Isolation of iron-containing superoxide dismutase from Bacteroides fragilis: reconstitution as a Mn-containing enzyme

Superoxide dismutase from the anaerobe Bacteroides fragilis has been purified to apparent homogeneity. The protein, Mr 42,000, is a dimer of equally sized subunits joined by noncovalent interactions. Metal analysis of the native enzyme revealed 1.8-1.9 g-atoms Fe, 0.2 g-atoms Zn, and less than 0.05 g-atoms Mn per mole dimer in a preparation whose specific activity was 1200 U/mg. Exposure of the enzyme to guanidinium chloride plus 8-hydroxyquinoline (T. Kirby, J. Blum, I. Kahane, and I. Fridovich, 1980, Arch. Biochem. Biophys. 201, 551-555) resulted in complete loss of enzymatic activity. Activity could be restored by dialysis of the denatured apoprotein against Tris buffer containing either ferrous ammonium sulfate or manganous chloride. The Fe-reconstituted enzyme was inhibited by 1 mM azide and inactivated by H2O2 in a manner similar to the native enzyme. Mn-reconstituted enzyme was inhibited by azide but resisted inactivation by H2O2 comparable to other purified manganese-containing superoxide dismutases. The manganese reconstituted protein contained approximately 1 gm-atom Mn/mol dimer. Zn ion potently inhibited reconstitution of the denatured apoprotein by either Mn or Fe and bound to the protein with a stoichiometry of 2-3 g-atoms/mol dimer.     

Isolation and reconstitution of iron- and manganese-containing superoxide dismutases from Bacteroides thetaiotaomicron.

Superoxide dismutase (SOD) from extracts of anaerobically maintained Bacteroides thetaiotaomicron was a dimer of equally sized 23,000-molecular-weight monomers joined noncovalently. A preparation with a specific activity of 1,200 U/mg contained 1.1 g-atom of Fe, 0.6 g-atom of Zn, and less than 0.05 g-atom of Mn per mol of dimer. The apoprotein, prepared by dialysis of iron-SOD in 5 M guanidinium chloride-20 mM 8-hydroxyquinoline, had no superoxide-scavenging activity when renatured without exogenous metal. Enzymatic activity was restored to the denatured apoprotein by dialysis against either 1 mM Fe(NH4)2 or 1 mM MnCl2 in 20 mM Tris (pH 7.0). The Fe-reconstituted enzyme and the native enzyme were inhibited approximately 50% by 0.2 mM NaN3, whereas the Mn-reconstituted enzyme was inhibited 60% by 10 mM NaN3. Aeration of the anaerobic cells resulted in a fourfold induction of an azide-resistant SOD. The enzyme (43,000 molecular weight) isolated from aerated cells was a dimer of equally sized subunits. The metal content was 1.0 g-atom of Mn, 0.55 g-atom of Fe, and 0.3 g-atom of Zn per mol of dimer. Enzymatic activity of the denatured apoprotein from this enzyme was also restored on addition of either iron or manganese. The constitutive Fe-SOD and the O2-induced Mn-SOD, tested alone and in combination, migrated identically on acrylamide gels, had similar amino acid compositions, and had alanine as the sole N-terminal amino acid. These data are consistent with the synthesis of a single apoprotein in either anaerobically maintained or oxygenated cells. We have observed a similar phenomenon with SOD from Bacteroides fragilis (E. M. Gregory, Arch. Biochem. Biophys. 238:83-89, 1985).     

Characterization of the O2-induced manganese-containing superoxide dismutase from Bacteroides fragilis

