Superoxide Dismutases WAYNEBEYER, IMLAY AND IRWIN FRIDOVICH~ JAMES
Department of Biochemistry Duke Unioersity Medical Center Durham, North Carolina 27710
I. Superoxide and Superoxide Dismutases . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Brief History . . B. Dangers of the ................................. C. SODS:Varieties ........,.. D. X-Ray Crystallograp F. Reversible Resolution , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . Catalytic Mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. Reactions with H 2 0 z . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Superoxide and Oxidative DNA Damage . . . . . . . . . . . . . . . . . . . . . . . . . . . A. DNA Damage Products B. Enzymatic Repair of Oxi amage .................... C. How Prevalent Is Oxidative 111. Cellular Regulation of SOD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Inductions B. SOD Muta C. Additional Regulons Pertinent to Oxy-radical Damage . . . . . . . . . . . . . IV. Epilogue . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . , . . . . . . . . . . . . . . . . . , . . . . . . . . , . . . .
221 221 222 224 226 230 234 236 237 238 239 240 242 242 242 245 246 248 248
1. Superoxide and Superoxide Dismutases A. Brief History Enzymes are usually discovered and subsequently isolated on the basis of their catalytic activities. Physicochemical characterization, which follows isolation, thus lags behind discovery of the activity. In the case of SOD,2 this sequence of events was reversed. David Keilin had embarked on the isolation of carbonic anhydrase, which he suspected might contain copper. All of the protein fractions obtained from the hemolysate were therefore being assayed both for catalysis of the hydration of CO, and for copper content. Given that carbonic anhydrase contains zinc, not copper, it was inevitable To whom correspondence may he addressed. Superoxide dismutase (EC 1.15.1. l), which catalyzes the conversion of superoxide ( 0 2 - ) 2 and HzOz. [Eds.]
22 1 Progress in Nucleic Acid Research and Molecular Biology, Vol. 40
Copyright 8 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
WAYNE BEYER ET AL.
that the copper and the catalytic activity should part company. The coppercontaining protein, which had been encountered during these fractionations, was isolated on the basis of enrichment for copper. Since it had no known activity, it was named hemocuprein and tentatively assigned the function of copper storage (I).Virtually identical proteins were subsequently isolated from a variety of sources and were given names that reflected their tissue of origin and copper content. Hence, the literature dealing with erythrocuprein, hepatocuprein, cerebrocuprein, and cytocuprein (2-9) burgeoned. Independent studies of the peculiar oxygen-dependent properties of milk xanthine oxidase led to the realization that this enzyme produces 0,as well as H,O,. Having an enzymatic source of 0 2 - which , was demonstrably responsible for several easily measurable chemical changes, led to the discovery of SOD activity, and this activity guided the isolation of the enzyme from bovine erythrocytes (10, 11). Once the enzyme was in hand, its physicochemical similarities to the cupreins were obvious. The availability of a spectrophotometric assay for SOD activity and of an activity stain applicable to polyacrylamide gels (12)facilitated the isolation of SODS from a variety of sources. A manganese-containing SOD (Mn-SOD) was rather quickly isolated from Escherichia coli (13)and from chicken liver mitochondria (14), and an iron-containing SOD (Fe-SOD) was isolated from E. coli (15).The history of these events has been described in considerable detail (16-18). These SODS are widely distributed, abundant, and stable, and thus are well-suited to studies of structure, function, and evolution.
