The primary structure of human liver manganese superoxide dismutase

The complete amino acid sequence of manganese superoxide dismutase from human liver was determined. The sequence was deduced following characterization of the peptides obtained from tryptic, chymotryptic, and Staphylococcus aureus digests of the apoprotein. Chemical cleavage with dimethyl sulfoxide-hydrobromic acid was also carried out. The amino acid sequence listed below is made up of 196 amino acids and the two subunit polypeptides in the native enzyme appear to be identical. No homology was observed with copper/zinc containing class of superoxide dismutase. Lys-His-Ser-Leu-Pro-Asp-Leu-Pro-Tyr-Asp-Tyr-Gly-Ala-Leu-Glu-Pro-His-Il e -Asn-Ala-Gln-Ile-Met-Gln-Leu-His-His-Ser-Lys-His-His-Ala-Ala-Tyr-Val-Asn -Asn-Leu-Asn-Val-Thr-Gln-Glu-Lys-Tyr-Gln-Glu-Ala-Leu-Ala-Lys-Gly-Asp-Val -Thr-Ala-Gln-Ile-Ala-Leu-Gln-Pro-Ala-Leu-Lys-Phe-Asn-Gly-Gly-Gly-His-Ile -Asn-His-Ser-Ile-Phe-Trp-Thr-Asn-Leu-Ser-Pro-Asn-Gly-Gly-Gly-Gln-Pro-Lys -Gly-Glu-Leu-Leu-Glu-Ala-Ile-Lys-Arg-Asp-Phe-Gly-Ser-Phe-Asp-Lys-Phe-Lys -Gln-Lys-Leu-Thr-Ala-Ala-Ser-Val-Gly-Val-Gln-Gly-Ser-Gly-Trp-Leu-Gly-Phe -Asn-Lys-Gln-Arg-Gly-His-Leu-Gln-Ile-Ala-Ala-Cys-Pro-Asn-Gln-Asp-Pro-Leu -Gln-Gly-Thr-Thr-Gly-Leu-Ile-Pro-Leu-Leu-Gly-Ile-Asp-Val-Trp-Glu-His-Ala -Tyr-Tyr-Leu-Gln-Tyr-Lys-Asn-Val-Arg-Pro-Asp-Tyr-Leu-Lys-Ala-Ile-Trp-Asn -Val-Ile-Asn-Trp-Glu-Asn-Val-Thr-Glu-Arg-Tyr-Met-Ala-Cys-Lys-Lys.     

Role of a glutamate bridge spanning the dimeric interface of human manganese superoxide dismutase

The function in the structure, stability, and catalysis of the interfaces between subunits in manganese superoxide dismutase (MnSOD) is currently under scrutiny. Glu162 in homotetrameric human MnSOD spans a dimeric interface and forms a hydrogen bond with His163 of an adjacent subunit which is a direct ligand of the manganese. We have examined the properties of two site-specific mutants of human MnSOD in which Glu162 is replaced with Asp (E162D) and Ala (E162A). The X-ray crystal structures of E162D and E162A MnSOD reveal no significant structural changes compared with the wild type other than the removal of the hydrogen bond interaction with His163 in E162A MnSOD. In the case of E162D MnSOD, an intervening solvent molecule fills the void created by the mutation to conserve the hydrogen bond interaction between His163 and residue 162. These mutants retain their tetrameric structure and their specificity for manganese over iron. Each has catalytic activity in the disproportionation of superoxide that is typically 5-25% of that of the wild-type enzyme and a level of product inhibition greater by approximately 2-fold. Differential scanning calorimetry indicates that the hydrogen bond between Glu162 and His163 contributes to the stability of MnSOD, with the major unfolding transition occurring at 81 degrees C for E162A compared to 90 degrees C for wild-type MnSOD. These results suggest that Glu162 at the tetrameric interface in human MnSOD supports stability and efficient catalysis and has a significant role in regulating product inhibition.     