A manganese-containing superoxide dismutase (MnSOD) has been isolated from extracts of O2-induced Bacteroides fragilis. The enzyme, Mr 43,000, was a dimer composed of noncovalently associated subunits of equal size. A preparation whose specific activity was 1760 U/mg had 1.1 g-atoms Mn, 0.3 g-atoms Fe, and 0.2 g-atoms Zn per mol dimer. Exposing the enzyme to 5 M guanidinium chloride, 20 mM 8-hydroxyquinoline abolished enzymatic activity. Dialysis of the denatured apoprotein in buffer containing either Fe (NH4)2(SO4)2 or MnCl2 restored O2-. scavenging activity. The iron-reconstituted enzyme was inhibited 89% by 2 mM NaN3, similar to other Fe-containing superoxide dismutases. The Mn-reconstituted and native MnSOD were inhibited approximately 50% by 20 mM NaN3. Addition of ZnSO4 to dialysis buffer containing either the iron or manganese salt inhibited restoration of enzymatic activity to the denatured apoprotein. MnSOD migrated as a single protein band coincident with a single superoxide dismutase activity band in 7.5 or 10% acrylamide gels. Isoelectric focusing resulted in a major isozymic form with pI 5.3 and a minor form at pI 5.0. Mixtures of the MnSOD and the iron-containing superoxide (FeSOD), isolated from anaerobically maintained B. fragilis [E. M. Gregory and C. H. Dapper (1983) Arch. Biochem. Biophys. 220, 293-300], migrated as a single band on acrylamide gels and isoelectrically focused to a major protein band (pI 5.3) and a minor band at pI 5.0. The amino acid composition of MnSOD was virtually identical to that of the FeSOD. The data are consistent with synthesis of a single superoxide dismutase apoprotein capable of accepting either Mn or Fe to form the holoenzyme.     

A cambialistic SOD in a strictly aerobic hyperthermophilic archaeon, Aeropyrum pernix.

The superoxide dismutase (SOD) gene of Aeropyrum pernix, a strictly aerobic hyperthermophilic archaeon, was cloned and expressed in Escherichia coli, and its gene product was characterized. The molecular mass of the protein, based on the deduced amino acid sequence, was 24.6 kDa. The sequence showed overall similarity to the sequences of known Mn- and Fe-SODs. The metal binding residues conserved in Mn- and Fe-SODs were also found in A. pernix SOD. When the SOD gene was expressed in E. coli cells, the product formed a homodimer, and contained both Mn and Fe. Metal reconstitution experiments showed that A. pernix SOD is cambialistic, i.e. active with either Fe or Mn. The specific activities were 906 U/mg with Mn and 175 U/mg with Fe. No loss of activity of Mn-reconstituted SOD was observed at 105 degrees C even after 5 h incubation. Sodium azide, an inhibitor of SODs, did not inhibit the Mn-reconstituted SOD from A. pernix even at concentrations up to 400 mM. This SOD from an aerobic hyperthermophilic archaeon, Aeropyrum pernix, was extremely thermostable and active with either Mn or Fe. With Mn as a metal cofactor, it was more thermostable, and less sensitive to sodium azide and sodium fluoride than with Fe.     

Iron- and manganese-containing superoxide dismutases from Methylomonas J: identity of the protein moiety and amino acid sequence.

Mn-superoxide dismutase (SOD) and Fe-SOD were isolated from Methylomonas J, an aerobic methylotrophic bacterium, grown in methylamine media containing either manganese (Mn-rich medium) or iron (Fe-rich medium), respectively. The specific activity of the Mn-SOD was 2250 units mg-1 (mol of Mn)-1 (mol of dimer)-1, and the metal content of the enzyme was 0.98 mol of Mn and 0.12 mol of Fe per mole of dimer, while those of Fe-SOD were 88.5 units mg-1 (mol of Fe)-1 (mol of dimer)-1 and 1.04 mol of Fe and 0.02 mol of Mn. The electrophoretic mobilities in the presence of sodium dodecyl sulfate, with or without urea, and the chromatographic behavior on an HPLC column using an octadodecyl silicated column and a gel permeation column were identical. Amino acid compositions were practically indistinguishable in both SODs. The enzyme activity was restored by dialysis of an apoprotein obtained from the Mn-enzyme with either manganese sulfate or ferrous ammonium sulfate up to an activity level similar to that for the native Mn-SOD and the native Fe-SOD, respectively. The same result has been reported with the reconstitution using an apoprotein obtained from the Fe-enzyme [Yamakura, F., Matsumoto, T., & Terauchi, K. (1990) Free Radical Res. Commun. (in press)]. These results suggest the possibility that both types of SODs are composed of a single apoprotein synthesized in cells grown in either the Fe-rich medium or the Mn-rich medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tetrameric manganese superoxide dismutases from anaerobic Actinomyces

