Isolation and characterization of an iron-containing superoxide dismutase from a eucaryote, Brassica campestris

A cyanide-insensitive superoxide dismutase was purified from mustard leaves, Brassica campestris. The protein had a molecular weight of 41,000 and was composed of two equally sized subunits. Metal analysis revealed that the enzyme contained 1.6 g atoms of iron per dimer. The isolation of an iron-containing superoxide dismutase from mustard leaves represents the first report of this enzyme in a multicellular eucaryotic organism.     

Identification of catalytic residues involved in iron uptake by L-chain ferritins

When either horse spleen apoferritin (containing more than 90% of L chains) or recombinant horse L apoferritin are modified with glycineamide or taurine in the presence of a water-soluble carbodiimide, a total of 11 to 12 carboxyl groups per subunit are modified, and iron incorporation is effectively abolished. In contrast, when horse spleen ferritin (containing on average 2500 atoms per molecule) is modified under similar conditions, seven to eight carboxyl groups are modified. When apoferritin is prepared from this modified ferritin, it retains full iron incorporation activity. Apoferritin in which seven to eight carboxyls per subunit have been modified by glycineamide can subsequently be modified by taurine; a total of three to four carboxyl groups are modified accompanied by total loss of iron incorporation. Additional studies confirm that three carboxyl groups per subunit are protected from modification by glycineamide by Cr(III) inhibition of iron incorporation. Using tandem mass spectroscopy we have looked for taurine-labelled peptides in tryptic digests of succinylated apoferritins after taurine modification. In the sample where the residues involved in iron uptake have been modified with taurine, we have identified the peptide: This corresponds to residues 53–59 of the L subunit, where it is part of a region of the B-helix which is directed towards the inside of the apoferritin protein shell. The same peptide was identified using classical protein sequencing techniques after (1,2-³H)-taurine modification. We conclude that in L-chain apoferritins the Glu residues at positions 53, 56 and 57 are involved in the mechanism of iron incorporation. Glu 53 and 56 are conserved in L but not in H ferritins, and are located in close proximity to each other within the three-dimensional structure. There is ample room for rotation of Glu 57 to join with the other two to form an iron-binding site. This may represent a site of iron incorporation (most probably involving nucleation) unique to L-chain ferritins, and may explain the predominant L-chain involvement in conditions of iron overload.     

A Streptococcus mutans superoxide dismutase that is active with either manganese or iron as a cofactor

The superoxide dismutase produced by Streptococcus mutans OMZ176 during aerobic growth in a chemically defined medium (modified FMC) that was treated with Chelex 100 (to lower trace metal contamination) and supplemented with high purity manganese was purified (162-fold) by heat treatment, ammonium sulfate precipitation, and chromatofocusing chromatography. The superoxide dismutase produced during aerobic growth in the same medium, but without manganese and supplemented with high purity iron, was similarly purified (220-fold). The molecular masses of each holoenzyme were approximately 43,000 with a subunit mass of 20,700, indicating that the enzymes were dimers of two equally sized subunits. The superoxide dismutase from manganese-grown cells was a manganese enzyme (MnSOD) containing 1.2 atoms of manganese and 0.25 atoms of iron/subunit. The superoxide dismutase from iron-grown cells was an iron enzyme (FeSOD) containing 0.07 atoms of manganese and 0.78 atoms of iron/subunit. The amino acid compositions of the MnSOD and the FeSOD were virtually identical, and their amino-terminal sequences were identical through the first 22 amino acids. Dialysis of the FeSOD with o-phenanthroline and sodium ascorbate generated aposuperoxide dismutase with 94% loss of activity; subsequent dialysis of apoenzyme with either manganese sulfate or ferrous sulfate reconstituted activity (recoveries of 37 and 30%, respectively). Electrophoretic determination of cytoplasmic radioiron distribution indicated that (during aerobic growth) manganese prevented insertion of iron into superoxide dismutase, although the iron levels of at least two other cytoplasmic fractions were not altered by manganese. Therefore, S. mutans used the same aposuperoxide dismutase to form either FeSOD or MnSOD, depending upon which metal was available in the culture medium. Such "cambialistic" enzymes (those capable of making a cofactor substitution) may represent a previously unrecognized family of superoxide dismutases.     

