) ferredoxin and a rubredoxin have been isolated from the sulfur-reducing bacterium Desulfuromonas acetoxidans. Their amino acid compositions are reported and compared to those of other iron-sulfur proteins. The ferredoxin contains 8 cysteine residues, 8 atoms of iron and 8 atoms of labile sulfur per molecule; its minimum molecular weight is 6163. The protein exhibits an abosrbance ratio of A385/A283 = 0.74. Storage results in a bleaching of the chromophore; the denatured ferredoxin is reconstitutable with iron and sulfide. The instability temperature is 52 degrees C. The rubredoxin does not differ markedly from rubredoxins from other anaerobic bacteria.     

Chemical characterization and purification of the beta subunit of the DNA polymerase III holoenzyme from an overproducing strain

We have purified the beta subunit of the DNA polymerase III holoenzyme to homogeneity from an overproducing strain (Blanar, M., Sandler, S., Armengod, M., Ream, L., and Clark, A. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4622-4626). From this procedure we can obtain 100 mg quantities of protein. The beta isolated from the overproducer is indistinguishable from that isolated from wild-type cells in terms of its activity and molecular weight. Partial amino acid sequence analysis has confirmed the DNA sequence of the dnaN gene (Ohmori, H., Kimura, M., Nagata, T., and Sakakibara, Y. (1984) Gene (Amst.) 28, 159-170) and established the sites for initiation and termination of translation. No processing that removes amino acid residues from beta occurs since the active protein begins with the initiating methionine and terminates at the position predicted from the DNA sequence. Our knowledge of the precise amino acid composition has been used to determine the extinction coefficient of beta to be 17,900 and 18,700 cm-1 M-1 at 280 and 277 nm, respectively. The extinction coefficient at 280 nm is reduced to 14,700 cm-1 M-1 under denaturing conditions in guanidine HCl. Conditions have been optimized so that 1 N-ethylmaleimide residue can be incorporated per beta monomer with full preservation of activity.     

A new plant-type ferredoxin from halobacteria.

A stable, 2Fe-type ferredoxin has been prepared from Halobacterium halobium and purified by chromatography. A similar ferredoxin was also found in three other Halobacteria. The ferredoxin is present in large amounts-about 1 percent of the total soluble protein. From amino acid composition a molecular weight of 14800 +/- 200 was calculated. The ferredoxin was found to contain two atoms each of iron and sulphide. The midpoint redox potential of the protein is about -345 mV. The electron paramagnetic resonance spectrum of the reduced form shows much similarity to plant and algal ferredoxins with gx = 1.90, gy = 1.97 and gz = 2.07. The same similarity is observed in the optical absorption, optical rotatory dispersion and circular dichroism spectra. However it does not seem to mediate electron transport in the NADP-photoreduction system of chloroplasts. Extracts of the bacterial cells catalyze the reduction of the ferredoxin by NADH.     

Characterization and cloning of an extremely thermostable, Pyrococcus furiosus-type 4Fe ferredoxin from Thermococcus profundus.

An extremely thermostable [4Fe-4S] ferredoxin was isolated under anaerobic conditions from a hyperthermophilic archaeon Thermococcus profundus, and the ferredoxin gene was cloned and sequenced. The nucleotide sequence of the ferredoxin gene shows the ferredoxin to comprise 62 amino acid residues with a sequence similar to those of many bacterial and archaeal 4Fe (3Fe) ferredoxins. The unusual Fe-S cluster type, which was identified in the resonance Raman and EPR spectra, has three cysteines and one aspartate as the cluster ligands, as in the Pyrococcus furiosus 4Fe ferredoxin. Under aerobic conditions, a ferredoxin was purified that contains a [3Fe-4S] cluster as the major Fe-S cluster and a small amount of the [4Fe-4S] cluster. Its N-terminal amino acid sequence is the same as that of the anaerobically-purified ferredoxin up to the 26th residue. These results indicate that the 4Fe ferredoxin was degraded to 3Fe ferredoxin during aerobic purification. The aerobically-purified ferredoxin was reversibly converted back to the [4Fe-4S] ferredoxin by the addition of ferrous ions under reducing conditions. The anaerobically-purified [4Fe-4S] ferredoxin is quite stable; little degradtion was observed over 20 h at 100 degrees C, while the half-life of the aerobically-purified ferredoxin is 10 h at 100 degrees C. Both the anaerobically- and aerobically-purified ferredoxins were found to function as electron acceptors for the pyruvate-ferredoxin oxidoreductase purified from the same archaeon.