Superoxide dismutase was isolated from each of the anaerobically grown organisms Actinomyces naeslundii, Actinomyces strain E1S.25D, and Actinomyces odontolyticus. The enzymes were 100,000-110,000 mol wt acidic proteins (pI 4.3-4.6) and contained Mn and Zn, but no detectable Fe. The Mn and Zn content varied with the enzyme source. A. naeslundii superoxide dismutase, specific activity 2200 U/mg, contained 2.3 g atoms Mn and 1.4 g atoms Zn per mole tetramer whereas A. odontolyticus SOD, specific activity 700 U/mg, contained 1.4 g atoms Mn and 1.8 g atoms Zn per mole tetramer. Actinomyces strain E1S.25D, specific activity 1300 U/mg, contained 1.8 g atoms Mn and 1.2 g atoms Zn per mole tetramer. The amino acid compositions of the enzymes were comparable except for arginine, lysine, and tryptophan content. The enzymatic activity of each enzyme was stable in 5 mM H2O2 at 23 degrees C for 2 h. The enzymes were only modestly inhibited by 20 mM NaN3. The enzymatic activity was increased at low ionic strength but was markedly decreased at increased ionic strength with each salt tested except sodium perchlorate, which caused marked inhibition even at low ionic strength. Polyclonal antibodies to A. naeslundii and Actinomyces strain E1S.25D precipitated and inactivated their respective antigens whereas the precipitated A. odontolyticus superoxide dismutase-antibody complex retained virtually full catalytic activity. Immunological studies revealed that the native A. naeslundii and Actinomyces strain E1S.25D MnSODs share common epitopes and cross-reacted with precipitin lines of complete identity in Ouchterlony double diffusion gels. Antibody to the A. odontolyticus enzyme displayed only partial cross-reactivity with superoxide dismutase from the two other Actinomyces. Western blotting of the denatured antigens revealed reactivities of the antibodies that differed only slightly from the results of the Ouchterlony gels.     

Isolation of Mn-SOD and Low Active Fe-SOD from Methylomonas J; Consisting of Identical Proteins

Cultures of Methylomonas J. an aerobic methylotrophic bacterium, were grown both in Mn-rich and Fe-rich media. Crude extracts of the cultures from the Mn-rich and Fe-rich medium showed a specific activity of 12.2 and 0.6 units/mg by a cytochrome c-xanthine oxidase method and 19.4 and 1.3 units/mg by an ESR method, respectively. We isolated Mn-SOD and Fe-SOD from the bacteria grown in the Mn-rich and Fe-rich mediums, respectively. Specific activity and metal contents of the Mn-enzyme were 2,250 units/mg/g-atom Mn and Mn = 0.98 and Fe = 0.12 (g-atoms/mol dirner), while those of the Fe-enzyme were 61 units/mg/g-atom Fe and Mn = 0.02 and Fe = 1.08. No difference of physicochemical properties of the Fe-and Mn-enzymes were detected. Furthermore, enzyme activity was restored by dialysis of an apoprotein obtained from the Fe-enzyme with either manganese sulfate or ferrous ammonium sulfate.     

Molecular cloning,purification, and characterization of a superoxide dismutase from a fast-growing <Emphasis Type="Italic">Mycobacterium</Emphasis> sp. Strain JC1 DSM 3803

A cytosolic superoxide dismutase (SOD) was purified and characterized from a fast-growing Mycobacterium sp. strain JC1 DSM 3803 grown on methanol. The native molecular weight of the purified SOD was estimated to be 48 kDa. SDS-PAGE revealed a subunit of 23 kDa, indicating that the enzyme is a homodimer. The enzyme activity was inhibited by H₂O₂ and azide. The purified SOD contained 1.12 and 0.56 g-atom of Mn and Fe per mol of enzyme, respectively, suggesting that it may be a Fe/Mn cambialistic SOD. The apo-SOD reconstitution study revealed that Mn salts were more specific than Fe salts in the SOD activity. The gene encoding the SOD was identified from the JC1 cosmid genomic library by PCR screening protocol. The cloned gene, sodA, had an open reading frame (ORF) of 624 nt, encoding a protein with a calculated molecular weight of 22,930 Da and pi of 5.33. The deduced SodA sequence exhibited 97.6% identity with that of Mycobacterium fortuitum Mn-SOD and clustered with other mycobacterial Mn-SODs. A webtool analysis on the basis of SOD sequence and structure homologies predicted the SOD as a tetrameric Mn-SOD, suggesting that the protein is a dimeric Mn-SOD having tetramer-specific sequence and structure characteristics.     