Asymmetry of tyrosyl-tRNA synthetase in solution

The tyrosyl-tRNA synthetase from Bacillus stearothermophilus crystallizes as a symmetrical dimer with each subunit having a complete active site. The enzyme-substrate complexes, however, are known to be asymmetrical in solution because the enzyme exhibits half-of-the-sites activity by binding tightly only 1 mol of tyrosine or 1 mol of tyrosyl adenylate per mole of dimer. Evidence is now presented that the unligated enzyme is also asymmetrical in solution. Symmetry was investigated by construction of heterodimers containing one full-length subunit and one truncated subunit, allowing the introduction of different mutations into each monomer. Each dimer is active at only one site, but the site used is randomly distributed between the subunits. Each heterodimer thus consists of two equal populations, one activating tyrosine at a full-length subunit and the other at the truncated subunit. No detectable interconversion is found between active and inactive sites over several minutes either in the absence of substrates or when the enzyme is turning over in the steady state. Kinetic evidence implies that wild-type enzyme is inherently asymmetrical even in the absence of substrate.     

Manganese and iron superoxide dismutases are structural homologs

The crystal structure of a tetrameric manganese superoxide dismutase from a thermophilic bacterium, Thermus thermophilus HB8, has been determined at 4.4-A resolution by local averaging of electron density maps calculated by isomorphous replacement. The spatial arrangement of the principal secondary structural features of iron superoxide dismutase is conserved in manganese dismutase. The structural homology is displayed by orienting the polypeptide chain of Escherichia coli Fe dismutase in the electron density map of Mn dismutase. Densities corresponding to bound Mn3+ occur at locations equivalent to the Fe3+ positions in iron dismutase, indicating one metal binding site per chain, or four sites per tetramer. The Mn tetramer, with 222 symmetry, is approximately rectangular in shape and appears to be constructed with only two unique interfaces. One set of interchain contacts closely resembles the dimer interface of Fe dismutase, but the other interface utilizes an inserted polypeptide segment that has no equivalent in Fe dismutase.     

Content and localization of FMN, Fe-S clusters and nickel in the NAD-linked hydrogenase of Nocardia opaca 1b

By preparative polyacrylamide gel electrophoresis at pH 8.5, and in the absence of nickel ions, two types of subunit dimers of the NAD-linked hydrogenase from Nocardia opaca 1b were separated and isolated, and their properties were compared with each other as well as with the properties of the native enzyme. The intact hydrogenase contained 14.3 +/- 0.4 labile sulphur, 13.6 +/- 1.1 iron and 3.8 +/- 0.1 nickel atoms and approximately 1 FMN molecule per enzyme molecule. The oxidized hydrogenase showed an absorption spectrum with maxima (shoulders) at 380 nm and 420 nm and an electron spin resonance (ESR) spectrum with a signal at g = 2.01. The midpoint redox potential of the Fe-S cluster giving rise to this signal was +25 mV. In the reduced state, hydrogenase gave characteristic low-temperature (10-20 K) and high-temperature (greater than 40 K) ESR spectra which were interpreted as due to [4Fe-4S] and [2Fe-2S] clusters, respectively. The midpoint redox potentials of these clusters were determined to be -420 mV and -285 mV, respectively. The large hydrogenase dimer, consisting of subunits with relative molecular masses Mr, of 64000 and 31000, contained 9.9 +/- 0.4 S2- and 9.3 +/- 0.5 iron atoms per protein molecule. This dimer contained the FMN molecule, but no nickel. The absorption and ESR spectra of the large dimer were qualitatively similar to the spectra of the whole enzyme. This dimer did not show any hydrogenase activity, but reduced several electron acceptors with NADH as electron donor (diaphorase activity). The small hydrogenase dimer, consisting of subunits with Mr of 56000 and 27000, was demonstrated to have substantially different properties. For iron and labile sulphur average values of 3.9 and 4.3 atoms/dimer molecule have been determined, respectively. The dimer contained, in addition, about 2 atoms of nickel and was free of flavins. In the oxidized state this dimer showed an absorption spectrum with a broad band in the 400-nm region and a characteristic ESR signal at g = 2.01. The reduced form of the dimer was ESR-silent. The small dimer alone was diaphorase-inactive and did not reduce NAD with H2, but it displayed high H2-uptake activities with viologen dyes, methylene blue and FMN, and H2-evolving activity with reduced methyl viologen. Hydrogen-dependent NAD reduction was fully restored by recombining both subunit dimers, although the reconstituted enzyme differed from the original in its activity towards artificial acceptors and the ESR spectrum in the oxidized state.     

