J. Mol. Biol. (1976) 107, 175-178
LETTERS TO THE EDITOR
Superoxide Dismutase from Bacillus stearothermophilus. Preparation of Stable Apoprotein and Reconstitution of Full}, Active Mn Enzyme A stable metal-free apoprotein has been obtained from Bacillus stearothermophilus Ma superoxide dismutase following exposure to 8M-urea at acid pH. The apoenzyme retains a dimeric structure but is inactive. Total reeonstitution of fully active Mn enzyme was achieved by addition of Mn 2+ to the apoprotein in the presence of urea. Reconstitution has been monitored by absorbtion spectroscopy, gel electrophoresis, Mn analysis and regain of activity. By these criteria the reconstituted enzyme appears to be indistinguishable from native Mn superoxide dismutase. The greater stability of the thermophile apo- and Mn enzymes is of advantage for studies of the structure and mechanism of action of superoxide dismutase. Superoxide dismutases are widely distributed enzymes which catalyse the dismutation of superoxide radicals to hydrogen peroxide and molecular oxygen (for review see Fridovich, 1975). The enzyme appears to occur in two evolutionarily distinct forms. Cu/Zn enzymes found in the cytoplasm of eukaryotic organisms are dimers consisting of two chemieally identical subunits each containing one a t o m of Cu and Zn. Removal of the two metals with chelating agents causes total inactivation but activity is partially restored b y replacement of Cu alone, and fully restored b y replacement of both metals (Beem et al., 1974). Superoxide dismutases from eukaryotic mitochondria and from prokaryotes, on the other hand, contain either Mn or Fe and possess either dimeric or tetrameric structures comprising of chemically identical subunits. Moreover the two different classes of dismutase appear to be of independent origin (Steinman & Hill, 1973; Bridgen el al., 1975). The three-dimensional structure of the Cu/Zn enzyme from bovine erythrocytes has been elucidated (Richardson et al., 1975} and the protein ligands to the two metals have been identified. The three-dimensional structures of the Mn-eontaining enzymes from Escherichia coli (Beem et al., 1976) and Bacillus stearothermophilus (Bridgen et al., 1976) are also being investigated, and in order to complement these studies we have sought to prepare a Mn-free apoprotein as an aid to investigations of the mode of binding of the metal and of its role in the catalytic reaction. A t t e m p t s to prepare a M_n (or Fe)-free apoenzyme for independent study have hitherto been frustrated b y the instability of the putative apoenzyme under conditions t h a t ensure complete removal of the metal from the native enzymes (cf. Keele et al., 1970; Puget & Michelson, 1974). More recently, however, Ose & Fridovieh (1976) have reported the reversible removal of manganese from E. coli superoxide dismutase. Nevertheless, the apoprotein as such was again found to be unstable in solution and reconstitution of active Mn enzyme could only be achieved in situ b y addition of excess MnCl2 in the presence of the metal chelating agent, 8-hydroxyquinoline, and 2 M-guanidinium hydrochloride; i.e. under conditions t h a t allow the metal to be released from the native enzyme. 175
176
C. BROCK E T A L .
Enzymes from thermophilic bacteria are known to be more stable than their counterparts in mesophiles. Thus from B. stearothermophilu8 it has been possible to prepare a stable crystalline apo- (NAD-free) glyceraldehyde 3-phosphate dehydrogenase suitable for X-ray crystallographic study (Suzuki & Harris, 1971) as well as a stable but inactive metal-free class II aldolase (Jack, 1973) from which active metallo enzyme could be reconstituted by addition of the appropriate divalent metal (Hill et al., 1976). In this paper we report the preparation and charactel%ation of a stable but inactive metal-free apoprotein from B. stearothermophilus superoxide dismutase, and the total reconstitution of native active enzyme with manganese. B. stearothermophilus ceils (strain NCA 1503) were grown, disrupted and extracted as described previously (Atkinson et al., 1972). Pure Mn superoxide dismutase was obtained in a yield of 1 g from 10 kg frozen cells from the 'phosphofructokinase rich' fraction (cL Hengartner & Harris, 1975) by gel filtration on a column of Sephadex G75 of the fraction that did not absorb to AMP-Sepharose. The pure enzyme has a molecular weight of 40,000 and comprises of two identical protein subunits (Bridgen et al., 1975). It contains firmly bound manganese and in contrast to the eukaryotie Cu/Zn enzyme the M_n enzyme from B. stearothermophilus is stable in EDTA even in 8 M-urea at neutral pH. However, in 8 M-urea, 10 mM-EDTA adjusted to pH 3 to 4 with either acetic acid or HC1 the purple-red colour disappears with concomitant loss of manganese and of enzyme activity. We have therefore used these conditions to prepare pure apoprotein. A 9 mg/ml solution of pure M_n superoxide dismutase in 50 mM-Tris.HC1 (pH 7.5) was dialysed for 12 hours at 4~ against 8 M-urea, 10 mM-EDTA adjusted to pH 3"7 with acetic acid. The resulting colourless solution was next dialysed for 6 to 12 hours against 8 M-urea, 10 mM-EDTA, 50 mM-Tris-HC1 (pH 7.5), followed by dialysis against the same buffer with 0.1 mM-EDTA but lacking urea. The slightly turbid solution was centrifuged (if the dialysis against 8 M-urea at neutral pH is omitted most of the protein has precipitated at this stage). The soluble protein (approx. 