B. Dangers of the Substrate
The ubiquity and abundance of SODSamong aerobes suggest that their substrate, 0, -, is frequently encountered and potentially damaging. In fact, the barrier to divalent reduction of O,, which is raised by the spin restriction (19), leaves univalent reduction as the most facile pathway. Although predominant in abiotic 0, reduction, this univalent free-radical pathway is usually minimized in living cells by enzymes that accomplish two- or fourelectron reductions of O,, without the release of intermediates. Nevertheless, 0,- is produced in cells during both enzymatic and spontaneous oxidations. Moreover, its rate of production can be markedly increased by photosensitizers in the light, and by viologens, quinones, and arylnitro compounds in the dark. These and related compounds undergo cycles of enzymatic reduction followed by auto-oxidation and thus mediate net 0, production. Biological sources of 0,- have been described (20, 21). Free radicals are usually reactive, and their reactions with diamagnetic molecules always produce new radicals. We may therefore anticipate the possibility of direct attack of 0,- on cellular targets, as well as indirect
effects due to other radicals engendered by 0,- . Several (Fe-S),-containing enzymes appear to be subject to direct attack by 0,-. Among these is the E . coli dihydroxy-acid dehydratase (EC 188.8.131.52),which is on the pathway of biosynthesis of branched-chain amino acids and which is exquisitely sensitive to 0,- (22, 23). Other enzymes directly inactivated by 0,- include catalases and peroxidases (24). Small molecules can also serve as targets for 0,-, which can initiate and sometimes also propagate free-radical oxidations of sulfite, enediolates, polyphenols, catecholamines, and tetrahydropterins. The ability of 0,- to act as an oxidant can be markedly augmented by association with cationic centers, such as protons or the metals Mn(I1) or V(V). Thus, 0,- per se does not oxidize NADH, but does so in the presence of Mn(I1) or V(V), due to the formation of Mn(I)O, or V(IV)O,. In the presence of V(V), 0,- initiates a free-radical chain oxidation of NAD(P)H, which results in the oxidation of many molecules of NAD(P)H per 0,- introduced (25). Similarly, 0,- does not directly oxidize polyunsaturated fatty acids, but protonation (pK, = 4.7) yields HO,, which does so (26). H,O, is produced by the dismutation of 0,- and is therefore present whenever 0,- is being made in aqueous media. This sets the stage for the generation of devastatingly powerful oxidants. Thus, 0,- can reduce transition metals, such as Fe(II1) or Cu(II), whose reduced forms can, in turn, reduce H,O,, yielding HO- or metal derivatives of HO.. This process, which is sometimes called the metal-catalyzed Haber-Weiss reaction, can be represented as follows: Fe(II1) + 02-+ Fe(I1) + O2 Fe(I1) + HOOH .+ F e ( I W 0 H + H + Fe(I)-OOH Fe(II)==O HOFe(I1)O + H + -+ Fe(II1-H Fe(II1) + HO.
Since the metal reduced in reaction (a) is reoxidized during reactions (b)(d), its role is catalytic. This allows low levels of the metal to mediate substantial production of HO.. Indeed, an early report (27)of the production of HO. by an enzymatic source of 0,- and HzO, failed to recognize the catalytic role of the metal, which was present as an impurity in the phosphate buffer. HO. and Fe(I1)O are extremely reactive and would not survive many collisions with cellular constituents without reacting. This leads to the expectation that HO., generated in free solution, would not be very damaging because it would expend itself on easily replaced sugar phosphates, amino acids, nucleotides, or metabolic intermediates. However, since the catalytic metals are likely to be bound onto the polyanionic DNA or cell membranes,
WAYNE BEYER ET AL.