Structure of yeast hexokinase: IV. Low-resolution structure of enzyme—Substrate complexes revealing negative co-operativity and allosteric interactions

We conclude from X-ray diffraction studies at low resolution (7 Å) that the binding of sugar and nucleotide substrates to dimeric yeast hexokinase BII crystals exhibits both negative co-operativity and positive allosteric co-operativity. Difference electron density maps show the positions of sugar and nucleotide binding sites and extensive substrate-induced structural changes in the protein. Sugar substrates and inhibitors bind in the deep cleft that divides each subunit into two lobes and nucleotide substrates bind nearby to one site per dimer, which lies between the subunits and on the molecular symmetry axis. Although the inhibitors o- and p-iodobenzoylglucosamine and o-toluoylglucosamine bind equally to both subunits, the degree of substitution of glucose or xylose is very different for the two subunits. The substrate analog β, γ-imido ATP shows only one strong binding site per dimer. This negative co-operativity in substrate binding may result from the heterologous or non-equivalent association of the two subunits (Anderson et al., 1974), which provides non-equivalent environments for the two chemically identical subunits.Further, there is a positive allosteric interaction between the sugar and nucleotide binding sites. Sugar binding is required for nucleotide binding at the intersubunit site and the binding of nucleotide modifies the binding of sugars. These positive heterotropic interactions appear to be mediated by extensive substrate-induced structural changes in the enzyme.     

Refined 1.83 A structure of trypanosomal triosephosphate isomerase crystallized in the presence of 2.4 M-ammonium sulphate. A comparison with the structure of the trypanosomal triosephosphate isomerase-glycerol-3-phosphate complex

Triosephosphate isomerase (TIM) is a dimeric glycolytic enzyme. TIM from Trypanosoma brucei brucei has been crystallized at pH 7.0 in 2.4 M-ammonium sulphate. The well-diffracting crystals have one dimer per asymmetric unit. The structure has been refined at 1.83 A resolution with an R-factor of 18.3% for all data between 6 A and 1.83 A (37,568 reflections). The model consists of 3778 protein atoms and 297 solvent atoms. Subunit 1 is involved in considerably more crystal contacts than subunit 2. Correlated with these differences in crystal packing is the observation that only in the active site of subunit 2 is a sulphate ion bound. Furthermore, significant differences with respect to structure and flexibility are observed in three loops near the active site. In particular, there is a 7 A positional difference of the tip of the flexible loop (loop 6) when comparing subunit 1 and subunit 2. Also, the neighbouring loops (loop 5 and loop 7) have significantly different conformations and flexibility. In subunit 1, loop 6 is in an "open" conformation, in subunit 2, loop 6 is in an "almost closed" conformation. Only in the presence of a phosphate-containing ligand, such as glycerol-3-phosphate, does loop 6 take up the "closed" conformation. Loop 6 and loop 7 (and also to some extent loop 5) are rather flexible in the almost closed conformation, but well defined in the open and closed conformations. The closing of loop 6 (167 to 180), as observed in the almost closed conformation, slightly changes the main-chain conformation of the catalytic glutamate, Glu167, leading to a change of the chi 1 angle of this residue from approximately -60 degrees to approximately 60 degrees and the weakening of the hydrogen bonds between its polar side-chain atoms and Ser96. In the closed conformation, in the presence of glycerol-3-phosphate, the main-chain atoms of Glu167 remain in the same position as in the almost closed conformation, but the side-chain has rotated around the CA-CB bond changing chi 1 from approximately 60 degrees to approximately -60 degrees. In this new position the hydrogen bonding to Ser96 is completely lost and also a water-mediated salt bridge between OE2(Glu167) and NE(Arg99) is lost. Comparison of the two independently refined subunits, showed that the root-mean-square deviation for all 249 CA atoms is 0.9 A; for the CA atoms of the beta-strands this is only 0.2 A. The average B-factor for all subunit 1 and subunit 2 atoms is 20 A2 and 25 A2, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis. A novel iron-sulfur protein.