9 mg/ml) contained less than 0.01 atom Mn/mol, did not absorb at 478 to 480 nm and was completely inactive when assayed for superoxide dismutase activity by two different methods (i~IcCord & Fridovich, 1969; Rotiho et al., 1972); Table 1. The apoprotein was indistinguishable from the native enzyme when it was analysed by acrylamide gel electrophoresis at pH 8.9, or by gel-filtration on Sephadex G75, showing that the dimerie structure is maintained in the absence of the metal. Reconstitution of active Mn enzyme does not occur by addition of MnC12 to the apoprotein in buffer at neutral pH. Total reconstitution of fully active superoxide dismutase was, however, found to occur following exposure of the apoprotein to excess MnC12 in the presence of 8 ~-urea at acid pH. In a typical experiment the apoprotein (5 mg/ml) in 50 mM-Tris.HC1 (pH 7.5), 0.1 mM-EDTA was dialysed successively for periods of up to 12 hours at 4~ against: (a) 8 M-urea, 10 m~-MnCl~, adjusted to pH 3-7 with acetic acid; (b) 8 M-urea, 50 m~-Tris.HCl (pH 7.5), 10 mM-MnC12;
(e) 50 mM-Tris.HC1 (pH 7.5), 1 mM-MnC12. The resulting purple-red solution was next dialysed against several changes of the same Tris.HC1 buffer containing 0.1 mM-EDTA; or alternatively the protein solution was gel-filtered on Sephadex G50, in order to remove excess MnC12.
177
L E T T E R S TO THE E D I T O R TABLE 1
Reconstitution of Mn superoxide dismutase Enzyme
Protein concentration (mg/ml)
Native
9
37
3.9 x l0 s
0.9
Apo
9
200
LETTERS TO THE EDITOR
Superoxide Dismutase from Bacillus stearothermophilus. Preparation of Stable Apoprotein and Reconstitution of Full}, Active Mn Enzyme A stable metal-free apoprotein has been obtained from Bacillus stearothermophilus Ma superoxide dismutase following exposure to 8M-urea at acid pH. The apoenzyme retains a dimeric structure but is inactive. Total reeonstitution of fully active Mn enzyme was achieved by addition of Mn 2+ to the apoprotein in the presence of urea. Reconstitution has been monitored by absorbtion spectroscopy, gel electrophoresis, Mn analysis and regain of activity. By these criteria the reconstituted enzyme appears to be indistinguishable from native Mn superoxide dismutase. The greater stability of the thermophile apo- and Mn enzymes is of advantage for studies of the structure and mechanism of action of superoxide dismutase. Superoxide dismutases are widely distributed enzymes which catalyse the dismutation of superoxide radicals to hydrogen peroxide and molecular oxygen (for review see Fridovich, 1975). The enzyme appears to occur in two evolutionarily distinct forms. Cu/Zn enzymes found in the cytoplasm of eukaryotic organisms are dimers consisting of two chemieally identical subunits each containing one a t o m of Cu and Zn. Removal of the two metals with chelating agents causes total inactivation but activity is partially restored b y replacement of Cu alone, and fully restored b y replacement of both metals (Beem et al., 1974). Superoxide dismutases from eukaryotic mitochondria and from prokaryotes, on the other hand, contain either Mn or Fe and possess either dimeric or tetrameric structures comprising of chemically identical subunits. Moreover the two different classes of dismutase appear to be of independent origin (Steinman & Hill, 1973; Bridgen el al., 1975). The three-dimensional structure of the Cu/Zn enzyme from bovine erythrocytes has been elucidated (Richardson et al., 1975} and the protein ligands to the two metals have been identified. The three-dimensional structures of the Mn-eontaining enzymes from Escherichia coli (Beem et al., 1976) and Bacillus stearothermophilus (Bridgen et al., 1976) are also being investigated, and in order to complement these studies we have sought to prepare a Mn-free apoprotein as an aid to investigations of the mode of binding of the metal and of its role in the catalytic reaction. A t t e m p t s to prepare a M_n (or Fe)-free apoenzyme for independent study have hitherto been frustrated b y the instability of the putative apoenzyme under conditions t h a t ensure complete removal of the metal from the native enzymes (cf. Keele et al., 1970; Puget & Michelson, 1974). More recently, however, Ose & Fridovieh (1976) have reported the reversible removal of manganese from E. coli superoxide dismutase. Nevertheless, the apoprotein as such was again found to be unstable in solution and reconstitution of active Mn enzyme could only be achieved in situ b y addition of excess MnCl2 in the presence of the metal chelating agent, 8-hydroxyquinoline, and 2 M-guanidinium hydrochloride; i.e. under conditions t h a t allow the metal to be released from the native enzyme. 175