the HO. or Fe(I1)O would be generated adjacent to, and would preferentially attack, these critical targets. This has been called the site-specific Haber-Weiss reaction (28, 29). Metals such as Zn(II), which can compete with Fe(II1) or Cu(1I) for binding sites, but which cannot participate in redox reactions, should protect. This has been demonstrated (30),as has the anticipated protection by SOD or catalase, or by chelating agents, which would displace the bound metal (31). Desferal, because of its selective affinity for Fe(III), hinders redox cycling of iron and thus prevents this metal from catalyzing HO. production from 0,- plus H,O, (32). Diethylenetriamine pentaacetic acid (pentetic acid) exerts a similar effect (33). Metal-catalyzed production of HO. from 0,- plus H,O, appears to occur in uiuo. DNA scissions within fibroblasts, caused by H,O,, can be prevented by the chelating agents o-phenanthroline or a,&’-bipyridyl. Moreover, HO. scavengers, such as thiourea, also protect, whereas inactivation of the intracellular Cu,Zn-SOD with diethyldithiocarbamate augments the DNA damage (34). Similar studies of the effect of H,O, on the viability of hepatocytes established that Desferal, SOD, catalase, and thiourea protect, in accord with the view that HO. generated from 0,- + H,O, is the lethal species (35). Mutants of E. coli with defects in both SOD genes were hypersensitive to aerobic H,O,, even though they contained normal levels of catalases, an indication that 0,- can augment the damaging effect of H,O, (36). Hydroxylated purines and pyrimidines are among the products of attack on DNA by HO. (37).These products are normally excreted in urine and can be found in both nuclear and mitochondria1 DNA (38, 39). Their rate of production appears to be inversely related to life span (40). All of this supports the view that the metal-catalyzed Haber-Weiss reaction occurs in uiuo. The theoretical basis of this reaction has been considered (41).
C. SODs: Varieties and Distributions Common selection pressures applied to varied biotas are apt to elicit multiple solutions to the same problem. One need only consider the manifold types of protective coloration, mimicry, and other forms of camouflage evolved in response to the pressures of predation in order to appreciate the validity of this statement. The oxygenation of the earth, by the earliest water-splitting photosynthetic organisms, must have imposed such a selection pressure on a biota composed of anaerobes. It is thus not surprising that we now find more than one type of SOD. We know of two independently evolved groups of SODs, the Cu,ZnSODS, on the one hand, and the Mn-SODs/Fe-SODS, on the other. Similar multiplicity is seen with other enzymes providing antioxidative defense. There are catalases based on heme and others based on binuclear clusters of
manganese ions, and some lactobacilli are capable of producing one or the other, depending on conditions of growth. There are also peroxidases based on heme and others that contain selenocysteine at the active site. In some cases, the distribution of SODS provides a clear indication of evolutionary history, while other cases remain enigmatic. Mitochondria contain a Mn-SOD, while the cytosols of eukaryotic cells contain a Cu,Zn-SOD. The mitochondria1 Mn-SOD is strikingly homologous to the Mn-SODs/FeSODs found in prokaryotes, but bears no resemblance to the cytosolic Cu,Zn-SOD, which supports the idea of an endosymbiotic origin for this organelle (42, 43). Among the enigmas is why a few bacteria contain Cu,ZnSODs, which are typically found in eukaryotic cytosols. These bacterial Cu,Zn-SODS, usually referred to as bacteriocupreins, do show homology to their eukaryotic counterparts (44),but contain a leader peptide indicative of a periplasmic localization (45).Another puzzle is the occurrence of Fe-SODS in certain plants (46, 47).
1. CU,ZN-SODS Although found most often in the cytosols of eukaryotic cells, Cu,ZnSODS have been identified in, and isolated from, several bacteria, including Photobacter leiognathi (48),Caulobacter crescentis (49),two pseudomonads (50), and Paracoccus denitrijicans (51). There is, moreover, a tetrameric, glycosylated Cu,Zn-SOD found in mammalian extracellular fluids (52). Cu,Zn-SODS have been thoroughly reviewed (43, 53-55) and are not described here in further detail.
2. MN-SODS AND FE-SODS Mn-SODS and Fe-SODS were first isolated from E . coli (13,15)and have since been found in, and isolated from, a variety of prokaryotes. Table I summarizes the properties of the Mn-SOD and Fe-SOD of E . coli. Although most frequently encountered as homodimers with a subunit M , of -22,000, some bacterial Mn-SODS (56, 57) and Fe-SODS (58-60) occur as tetramers. Mitochondria contain a homotetrameric Mn-SOD (61-63) with extensive sequence homology to the corresponding bacterial enzymes (42),in keeping with a prokaryotic ancestry and an endosymbiotic origin for this organelle (64). Anaerobes, or anaerobically grown facultative bacteria, usually contain a Fe-SOD, while aerobic growth is often associated with induction of a MnSOD. In spite of extensive sequence homologies, Mn-SODS and Fe-SODS are usually active only when the catalytic site is occupied with the metal found in the native enzyme. Nevertheless, a few species of Bacteroides produce a single SOD protein which contains, and is active with, Fe when grown anaerobically and with Mn when grown in the presence of 0, (65-68).