Glutamine phosphoribosylpyrophosphate amidotransferase, purifed to better than 98% purity from derepressed Bacillus subtilis, exists as a tetramer and as a dimer of apparently identical subunits with a molecular weight of 50,000 each. The enzyme contains 3 atoms of iron and 2 atoms of inorganic sulfide per subunit and has a yellow-brown color. The absorption spectrum is not altered by dithionite, but exposure to oxygen causes inactivation and partial bleaching of the visible spectrum. Thus, the Bacillus amidotransferase exhibits novel structural features and a new reaction type of proteins of the iron-sulfur group.     

Structures of D-xylose isomerase from Arthrobacter strain B3728 containing the inhibitors xylitol and D-sorbitol at 2.5 A and 2.3 A resolution, respectively

The structures of D-xylose isomerase from Arthrobacter strain B3728 containing the polyol inhibitors xylitol and D-sorbitol have been solved at 2.5 A and 2.3 A, respectively. The structures have been refined using restrained least-squares refinement methods. The final crystallographic R-factors for the D-sorbitol (xylitol) bound molecules, for 43,615 (32,989) reflections are 15.6 (14.7). The molecule is a tetramer and the asymmetric unit of the crystal contains a dimer, the final model of which, incorporates a total of 6086 unique protein, inhibitor and magnesium atoms together with 535 bound solvent molecules. Each subunit of the enzyme contains two domains: the main domain is a parallel-stranded alpha-beta barrel, which has been reported in 14 other enzymes. The C-terminal domain is a loop structure consisting of five helical segments and is involved in intermolecular contacts between subunits that make up the tetramer. The structures have been analysed with respect to molecular symmetry, intersubunit contacts, inhibitor binding and active site geometry. The refined model shows the two independent subunits to be similar apart from local deviations due to solvent contacts in the solvent-exposed helices. The enzyme is dependent on a divalent cation for catalytic activity. Two metal ions are required per monomer, and the high-affinity magnesium(II) site has been identified from the structural results presented here. The metal ion is complexed, at the high-affinity site, by four carboxylate side-chains of the conserved residues, Glu180, Glu216, Asp244 and Asp292. The inhibitor polyols are bound in the active site in an extended open chain conformation and complete an octahedral co-ordination shell for the magnesium cation via their oxygen atoms O-2 and O-4. The active site lies in a deep pocket near the C-terminal ends of the beta-strands of the barrel domain and includes residues from a second subunit. The tetrameric molecule can be considered to be a dimer of "active" dimers, the active sites being composed of residues from both subunits. The analysis has revealed the presence of several internal salt-bridges stabilizing the tertiary and quaternary structure. One of these, between Asp23 and Arg139, appears to play a key role in stabilizing the active dimer and is conserved in the known sequences of this enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification of a manganese-containing superoxide dismutase from Halobacterium halobium