L-histidinol dehydrogenase, a Zn2+-metalloenzyme

The enzymatic activity of L-histidinol dehydrogenase from Salmonella typhimurium was stimulated by the inclusion of 0.5 mM MnCl2 in the assay medium. At pH 9.2 the stimulation was correlated with binding of 1 g-atom of 54Mn2+/mol dimer, KD = 37 microM. ZnCl2, which prevented the MnCl2 stimulation, also bound to the enzyme, 1.2 g-atom/mol dimer, KD = 51 microM, and prevented Mn2+ binding. Enzyme activity was lost when histidinol dehydrogenase was incubated in 8 M urea. Reactivation was observed when urea-denatured enzyme was diluted into buffer containing 2-mercaptoethanol but required either MnCl2 or ZnCl2. Histidinol dehydrogenase was inactivated by the transition metal chelator 1,10-phenanthroline or by high levels of 2-mercaptoethanol. The nonchelating 1,7-phenanthroline was not an inactivator, and inactivation by either 1,10-phenanthroline or 2-mercaptoethanol was prevented by MnCl2. Enzyme inactivated by 1,10-phenanthroline could be reactivated by addition of MnCl2 or ZnCl2 in the presence of 2-mercaptoethanol. Reactivation was correlated with the binding of 1.5 g-atom 54Mn2+/mol dimer. Atomic absorption analysis of the native enzyme indicated the presence of 1.65 g-atom Zn/mol dimer, and no Mn was detected. The results demonstrate, therefore, that histidinol dehydrogenase contains two metal binding sites per enzyme dimer, which normally bind Zn2+, but which may bind Mn2+ while retaining enzyme function. Histidinol dehydrogenase is thus the third NAD-linked oxidoreductase in which Zn2+ fulfills an essential structural and/or catalytic role.     

Apoprotein formation and heme reconstitution of cytochrome P-450cam

Apoprotein suitable for heme reconstitution has been prepared by an acid/butanone extraction of cytochrome P-450cam at pH 2.5. Absorption spectra of apo-P-450cam indicate less than 2% residual holoenzyme. Four tryptophan residues per molecule were estimated from the aromatic absorbance region of denatured apoprotein. Heme-reconstituted holoprotein was purified in 30% yield to a specific activity equivalent to the native enzyme. Absorption and EPR spectra of 57Fe- and 54Fe-heme-enriched P-450cam reveal complete restoration of the native active site.     

Characterization of superoxide dismutases purified from either anaerobically maintained or aerated Bacteroides gingivalis.

Superoxide dismutases (SODs) were purified from extracts of either anaerobically maintained or aerated Bacteroides gingivalis. Each purified enzyme (molecular weight, 46,000) was a dimer composed of two subunits of equal sizes. SOD from anaerobically maintained cells (anaero-SOD) contained 1.79 g-atom of Fe and 0.28 g-atom of Mn, and SOD from aerated cells (aero-SOD) contained 1.08 g-atom of Mn and 0.36 g-atom of Fe. Spectral analysis showed that anaero-SOD had the characteristic of Fe-SOD and that aero-SOD had that of Mn-SOD. Both enzyme preparations contained three isozymes with identical isoelectric points. On the basis of inactivation of SOD by H2O2, it was found that aero-SOD consisted of one Mn-SOD and a small quantity of two Fe-SODs, whereas anaero-SOD contained only Fe-SOD. However, each apoprotein from anaero-SOD and aero-SOD, prepared by dialysis in guanidinium chloride plus 8-hydroxyquinoline, showed only one protein band each with the same isoelectric point on an isoelectric focusing gel. Subsequent dialysis of both apoenzymes with either MnCl2 or Fe(NH4)2(SO4)2 restored the activity. These reconstituted SODs showed only one protein band with SOD activity on native polyacrylamide gel electrophoresis. Furthermore, the two enzymes had similar amino acid compositions, and their amino-terminal sequences were identical through the first 12 amino acids. These results suggest that the three isozymes of anaero-SOD and aero-SOD in B. gingivalis are formed from a single apoprotein.     