WAYNE BEYER ET AL.
TABLE I COMPARISON OF E . coli Mn-
Oligomeric state Subunit M Number of amino acids Isoelectric point Metal content (per subunit) Absorption Visible (Amax [el" UV (A, [A1%Ib Inhibitors Specific activity (U/mg) Stokes radius (A)
Dimeric 21,100 192 4.454.93 0.8-1.2 (Fe)
Dimeric 22,900 205 6.64, 6.90 0.6-0.9 (Mn)
s20 ( ~ 1 0 - 1 3 )
v (partial specific volume)
350 nm (1850 M-lcm-l 280 nm (25.4) H202,N3- (-2 mM) 50% 4200-5000 28.5 2 1.3 3.22 0.3 0.733
470 nm (610 M-'em-1) 282 nm (18.9) N3- (-30 mM) 50% 2800-3800 29.0 k 1.7 3.55 f 0.3 0.735
"Based on metal. bBased on protein.
A few species of plants contain Fe-SODS (69-71). There is, as yet, no adequate explanation for the presence of Cu,Zn-SODS in some bacteria, and of Fe-SODS in a few plants.
D. X-Ray Crystallography of Mn-SODS and Fe-SODS Mn-SODS from yeast (72),E . coli (72), Thermus thermophilus (73),and Bacillus stearothermophilus (74)have been examined by X-ray crystallography, in the latter two cases to a resolution of 2.4 A. Studies of the B . stearothermophilus Mn-SOD were actually initiated in the mid-1970s (75), but were discontinued because of the report of preliminary studies of the E . coli Mn-SOD (72).The latter studies have not yet been carried to fruition, but structural analysis of the B . stearothemphilus enzyme was subsequently pursued to 2.4 A resolution (74). X-ray crystallographic analysis has also been applied to the Fe-SODS from Thermoplasmu acidophilus (76), Pseudomonas ovalis (77), and E . coli (78). The Fe-SODS from P. ovalis and E . coli exhibit practically identical structures. Crystals of the P. oualis Fe-SOD were unstable in the x-ray beam. This problem was overcome by using the azide or fluoride derivatives. The instability may have been due to radiochemical production of H,O,, which is known to inactivate Fe-SODS (79, 80). That Fe-SODS and Mn-SODS also exhibit similar structures might be expected from their sequence homologies (81).Comparison of the tetrameric Mn-SOD from T. thermophilus with the dimeric E . coli Fe-SOD revealed one metal atom per subunit in comparable binding sites (82).The tetrameric enzyme showed two sets of subunit con-
tacts. One of these corresponded with the dimer interface of the E. coli FeSOD, while the other involved an inserted peptide segment absent from the E. coli Fe-SOD. The conformations of the Mn-SOD/Fe-SOD family of enzymes is unrelated to that of the Cu,Zn-SOD enzyme family (78), as expected from the lack of sequence homology between these families of SODs. The structure of one subunit of the Mn-SOD from B. s t e u ~ ~ t ~ e ~ p ~ i ~ u (74) is shown in Fig. 1. Fitting amino-acid sequences to electron-density maps allowed detailed structural analysis, which shows that both Mn-SOD (73) and Fe-SOD (81) ligate the metal at the active site through three histidine imidazoles and one
FIG. 1. The B. stearothennophilus Mn-SOD monomer. a-Helices are labeled al-a7 and P-strands Pl-P3. The active-site manganese and its ligands are indicated. A probable pathway of approach of the 0 2 - ion and the proximity of Tyr-34 and Lys-29 are shown. (Reproduced from 74.)