An oxygen-induced superoxide dismutase was purified from the halophilic bacterium, Halobacterium halobium, strain NRL. Due to the high salt requirement for enzyme stability, the purification had to be performed in the presence of 2 M NaCl. The pI of the protein was 4.95. The approximate Mr was 38,500. The subunit size as determined by sodium dodecyl sulfate-electrophoresis was approximately 19,000. Metal analysis showed 1.5 atoms of manganese per dimer, 0.5 atom zinc, and 1.54 atoms copper. The N-terminal sequence of amino acids was determined, and based upon the first 26 amino acids significant homology to other manganese- and iron-containing superoxide dismutases was revealed.     

菠菜铁型超氧化物歧化酶的纯化及性质

用聚丙烯胺梯度凝胶电泳法检测出菠菜ＳＯＤ同工酶谱带中含３条Ｆｅ－ＳＯＤ活性带,菠菜叶Ｆｅ－ＳＯＤ粗提取液经硫酸铵分部沉淀,ＤＥＡＥ－纤维素－Ａ５２和ＳｅｐｈａｄｅｘＧ－１００柱层析,纯化出单一的Ｆｅ－ＳＯＤ活性带,纯化酶的分子量为４２．６ｋＤ,亚基分子量为２１ｋＤ。对金属元素的分析表明,该酶每分子含２．６个Ｆｅ原子,该酶紫外区最大吸收峰为２７８ｎｍ,等电点为４．６,氨基酸组成和其它来源的Ｆｅ－ＳＯ     

The refined crystal structure of Pseudomonas putida lipoamide dehydrogenase complexed with NAD+ at 2.45 A resolution.

The three-dimensional structure of one of the three lipoamide dehydrogenases occurring in Pseudomonas putida, LipDH Val, has been determined at 2.45 A resolution. The orthorhombic crystals, grown in the presence of 20 mM NAD+, contain 458 residues per asymmetric unit. A crystallographic 2-fold axis generates the dimer which is observed in solution. The final crystallographic R-factor is 21.8% for 18,216 unique reflections and a model consisting of 3,452 protein atoms, 189 solvent molecules and 44 NAD+ atoms, while the overall B-factor is unusually high: 47 A2. The structure of LipDH Val reveals the conformation of the C-terminal residues which fold "back" into the putative lipoamide binding region. The C-terminus has been proven to be important for activity by site-directed mutagenesis. However, the distance of the C-terminus to the catalytically essential residues is surprisingly large, over 6 A, and the precise role of the C-terminus still needs to be elucidated. In this crystal form LipDH Val contains one NAD+ molecule per subunit. Its adenine-ribose moiety occupies an analogous position as in the structure of glutathione reductase. However, the nicotinamide-ribose moiety is far removed from its expected position near the isoalloxazine ring and points into solution. Comparison of LipDH Val with Azotobacter vinelandii lipoamide dehydrogenase yields an rms difference of 1.6 A for 440 well defined C alpha atoms per subunit. Comparing LipDH Val with glutathione reductase shows large differences in the tertiary and quaternary structure of the two enzymes. For instance, the two subunits in the dimer are shifted by 6 A with respect to each other. So, LipDH Val confirms the surprising differences in molecular architecture between glutathione reductase and lipoamide dehydrogenase, which were already observed in Azotobacter vinelandii LipDH. This is the more remarkable since the active sites are located at the subunit interface and are virtually identical in all three enzymes.     