Manganese superoxide dismutase from a higher plant

A manganese-containing superoxide dismutase (EC 1.15.1.1) was purified to homogeneity from a higher plant for the first time. The enzyme was isolated fromPisum sativum leaf extracts by thermal fractionation, ammonium sulfate salting out, ion-exchange and gel-filtration column chromatography, and preparative polyacrylamide gel electrophoresis. Pure manganese superoxide dismutase had a specific activity of about 3,000 U mg^-1 and was purified 215-fold, with a yield of 1.2 mg enzyme per kg whole leaf. The manganese superoxide dismutase had a molecular weight of 94,000 and contained one g-atom of Mn per mol of enzyme. No iron and copper were detected. Activity reconstitution experiments with the pure enzyme ruled out the possibility of a manganese loss during the purification procedure. The stability of manganese superoxide dismutase at-20°C, 4°C, 25°C, 50°C, and 60°C was studied, and the enzyme was found more labile at high temperatures than bacterial manganese superoxide dismutases and iron superoxide dismutases from an algal and bacterial origin.Abbreviations NBT nitro blue tetrazolium - SOD superoxide dismutase (EC 1.15.1.1)     

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.     

Potentiometric titrations and oxidation-reduction potentials of manganese and copper-zinc superoxide dismutases.

Bovine erythrocyte superoxide dismutase and two manganese-containing superoxide dismutases have been reduced by the indirect coulometric titration method with methylviologen as the mediator-titrant. On the basis of the titration data the manganese-containing superoxide dismutases contain 1 g-atom of metal per mol of enzyme (dimer). E0' = +0.31 V for the enzyme from Escherichia coli which exhibits a complicated pH dependence above neutral pH. The Bacillus stearothermophilus manganese-containing enzyme has an E0' = +0.26 V and delta Em/pH is 50 mV. Bovine erythrocyte superoxide dismutase exhibits anomalous behavior in the coulometric titration curves, which is indicative of two nonequivalent copper centers in the enzyme. Addition K3Fe(CN)6 or K2IrCl6 to the enzyme solution, prior to coulometric titration, indicates that these anions bind preferentially to one of the copper centers.     

Iron and aconitase activity

Aconitase activated with Fe(2+), cysteine and ascorbate incorporates 1 g-atom of Fe(2+)/mol. Loss of this Fe(2+) by transfer to ferrozine, a Fe(2+) chelator, results in loss of activity. Ascorbate increases the rate of transfer of the essential Fe(2+) whereas citrate retards the rate of transfer. Transfer of Fe(2+) from inactive aconitase, 2 g-atoms of Fe/mol, can be accomplished in the presence of urea and ascorbate. The correlation of activity with the presence of an added g-atom of Fe(2+)/mol leads to the conclusion that active aconitase has only one active site per mol.     

Purification and properties of a unique superoxide dismutase from Nocardia asteroides

A unique form of superoxide dismutase was isolated and characterized from Nocardia asteroides GUH-2. This enzyme contains 1 to 2 g atoms each of Fe, Mn, and Zn per mol and exhibits spectral properties suggestive of Fe- or Mn-containing superoxide dismutases. Its Mr = 100,000, and it is composed of four subunits of equal size which are not covalently joined. The amino acid composition of the enzyme was more closely related to the Mn- or Fe-containing enzymes of Mycobacterium species and was least related to the Cu-Zn enzyme of eukaryotes. Azide at 1 and 20 mM inhibits the activity 10 and 41%, respectively, and 5 mM H2O2 inhibits 40%, but 1 or 5 mM cyanide caused trivial effect. The immunofluorescent staining, which was specific for superoxide dismutase of N. asteroides, indicated the association of this enzyme to the outer cell wall of the organism. Further, the enzyme was shown to be selectively secreted into the medium.     

Regulation of the synthesis of superoxide dismutases in rat lungs during oxidant and hyperthermic stresses