WAYNE BEYER ET AL.
aspartate carboxylate, as specified in Table 11. A liganding water molecule was also evident in the E . coli Fe-SOD and the T.thermophilus Mn-SOD, but not in the B. stearothermophiluv Mn-SOD (74, 81). The coordination geometry was described as tetrahedral, with distortion toward either a trigonal bipyramid (73, 81) or a trigonal pyramid (74).It is a curious coincidence that the Zn(I1) in the structurally unrelated Cu,Zn-SOD is also ligated to three histidines and one aspartate (83). The active site in Mn-SOD and Fe-SOD is surrounded by hydrophobic residues, including three Tyr, three Trp, and two Phe within 10 A of the metal center, with extensive aromatic stacking. Mn(II1) is potentially a strong oxidant; in water it dismutes to Mn(I1) plus MnO,. The electrodative ligand field and the surrounding shell of hydrophobic residues may serve to stabilize Mn(II1) at the active site of Mn-SOD. The importance of the residues in the hydrophobic shell around the active site is supported by their conservation in all Mn-SODS and Fe-SODS for which sequence data are available (84). It should be noted that Fe-SODS contain a Tyr at position 76, whereas in Mn-SODS we find a corresponding Phe (74, 81, 85). Fe-SOD and Mn-SOD subunits exhibit a two-domain structure in which one domain contains a-helices while the second is composed of both ahelices and P-sheets. The metal binding site occurs between these two domains. The hinge region exhibits both insertions and deletions (74).In the case of the Mn-SOD from B.stearothennophilus, each subunit approximates a triangular prism, whose dimensions are 43 x 53 x 45 A. One domain of this subunit contains a pair of long, antiparallel a-helices, designated a1 and a3 in Fig. 1, which diverge at a 35" angle. A kink in al, in a histidine-rich TABLE I1
GEOMETRY OF THE MANGANESE-BINDING SITE IN B . stearothermophilus Mn-SODa Bond His-26 (Nep)-Mn His-81 (Ned-Mn His-167 (Nez)-Mn Asp-163 (OSI)-Mn
Distance 2.08 2.38 1.94 1.94
His-26 (Nez)-Mn-His-81 (Nez) His-26 (Nsz)-Mn-Asp-163 (08,) His-26 (Nez)-Mn-His-167 (Nez) His-81 (Nez)-Mn-His-167 (NQ) His-81 (Nez)-Mn-Asp-163(06,) His-163 (Nez)-Mn-His-167 (Nez)
96" 88" 97" 134O 92"
region, has the effect of positioning a conserved tyrosine residue (Tyr-34) closer to the metal center (73, 74). A two-turn helical segment, designated 012, is found in Mn-SODS, but not in Fe-SODS (73, 74, 81). In the T. t h e m p h i l u s Mn-SOD, a2 supplies residues that stabilize the tetrameric structure. The human and mouse tetrameric Mn-SODS, however, exhibit seven residue deletions in this region. This has led to the suggestion that a2 and the basis of tetramer assembly are species-specific (73, 74). Both a1 and a3 each contribute one histidine ligand to the metal center, while the second domain provides one histidine and one aspartate. The second domain may be described as a sandwich, with three helices (ad, a5, and 016) on one side, a @sheet in the middle, and an extended non-helical chain on the other side. The interdomain hinge joins 013 to a 4 and follows an unusual path that may constrain the manner of folding (73). Ligation of the metal by residues from both domains allows the metal to provide for both stabilization and catalysis. The active-site metals are 18 A apart in the dimeric Mn-SODs/Fe-SODS, There is no main-chain interpenetration across the dimer interface of the dimeric Mn-SOD from B . stearothermophilus, but in the corresponding tetrameric enzyme from T. thermophilus, Tyr-172 and Glu-168 from one subunit penetrate into the active-site region of the neighboring subunit, perhaps contributing to stability. Table I11 (74) summarizes intersubunit contacts. E . coli Mn-SOD and Fe-SOD must have subtle differences in their dimer contact regions. Thus, removal of the metal leaves the apo-Mn-SOD as a dimer, whereas the apo-Fe-SOD dissociates into subunits (85). The suggestion that insertion of metal into the apoenzymes requires dissociation TABLE I11 MONOMER-MONOMERCONTACTS OBSERVED IN THE B . stearothennophilus Mn-SODa Monomer 1 Atom
His-30b Ser-1286 Glu-1666 Glu-1666 Hi~-167~ Hi~-167~ Tyr-1706 Phe-126 Trp-1656
0% N N81 OH
aFrom 74. bhvariant residue. CManganese ligand.