Two crystal structures of the isochorismate pyruvate lyase from Pseudomonas aeruginosa

Enzymatic systems that exploit pericyclic reaction mechanisms are rare. A recent addition to this class is the enzyme PchB, an 11.4-kDa isochorismate pyruvate lyase from Pseudomonas aeruginosa. The apo and pyruvate-bound structures of PchB reveal that the enzyme is a structural homologue of chorismate mutases in the AroQalpha class despite low sequence identity (20%). The enzyme is an intertwined dimer of three helices with connecting loops, and amino acids from each monomer participate in each of two active sites. The apo structure (2.35 A resolution) has one dimer per asymmetric unit with nitrate bound in an open active site. The loop between the first and second helices is disordered, providing a gateway for substrate entry and product exit. The pyruvate-bound structure (1.95 A resolution) has two dimers per asymmetric unit. One has two open active sites like the apo structure, and the other has two closed active sites with the loop between the first and second helices ordered for catalysis. Determining the structure of PchB is part of a larger effort to elucidate protein structures involved in siderophore biosynthesis, as these enzymes are crucial for bacterial iron uptake and virulence and have been identified as antimicrobial drug targets.     

Reactivation of apo horse liver alcohol dehydrogenase with the monovalent metal ion Ag(I)

Each subunit of the liver alcohol dehydrogenase dimer contains one catalytic and one structural Zn(II) atom. Enzyme with the catalytic metal atoms selectively removed is inactive but can be partly reactivated in the presence of Ag(I) ions. Reactivation results from Ag(1) ions entering the empty metal-binding site in the catalytic center. The specific activity of this silver enzyme reached 24% of the native enzyme. Atomic absorption analysis gave equal amounts of Ag(I) and Zn(II), corresponding to one mole of each metal per monomer. Metal-directed affinity labelling using bromo-imidazolyl propionate showed that the properties of the silver-reactivated enzyme were distinct from those of the native enzyme.     

Crystal structure of guanidinoacetate methyltransferase from rat liver: a model structure of protein arginine methyltransferase

Guanidinoacetate methyltransferase (GAMT) is the enzyme that catalyzes the last step of creatine biosynthesis. The enzyme is found in abundance in the livers of all vertebrates. Recombinant rat liver GAMT has been crystallized with S-adenosylhomocysteine (SAH), and the crystal structure has been determined at 2.5 A resolution. The 36 amino acid residues at the N terminus were cleaved during the purification and the truncated enzyme was crystallized. The truncated enzyme forms a dimer, and each subunit contains one SAH molecule in the active site. Arg220 of the partner subunit forms a pair of hydrogen bonds with Asp134 at the guanidinoacetate-binding site. On the basis of the crystal structure, site-directed mutagenesis on Asp134, and chemical modification and limited proteolysis studies, we propose a catalytic mechanism of this enzyme. The truncated GAMT dimer structure can be seen as a ternary complex of protein arginine methyltransferase (one subunit) complexed with a protein substrate (the partner subunit) and the product SAH. Therefore, this structure provides insight into the structure and catalysis of protein arginine methyltransferases.     

Direct observation of the iron binding sites in a ferritin

X-Ray analysis of the ferritin of Escherichia coli (Ec-FTN) and of Ec-FTN crystals soaked in (NH₄)₂Fe(SO₄)₂ has revealed the presence of three iron-binding sites per subunit. Two of these form a di-iron site in the centre of the subunit as has been proposed for the ‘ferroxidase centres’ of human ferritin H chains. This di-iron site, lying within the 4-alpha-helix bundle, resemble those of ribonucleotide reductase, methane monoxygenase and haemerythrin. The third iron is bound by ligands unique to Ec-FTN on the inner surface of the protein shell. It is speculated that this state may represent the nucleation centre of a novel type of Fe(III) cluster, recently observed in Ec-FTN.     