Heat shock proteins are induced at normal temperatures by oxidants and during reoxygenation following hypoxia. We now report cyanide-resistant O2 consumption increased 30-50% in rat lungs exposed to heat shock or reoxygenation following hypoxia. The synthesis of Cu,Zn superoxide dismutase, but not Mn superoxide dismutase, was increased in rat lung slices by in vivo hyperthermia (39 degrees C), by in vitro heat shock (41 degrees C), and during incubation of lung slices with the Cu chelator diethyldithiocarbamate, which decreased the activity of Cu,Zn superoxide dismutase. The heat shock-induced increase in Cu,Zn superoxide dismutase developed 2 h later than the induction of heat shock proteins and was not blocked by actinomycin D. The rates of synthesis of both superoxide dismutases were decreased 50% by hypoxia and failed to increase during reoxygenation. During hypoxia the activity of Cu,Zn superoxide dismutase decreased about 50%, but the activity of Mn superoxide dismutase remained unchanged. We conclude that hyperthermia increases the synthesis of Cu,Zn superoxide dismutase, the synthesis of Cu,Zn superoxide dismutase and Mn superoxide dismutase are not coordinately regulated by hyperthermia or by the oxidant stress produced by lowering the activity of Cu,Zn superoxide dismutase, and the synthesis of heat shock proteins and Cu,Zn superoxide dismutase are regulated at different levels of gene expression.     

The functional role of zinc in angiotensin converting enzyme: implications for the enzyme mechanism

Zinc is essential to the catalytic activity of angiotensin converting enzyme. The enzyme contains one g-atom of zinc per mole of protein. Chelating agents abolish activity by removing the metal ion to yield the inactive, metal-free apoenzyme. Zinc does not stabilize protein structure since the native and apoenzymes are equally susceptible to heat denaturation. Addition of either Zn2+, Co2+, or Mn2+ to the apoenzyme generates an active metalloenzyme; Fe2+, Ni2+, Cu2+, Cd2+, and Hg2+ fail to restore activity. The activities of the metalloenzymes follow the order Zn greater than Co greater than Mn. The protein binds Zn2+ more firmly than it does Co2+ or Mn2+. Hydrolysis of the chromophoric substrate, furanacryloyl-Phe-Gly-Gly, by the active metalloenzymes is subject to chloride activation; the activation constant is not metal dependent. Metal replacement mainly affects Kcat with very little change in Km, indicating that the role of zinc is to catalyze peptide hydrolysis.     

In Vivo Metal Substitution in Bacteroides Fragilis Superoxide Dismutase

Bacteroides fragilis. an obligate anaerobe, synthesizes an azide-inhibitable iron-containing superoxide dismutase when grown in complex medium. Cells grown anaerobically in complex media containing dcsferrioxamine (Desferal^TM, Ciba-Geigy) and graded concentrations of Mn synthesize the azide-resistant manganese-containing SOD. The fraction of MnSOD activity in dialyzed cell extracts increased prograsively as the Mn concentration in the medium increased. The fraction of MnSOD activity also increased in extracts of cells grown in the medium with I mM Mn but with graded concentrations of desferrioxamine (0–10 micromolar). The SOD activity in the cells grown under the various conditions varied but not in a causal relationship with either Mn or desferrioxamine concentration. Electrophoresis reveaicd that the SOD activity in cells grown in the absence or presence of I mM Mn migrated with the same relative mobility and exhibited identical activity patterns when examined separately or as a mixture. These data are consistent with substitution of Mn for Fe in the B. fragilis apoprotein under anaerobic conditions and support the model of a single protein binding either Fe or Mn.     

Chemical and physical differentiation of superoxide dismutases in anaerobes.

Superoxide dismutase activity in crude or partially purified cell extracts from several species and strains of obligate anaerobe Bacteroides was inhibited instantaneously by NaN3 and was inactivated rapidly upon incubation with H2O2. The extent of NaN3 inhibition varied from 41 to 93%, and the half-life of the enzymatic activity in 5 mM H2O2 ranged from 1.2 to 6.1 min, depending upon the organism tests. When grown in a defined medium containing 59Fe, Bacteroides fragilis (VPI 2393) incorporated radiolabel into a 40,000-molecular-weight NaN3- and H2O2-sensitive superoxide dismutase but did not incorporate 54Mn into that protein under similar growth conditions. The anaerobe Actinomyces naeslundii (VPI 9985) incorporated 54Mn but not 59Fe into a NaN3-insensitive and H2O2-resistant superoxide dismutase. The apparent molecular weight of the superoxide dismutase from this and several other Actinomyces spp. was estimated to be 110,000 to 140,000. Comparison of these data with studies of homogeneous metallosuperoxide dismutases suggests that the Bacteroides spp. studied contain a ferrisuperoxide dismutase, whereas Actinomyces spp. contain a managanisuperoxide dismutase.