Monomer 2 Atom OH
OY N Nai OE1 OE2 NEZ
Residue T1y-170~ Se~128~ Hi~-167~ Hi~-167~ Glu-1tX6 Glu-1666 His-306 Trp-1656 Phe-126
Distance 2.66 3.06 2.86 3.02 2.86 3.02 2.66
of subunits (86) can be questioned on the grounds that native, dimeric apoMn-SOD can be reconstituted by the addition of Mn(I1) salts in the absence of chaotropic agents (87). A tyrosine residue (Tyr-36 in the 7’. thernwphihs Fe-SOD or Tyr-34 in the B. stearothermophilus Mn-SOD) places a phenolic hydroxyl within 5 A of the metal center (73, 74). Ionization of this phenolic hydroxyl may account for the activity-limiting ionization (PI(,= 9.5) responsible for a decline in activity with increasing pH (88-92). Electrostatic facilitation of the dismutation reaction has been demonstrated (93, 94) and may be provided by the
preponderance of cationic residues around the entrance to the active-site pocket. The effects of dithionite reduction of crystals of Mn-SOD (95) and of ascorbate reduction of crystals of Fe-SOD (95) were examined by difference Fourier methods. No significant conformational changes in the active-site region were noted upon reduction. It is possible that crystal lattice forces constrained changes which might otherwise take place upon reduction in free solution (95).
E. Evolutionary Relationships Figure 2 presents sequence data for Mn-SODS and Fe-SODS from a variety of sources. Much homology, suggesting a common evolutionary origin, is evident. Approximately 28 residues are invariant throughout, and many instances of conservative replacements at other positions indicate retention of conformation. Table IV summarizes the degrees of homology between pairs of Mn-SODS and Fe-SODs. The amino-terminal regions are particularly highly conserved, 21% of the invariant residues being found in residues 1-20. Possible explanations for this concentration of conserved residues in the amino-terminal region have been offered (74, 96). In spite of considerable sequence homology, the Mn-SOD and Fe-SOD of E. coli are not immunologically cross-reactive (97, 98), presumably because epitopes involve the least conserved surface residues. Most Fe-SODS are active only with iron at the active site and most MnSODs are correspondingly activity-specific for manganese, even though other metals can compete for occupancy of the active site (101). This specificity must depend on subtle structural differences (74, 96, 99, 100).The Bacteroides enzyme, which is active with either Mn or Fe at the active site (6568), may hold the key to this puzzle and should be carefully studied. Increasing ionic strength decreases the activities of all SODs, in keeping with an electrostatically facilitated catalytic process (93, 94, 102-104). Chemical modifications that eliminate positive charges in the active-site region also diminish activity (93, 94, 105).Numerous cationic residues are clustered near the entrance to the active-site pocket, including histidines 17,
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A K - K
F K A A I A -
FIG. 2. Aligned amino-acid sequences of nine Mn-SODS and four Fe-SODS. The metal ligands His-26, His-81, Asp-175, and His-179 are indicated by V.Invariant regions in the aligned sequences are indicatedby boxed areas. The references (metalion utilized and host organism)are [ l ] Mn H . halobium (204), Mn T.thennophihis (205),Mn maize (206), Mn mouse (209,  Mn rat (208), Mn human (209-211),  Mn S. cerevisiae (212,213), Mn B . stearothermophilus(121), MnE. coli(176,214),[lo] FeE. coli(81,215),[ l l ] FePs. ooalis(216), FeP. Zeiognathi (217),and  Fe Anacystic nidulans R2 (218).(Reproducedfrom 84.)