Amino acid substitution at the dimeric interface of human manganese superoxide dismutase

The side chains of His30 and Tyr166 from adjacent subunits in the homotetramer human manganese superoxide dismutase (Mn-SOD) form a hydrogen bond across the dimer interface and participate in a hydrogen-bonded network that extends to the active site. Compared with wild-type Mn-SOD, the site-specific mutants H30N, Y166F, and the corresponding double mutant showed 10-fold decreases in steady-state constants for catalysis measured by pulse radiolysis. The observation of no additional effect upon the second mutation is an example of cooperatively interacting residues. A similar effect was observed in the thermal stability of these enzymes; the double mutant did not reduce the major unfolding transition to an extent greater than either single mutant. The crystal structures of these site-specific mutants each have unique conformational changes, but each has lost the hydrogen bond across the dimer interface, which results in a decrease in catalysis. These same mutations caused an enhancement of the dissociation of the product-inhibited complex. That is, His30 and Tyr166 in wild-type Mn-SOD act to prolong the lifetime of the inhibited complex. This would have a selective advantage in blocking a cellular overproduction of toxic H2O2.     

Amino-acid sequence of a tetrameric, manganese superoxide dismutase from Thermus thermophilus HB8

The amino-acid sequence of a tetrameric manganese superoxide dismutase from Thermus thermophilus HB8 has been determined. The protein was cleaved with cyanogen bromide (BrCN) into four peptides and their alignment was deduced through the fragment of partial cleavage with BrCN and the peptides were produced by cleavage of the protein with o-iodosobenzoic acid. Most of the peptides were sequenced by solid phase Edman degradation. Some of the peptides were sequenced by the Edman dansyl method after sub-fragmentation by proteinase digestion. The amino-acid sequence consists of 203 residues corresponding to a subunit molecular weight of 23,144.     

Induction of mitochondrial manganese superoxide dismutase by interleukin 1

Interleukin 1 (IL 1) inhibits the growth of human melanoma A375 cells. To identify the subcellular events preceding inhibition of growth by IL 1, we have examined the effect of IL 1 on protein synthesis caused by A375 cells. IL 1 selectively and predominantly induced a 25-kDa polypeptide (p25) in A375 cells after 12 h. On subcellular fractionation, p25 was exclusively located in the 10,000 x g-pelleted (mitochondria-enriched) fraction. To identify the p25 moiety, it was purified to homogeneity by sequential chromatography on DEAE-Sephacel and reverse-phase, high-pressure liquid chromatography and its amino-terminal amino acid sequence was determined. The sequence of the 35 amino-terminal amino acids of the p25 moiety was identical to that of human manganese superoxide dismutase (Mn SOD). The enzymatic activities of SOD were induced only in the mitochondria-enriched fraction of IL 1-treated A375 cells. However, IL 1 also induced Mn SOD in normal human skin fibroblasts and peripheral blood mononuclear cells, whose growth was stimulated by IL 1. The results show that induction of Mn SOD by IL 1 is a common biochemical event in IL 1-responsive cells.     

Crystal structure of nitrated human manganese superoxide dismutase: mechanism of inactivation

A cellular consequence of the reaction of superoxide and nitric oxide is enhanced peroxynitrite levels. Reaction of peroxynitrite with manganese superoxide dismutase (MnSOD) causes nitration of the active-site residue Tyr34 and nearly complete inhibition of catalysis. We report the crystal structures at 2.4 A resolution of human MnSOD nitrated by peroxynitrite and the unmodified MnSOD. A comparison of these structures showed no significant conformational changes of active-site residues or solvent displacement. The side chain of 3-nitrotyrosine 34 had a single conformation that extended toward the manganese with O1 of the nitro group within hydrogen-bonding distance (3.1 A) of Nepsilon2 of the second-shell ligand Gln143. Also, nitration of Tyr34 caused a weakening, as evidenced by the lengthening, of a hydrogen bond between its phenolic OH and Gln143, part of an extensive hydrogen-bond network in the active site. Inhibition of catalysis can be attributed to a steric effect of 3-nitrotyrosine 34 that impedes substrate access and binding, and alteration of the hydrogen-bond network that supports proton transfer in catalysis. It is also possible that an electrostatic effect of the nitro group has altered the finely tuned redox potential necessary for efficient catalysis, although the redox potential of nitrated MnSOD has not been measured.     