Structural Basis of Heme Binding in the Cu,Zn Superoxide Dismutase from Haemophilus ducreyi

The Cu,Zn superoxide dismutase from Haemophilus ducreyi is characterized by the unique ability to bind heme at its dimer interface. Here we report the high-resolution crystal structures of this protein in the heme-loaded (holo) and heme-free (apo) forms. Heme is asymmetrically bound between the two enzyme subunits, where heme iron is coordinated by two histidine residues, His64 and His 124, provided by the two subunits. Moreover, the binding of heme to the protein is ensured by stabilizing contacts between the prosthetic group and a limited number of other residues, most of which are not present in other bacterial enzyme variants. We show that the introduction of only three mutations at the dimer interface of the enzyme from Haemophilus parainfluenzae, a closely related bacterial species, is sufficient to induce heme-binding ability by this enzyme variant. Heme binding does not alter protein activity. Moreover, the binding of the prosthetic group does not induce any significant structural perturbation at the subunit level and requires only limited local structural rearrangements that widen the cleft at the dimer interface and cause a limited shift in the relative orientation between the subunits. The presence of a preformed heme-binding pocket and the significant solvent exposure of the cofactor to the solvent are compatible with the suggested protective role of the enzyme against heme toxicity or with its involvement in heme trafficking in the periplasmic space.     

Iron-sulfur cluster interconversions in biotin synthase: dissociation and reassociation of iron during conversion of [2Fe-2S] to [4Fe-4S] clusters

Biotin synthase catalyzes the insertion of a sulfur atom into the saturated C6 and C9 carbons of dethiobiotin. This reaction has long been presumed to occur through radical chemistry, and recent experimental results suggest that biotin synthase belongs to a family of enzymes that contain an iron-sulfur cluster and reductively cleave S-adenosylmethionine, forming an enzyme or substrate radical, 5'-deoxyadenosine, and methionine. Biotin synthase (BioB) is aerobically purified as a dimer of 38 kDa monomers that contains two [2Fe-2S](2+) clusters per dimer. Maximal in vitro biotin synthesis requires incubation of BioB with dethiobiotin, AdoMet, reductants, exogenous iron, and crude bacterial protein extracts. It has previously been shown that reduction of BioB with dithionite in 60% ethylene glycol produces one [4Fe-4S](2+/1+) cluster per dimer. In the present work, we use UV/visible and electron paramagnetic resonance spectroscopy to show that [2Fe-2S] to [4Fe-4S] cluster conversion occurs through rapid dissociation of iron from the protein followed by rate-limiting reassociation. While in 60% ethylene glycol the product of dithionite reduction is one [4Fe-4S](2+) cluster per dimer, the product in water is one [4Fe-4S](1+) cluster per dimer. Further, incubation with excess iron, sulfide, and dithiothreitol produces protein that contains two [4Fe-4S](2+) clusters per dimer; subsequent reduction with dithionite produces two [4Fe-4S](1+) clusters per BioB dimer. BioB that contains two [4Fe-4S](2+/1+) clusters per dimer is rapidly and reversibly reduced and oxidized, suggesting that this is the redox-active form of the iron-sulfur cluster in the anaerobic enzyme.     

A highly stable cambialistic-superoxide dismutase from Antrodia camphorata: expression in yeast and enzyme properties

A cDNA encoding a putative superoxide dismutase (SOD) was identified in expressed sequence tags of Antrodia camphorata, a medicinal mushroom found only in Taiwan. The deduced protein was aligned with Mn-SODs and Fe-SODs from other organisms, this SOD showed greater homology to Mn-SOD. Functional A. camphorata SOD protein was overexpressed in yeast and purified. The purified enzyme showed two active forms on a 12.5% native PAGE, a dimer and a monomer. The dimeric protein's half-life of deactivation at 80 degrees C was 7 min, and its thermal inactivation rate constant K(d) was 9.87 x 10(-2)min(-1). The enzyme was stable in a broad pH range from 5-11; in the presence of 0.4M imidazole and 2% SDS. The atomic absorption spectrometric assay showed that 1.0 atom of manganese/iron (9:1) was present in each SOD subunit. The high stability of the enzyme make it better suited than other cambialistic-SODs for use in cosmetics. The SOD also documents its future utility in developing anti-inflammatory agent and in the treatment of chronic diseases.