N K N N N N N N A N A K
AMINO-ACID SEQUENCE IDENTITY BETWEEN
Mn Human Mouse Mn Human Mouse S. cerevisiae B. stearothemphilus T . themphilus E . coli Fe E . coli Ps. ovalis P . leiognathi
S. cerevisiae B . stearothemphilus
T . thennophilus E . coli E . coli Ps. ovalis P . Zeiognathi
43 48 48 42
39 42 39
40 42 37
40 42 38
34 34 34
49 52 49
40 41 37
42 43 40
aThe degree of sequence identity is expressed as a percentage. (From 96.)
27, 30, 31, and 78; lysines 20, 28, and 172; and arginines 125, 176, and 189 (74). Lys-28, thought to guide the approach of 0,- to the active site (96), is conserved in all Mn-SODS and Fe-SODS except the Mn-SOD from Halobacterium halobium, which functions at an ionic strength high enough to greatly weaken electrostatic interactions (106). Chan et al. (84) found that an arginine residue, most likely the conserved Arg-189, is important for the activities of Mn-SODS and Fe-SODS. The one exception was the Mn-SOD of Saccharomyces cerevisiae, in which Arg-189 is replaced by a lysine. As expected, this enzyme was very sensitive to lysine-modifying reagents.
1. HYBRIDSODs Given that the Mn-SOD and Fe-SOD from E . coli are homodimers with considerable sequence homologies, it is not surprising that a hybrid (HySOD) occurs. When first isolated (107),this E . coli Hy-SOD was found to contain 0.8 atoms of Fe per dimer and to be sensitive to H,O,. Its specific activity was low (350 U/mg protein). Because of their very different isoelectric points (4.9 for Fe-SOD and 6.6 for Mn-SOD), electrophoresis of the native E . coli enzymes readily separates Mn-SOD, Fe-SOD, and Hy-SOD. The isolated hybrid enzyme was unstable because of resegregation of the subunits. Reinvestigation of Hy-SOD (108) resulted in a preparation whose specific activity was 1900 U/mg and that contained 2.3 atoms Fe plus 0.41 of Mn per dimer. This Hy-SOD was inactivated to a limit of 70% by H,O,, whereas the corresponding figure for Fe-SOD is 90% and for Mn-SOD, 0%. Streptomycin, which had previously been seen to bind tightly to the E . coli Mn-SOD and Fe-SOD without eliminating catalytic activity ( l o g ) , similarly associated with Hy-SOD. Treatment of Hy-SOD with H,O, was accompanied by resegregation of the subunits and the formation of active Mn-SOD and inactive Fe-SOD (108).As expected for a hybrid, apo-Hy-SOD regained activity when treated with either Mn(I1) or Fe(I1). Separation of the subunits and the formation of hybrid molecules have also been reported for Cu,ZnSODS (110-112). 2. ACTIVITY WITH EITHERMN OR FE
A few SODs appear to be active with either Mn or Fe at the active site (65-68, 113, 114). Propionibacterium shermanii make either a Mn-SOD or an Fe-SOD, depending on the relative abundance of these two metals in the culture medium (114). The Mn-SOD, isolated from cells grown in Fe-poor medium, and the Fe-SOD, obtained from cells grown in Mn-poor medium, had identical amino-acid compositions and amino-terminal sequences (12 residues). The subunit M , of the P. s h e m n i i enzyme was reported to be -32,000, and the specific activity was 900 U/mg for the Mn-SOD and 400 U/mg for the Fe-SOD (115).These are aberrant in that well-characterized
W A Y N E BEYER ET AL.