Identification and characterization of a mitochondrial manganese superoxide dismutase of Spirometra erinacei

A gene encoding the manganese superoxide dismutase (Mn-SOD) of Spirometra erinacei was identified, and the biochemical properties of the recombinant enzyme were partially characterized. The S. erinacei Mn-SOD gene consisted of 669 bp, which encoded 222 amino acids. A sequence analysis of the gene showed that it had typical molecular structures, including characteristic metal-binding residues and motifs that were conserved in Mn-SODs. An analysis of the N-terminal presequence of S. erinacei Mn-SOD revealed that it had physiochemical characteristics commonly found in mitochondria-targeting sequences and predicted that the enzyme is located in the mitochondria. A biochemical analysis also revealed that the enzyme is a typical Mn-SOD. The enzyme was consistently expressed in both S. erinacei plerocercoid larvae and adult worms. Our results collectively suggested that S. erinacei Mn-SOD is a typical mitochondrial Mn-SOD and may play an important role in parasite physiology, detoxifying excess superoxide radicals generated in the mitochondria.     

Removing a hydrogen bond in the dimer interface of Escherichia coli manganese superoxide dismutase alters structure and reactivity

Among manganese superoxide dismutases, residues His30 and Tyr174 are highly conserved, forming part of the substrate access funnel in the active site. These two residues are structurally linked by a strong hydrogen bond between His30 NE2 from one subunit and Tyr174 OH from the other subunit of the dimer, forming an important element that bridges the dimer interface. Mutation of either His30 or Tyr174 in Escherichia coli MnSOD reduces the superoxide dismutase activity to 30--40% of that of the wt enzyme, which is surprising, since Y174 is quite remote from the active site metal center. The 2.2 A resolution X-ray structure of H30A-MnSOD shows that removing the Tyr174-->His30 hydrogen bond from the acceptor side results in a significant displacement of the main-chain segment containing the Y174 residue, with local rearrangement of the protein. The 1.35 A resolution structure of Y174F-MnSOD shows that disruption of the same hydrogen bond from the donor side has much greater consequences, with reorientation of F174 having a domino effect on the neighboring residues, resulting in a major rearrangement of the dimer interface and flipping of the His30 ring. Spectroscopic studies on H30A, H30N, and Y174F mutants show that (like the previously characterized Y34F mutant of E. coli MnSOD) all lack the high pH transition of the wt enzyme. This observation supports assignment of the pH sensitivity of MnSOD to coordination of hydroxide ion at high pH rather than to ionization of the phenolic group of Y34. Thus, mutations near the active site, as in the Y34F mutant, as well as at remote positions, as in Y174F, similarly affect the metal reactivity and alter the effective pK(a) for hydroxide ion binding. These results imply that hydrogen bonding of the H30 imidazole N--H group plays a key role in substrate binding and catalysis.     

Tissue-specific activity of two manganese superoxide dismutase promoters in transgenic tobacco.

In eukaryotes, manganese superoxide dismutase is a nuclear-encoded protein that scavenges superoxide radicals in the mitochondrial matrix. We have isolated two manganese superoxide dismutase genes from Nicotiana plumbaginifolia L. and fused the 5' upstream regulatory region of these genes to the beta-glucuronidase reporter gene. The two gene fusions displayed a differential tissue specificity in transgenic tobacco (Nicotiana tabacum). Promoter activity of the SodA1 gene fusion was found in the pollen, middle layer, and stomium of anthers, but was usually undetectable in vegetative organs of mature plants. The SodA2 gene fusion was expressed in the leaves, stems, roots, and flowers. SodA2 promoter activity was most prominent in the vascular bundles, stomata, axillary buds, pericycle, stomium, and pollen. Histochemical analysis of succinate dehydrogenase activity suggested that the spatial expression of the two gene fusions is generally correlated with mitochondrial respiratory activity.     