176
C. BROCK E T A L .
Enzymes from thermophilic bacteria are known to be more stable than their counterparts in mesophiles. Thus from B. stearothermophilu8 it has been possible to prepare a stable crystalline apo- (NAD-free) glyceraldehyde 3-phosphate dehydrogenase suitable for X-ray crystallographic study (Suzuki & Harris, 1971) as well as a stable but inactive metal-free class II aldolase (Jack, 1973) from which active metallo enzyme could be reconstituted by addition of the appropriate divalent metal (Hill et al., 1976). In this paper we report the preparation and charactel%ation of a stable but inactive metal-free apoprotein from B. stearothermophilus superoxide dismutase, and the total reconstitution of native active enzyme with manganese. B. stearothermophilus ceils (strain NCA 1503) were grown, disrupted and extracted as described previously (Atkinson et al., 1972). Pure Mn superoxide dismutase was obtained in a yield of 1 g from 10 kg frozen cells from the 'phosphofructokinase rich' fraction (cL Hengartner & Harris, 1975) by gel filtration on a column of Sephadex G75 of the fraction that did not absorb to AMP-Sepharose. The pure enzyme has a molecular weight of 40,000 and comprises of two identical protein subunits (Bridgen et al., 1975). It contains firmly bound manganese and in contrast to the eukaryotie Cu/Zn enzyme the M_n enzyme from B. stearothermophilus is stable in EDTA even in 8 M-urea at neutral pH. However, in 8 M-urea, 10 mM-EDTA adjusted to pH 3 to 4 with either acetic acid or HC1 the purple-red colour disappears with concomitant loss of manganese and of enzyme activity. We have therefore used these conditions to prepare pure apoprotein. A 9 mg/ml solution of pure M_n superoxide dismutase in 50 mM-Tris.HC1 (pH 7.5) was dialysed for 12 hours at 4~ against 8 M-urea, 10 mM-EDTA adjusted to pH 3"7 with acetic acid. The resulting colourless solution was next dialysed for 6 to 12 hours against 8 M-urea, 10 mM-EDTA, 50 mM-Tris-HC1 (pH 7.5), followed by dialysis against the same buffer with 0.1 mM-EDTA but lacking urea. The slightly turbid solution was centrifuged (if the dialysis against 8 M-urea at neutral pH is omitted most of the protein has precipitated at this stage). The soluble protein (approx. 9 mg/ml) contained less than 0.01 atom Mn/mol, did not absorb at 478 to 480 nm and was completely inactive when assayed for superoxide dismutase activity by two different methods (i~IcCord & Fridovich, 1969; Rotiho et al., 1972); Table 1. The apoprotein was indistinguishable from the native enzyme when it was analysed by acrylamide gel electrophoresis at pH 8.9, or by gel-filtration on Sephadex G75, showing that the dimerie structure is maintained in the absence of the metal. Reconstitution of active Mn enzyme does not occur by addition of MnC12 to the apoprotein in buffer at neutral pH. Total reconstitution of fully active superoxide dismutase was, however, found to occur following exposure of the apoprotein to excess MnC12 in the presence of 8 ~-urea at acid pH. In a typical experiment the apoprotein (5 mg/ml) in 50 mM-Tris.HC1 (pH 7.5), 0.1 mM-EDTA was dialysed successively for periods of up to 12 hours at 4~ against: (a) 8 M-urea, 10 m~-MnCl~, adjusted to pH 3-7 with acetic acid; (b) 8 M-urea, 50 m~-Tris.HCl (pH 7.5), 10 mM-MnC12;
(e) 50 mM-Tris.HC1 (pH 7.5), 1 mM-MnC12. The resulting purple-red solution was next dialysed against several changes of the same Tris.HC1 buffer containing 0.1 mM-EDTA; or alternatively the protein solution was gel-filtered on Sephadex G50, in order to remove excess MnC12.
177
L E T T E R S TO THE E D I T O R TABLE 1
Reconstitution of Mn superoxide dismutase Enzyme
Protein concentration (mg/ml)
Native
9
37
3.9 x l0 s
0.9
Apo
9
200