Mn-SODS and Fe-SODS have subunit M,s of -22,000 and specific activities in the range 4000-5000 U/mg. Re-examination of the properties of the P. s h e m n i i enzyme is clearly in order. Bacteroides fragilis produces an Fe-SOD when grown anaerobically. This Fe-SOD is a homodimer with a specific activity of 1200 U/mg. It has a M, of -42,000 and contains 1.8-1.9 atoms of Fe per dimer (65). The B. fragilis apo-SOD could be reactivated with either Mn(I1) or Fe(II), and its electrophoretic mobility was not influenced by which metal it contained. Exposure of anaerobically grown cells to low levels of 0, caused the appearance of a Mn-SOD whose polypeptide appeared identical to that of the Fe-SOD (66). Similar results have been obtained with other species of Bacteroides (67, 68). Streptococcus mutuns produces a Mn-SOD or an Fe-SOD, depending on which metal was most abundant in the culture medium, and the name “cambialistic” was suggested for enzymes capable of being active with more than one prosthetic group (113).Unfortunately, the data for this enzyme suggest that further study is also required. Thus, the Fe-SOD had a specific activity of only 176 Ulmg, whereas that of the Mn-SOD was 4500 U/mg. Moreover, earlier work (116) found only a Mn-SOD in S. mutans. It remains possible that a small impurity of active Mn-SOD could have been responsible for the low activity reported for the Fe-SOD of S. mutans (113).One must also allow for the frequent cross-contamination of iron salts with manganese; only ultrapure salts should be used for reconstitution of apoproteins prepared from “cambialistic” SODs. A useful diagnostic is sensitivity to H,O,, since Fe-SODS are inactivated by H,O, but Mn-SODS are not. This test was applied in the case of the Bacteroides enzymes, but not in the other cases reported. Table V summarizes metal contents for SODs reported to contain multiple metals. Since many metals compete with the native metal for occupancy of the active site (101),it seems likely that reports of SODS containing Fe + Mn Zn, or even Cu, are the result of such competition. In no case has the functional significance of these multiple metals been evaluated.
F. Reversible Resolution Specific procedures have been developed for the removal and restoration of the metal center from Mn-SOD and from Fe-SOD (80,85, 101, 117,118). Iron is removed from Fe-SOD, with loss of activity, by anaerobic treatment with dithiothreitol plus EDTA at alkaline pH (80,85,117,118).Subsequent dialysis against Fe(II), but not Fe(III), restored -80% of the original activity. Other procedures for the reversible resolution of Fe-SOD have been tried, but with a much lower restoration of activity (85, 113, 114, 119). Treatment of Mn-SOD with urea or guanidinium chloride, plus a chelat-
TABLE V METAL CONTENT"
Specific activity (Uh)
Mycobacterium phle i
Thennoplasma acidophilum Methanobacterium bryantii Norcardia asteroides
Y. Chikata, E. Kusonose, K. Ichihara and M. Kusunose, Osaka City Med J. 21, 127 (1975) 58
Bacteroides fragilis Pkctonemu boryanum
Bacteroides thetaiotaomicron Bacteroides fragilis Rhodococcus bronchialis
B. L. Beaman, S. M. Scaks, S. E. Moring, P. Deem and H. P. Misra, JBC 258, 91 (1983) 65 K. Asada, K. Yoshikawa, M. A. Takahashi, Y. Magday and K. Enmanji, JBC 250, 2801 (1975) 67
Ginko biloba Halobacterium halobium
2.2, 2.2 (Cu) 0.5, 1.5 (Cu)
nNR, Not reported.
65 K. Ichihara, I. Kasaoka, E. Kusunose and M. Kusunose, J . Gen. Appl. Microbiol. 26, 387 (1980) 69 106
WAYNE BEYER ET AL.
ing agent at low pH, removes the metal and causes loss of activity (120-123). The apo-Mn-SOD is then reconstituted by treatment with Mn(II), with a regain of -6040% of the initial activity. In most of the procedures devised to date, the apo-Mn-SOD is kept in the presence of the chaotrope until dialyzed against the reconstituting metal (101,120,122-124). We have noted that the apo form of E . coli Mn-SOD is stable in the absence of chaotropes only in dilute solutions (