Generation and characterization of a novel kidney-specific manganese superoxide dismutase knockout mouse

Inactivation of manganese superoxide dismutase (MnSOD), a mitochondrial antioxidant, has been associated with renal disorders and often results in detrimental downstream events that are mechanistically not clear. Development of an animal model that exhibits kidney-specific deficiency of MnSOD would be extremely beneficial in exploring the downstream events that occur following MnSOD inactivation. Using Cre-Lox recombination technology, kidney-specific MnSOD deficient mice (both 100% and 50%) were generated that exhibited low expression of MnSOD in discrete renal cell types and reduced enzymatic activity within the kidney. These kidney-specific 100% KO mice possessed a normal life-span, although it was interesting that the mice were smaller. Consistent with the important role in scavenging superoxide radicals, the kidney-specific KO mice showed a significant increase in oxidative stress (tyrosine nitration) in a gene-dose dependent manner. In addition, loss of MnSOD resulted in mild renal damage (tubular dilation and cell swelling). Hence, this novel mouse model will aid in determining the specific role (local and/or systemic) governed by MnSOD within certain kidney cells. Moreover, these mice will serve as a powerful tool to explore molecular mechanisms that occur downstream of MnSOD inactivation in renal disorders or possibly in other pathologies that rely on normal renal function.     

Role of tryptophan 161 in catalysis by human manganese superoxide dismutase.

Tryptophan 161 is a highly conserved residue that forms a hydrophobic side of the active site cavity of manganese superoxide dismutase (MnSOD), with its indole ring adjacent to and about 5 A from the manganese. We have made a mutant containing the conservative replacement Trp 161 --> Phe in human MnSOD (W161F MnSOD), determined its crystal structure, and measured the catalysis of the resulting mutant using pulse radiolysis to produce O(2)(*)(-). In the structure of W161F MnSOD the phenyl side chain of Phe 161 superimposes on the indole ring of Trp 161 in the wild type. However, in the mutant, the hydroxyl side chain of Tyr 34 is 3.9 A from the manganese, closer by 1.2 A than in the wild type. The tryptophan in MnSOD is not essential for the half-cycle of catalytic activity involving reduction of the manganese; the mutant W161F MnSOD had k(cat)/K(m) at 2.5 x 10(8) M(-)(1) s(-)(1), reduced only 3-fold compared with wild type. However, this mutant exhibited a strong product inhibition with a zero-order region of superoxide decay slower by 10-fold compared with wild type. The visible absorption spectrum of W161F MnSOD in the inhibited state was very similar to that observed for the inhibited wild-type enzyme. The appearance of the inhibited form required reaction of 2 molar equiv of O(2)(*)(-) with W161F Mn(III)SOD, one to form the reduced state of the metal and the second to form the inhibited complex, confirming that the inhibited complex requires reaction of O(2)(*)(-) with the reduced form of the enzyme. This work suggests that a significant role of Trp 161 in the active site is to promote the dissociation of product peroxide, perhaps in part through its effect on the orientation of Tyr 34.     

Superoxides from mitochondrial complex III: the role of manganese superoxide dismutase

In this report we show that ubiquinone cytochrome c reductase (complex III) from isolated rat heart mitochondria when inhibited with antimycin A, produces a large amount of superoxide as measured by the chemiluminescent probe coelenterazine. When mitochondria are inhibited with myxothiazol or stigmatellin, there is no detectable formation of superoxide. The antimycin A-sensitive free radical production can be dramatically reduced using either myxothiazol or stigmatellin. This suggests that the antimycin A-sensitive generation of superoxides originates primarily from the Q(o) semiubiquinone. When manganese superoxide dismutase depleted submitochondrial particles (SMP) were inhibited with myxothiazol or stigmatellin, a large superoxide signal was observed. These two inhibitors likely increase the concentration of the Q(i) semiquinone at the N center. The antimycin A-sensitive signal can, in the case of both the mitochondria and the SMP, be dissipated by the addition of copper zinc superoxide dismutase, suggesting that the measured coelenterazine signal was a result of superoxide production. Taken together, this data suggests that free radicals generated from the Q(i) species are more effectively eliminated by MnSOD in intact mitochondria.     

Characterization of crystals of genetically engineered human manganese superoxide dismutase

The genetically engineered human manganese superoxide dismutase crystallizes in space group P2(1)2(1)2 with a = 75.51 A, b = 79.00 A, c = 67.95 A. At room temperature the crystals are not stable against radiation, so we cooled them to 90 K and collected a data set to 3 A resolution at this temperature.     

The peroxidase activity of mitochondrial superoxide dismutase

Manganese superoxide dismutase (MnSOD) is an integral mitochondrial protein known as a first-line antioxidant defense against superoxide radical anions produced as by-products of the electron transport chain. Recent studies have shaped the idea that by regulating the mitochondrial redox status and H₂O₂ outflow, MnSOD acts as a fundamental regulator of cellular proliferation, metabolism, and apoptosis, thereby assuming roles that extend far beyond its proposed antioxidant functions. Accordingly, allelic variations of MnSOD that have been shown to augment levels of MnSOD in mitochondria result in a 10-fold increase in prostate cancer risk. In addition, epidemiologic studies indicate that reduced glutathione peroxidase activity along with increases in H₂O₂ further increase cancer risk in the face of MnSOD overexpression. These facts led us to hypothesize that, like its Cu,ZnSOD counterpart, MnSOD may work as a peroxidase, utilizing H₂O₂ to promote mitochondrial damage, a known cancer risk factor. Here we report that MnSOD indeed possesses peroxidase activity that manifests in mitochondria when the enzyme is overexpressed.     

Kinetic analysis of product inhibition in human manganese superoxide dismutase

Manganese superoxide dismutase (MnSOD) cycles between the Mn(II) and Mn(III) states during the catalyzed disproportionation of O(2)(*-), a catalysis that is limited at micromolar levels of superoxide by a peroxide-inhibited complex with the metal. We have investigated the role in catalysis and inhibition of the conserved residue Trp161 which forms a hydrophobic side of the active site cavity of MnSOD. Crystal structures of mutants of human MnSOD in which Trp161 was replaced with Ala or Phe showed significant conformational changes on adjacent residues near the active site, particularly Gln143 and Tyr34 which in wild-type MnSOD participate in a hydrogen bond network believed to support proton transfer during catalysis. Using pulse radiolysis and observing the UV absorbance of superoxide, we have determined rate constants for the catalytic dismutation of superoxide. In addition, the rates of formation and dissociation of the product-inhibited complex of these mutants were determined by direct observation of the characteristic visible absorption of the oxidized and inhibited states. Catalysis by W161A and W161F MnSOD was associated with a decrease of at least 100-fold in the catalytic rate of reduction of superoxide, which then promotes a competing pathway leading to product inhibition. The structural changes caused by the mutations at position 161 led to small changes, at most a 6-fold decrease, in the rate constant for formation of the inhibited complex. Solvent hydrogen isotope effects support a mechanism in which formation of this complex, presumably the peroxide dianion bound to the manganese, involves no rate-contributing proton transfer; however, the dissociation of the complex requires proton transfer to generate HO(2)(-) or H2O2.     

Redox properties of human manganese superoxide dismutase and active-site mutants

The redox potential of human manganese superoxide dismutase (MnSOD) has been difficult to determine because of the problem of finding suitable electron mediators. We have found that ferricyanide and pentacyanoaminoferrate can be used as electron mediators, although equilibration is very slow with a half-time near 6 h. Values of the midpoint potential were determined both by allowing enzyme and mediators to equilibrate up to 38 h and by reductive titration adding dithionite to enzyme and mediator. An overall value of the midpoint potential was found to be 393 +/- 29 mV. To elucidate the role of His30 and Tyr34 in the active site of human MnSOD, we have also measured the redox properties of the site-specific mutants His30Asn (H30N) and Tyr34Phe (Y34F) and compared them with the wild-type enzyme. Crystal structures have shown that each mutation interrupts a hydrogen bond network in the active site, and each causes a 10-fold decrease in the maximal velocity of catalysis of superoxide dismutation as compared with wild type. The present study shows that H30N and Y34F human MnSOD have very little effect, within experimental uncertainty, on the redox potential of the active-site metal. The redox potentials determined electrochemically were 365 +/- 28 mV for H30N and 435 +/- 30 mV for Y34F MnSOD. These results suggest that the role of His30 and Tyr34 is more in support of catalysis, probably proton transport, and not in the tuning of the redox potential.