Structural mobility in human manganese superoxide dismutase

Human manganese superoxide dismutase (MnSOD) is a homotetramer of 22 kDa subunits, a dimer of dimers containing dimeric and tetrameric interfaces. We have investigated conformational mobility at these interfaces by measuring amide hydrogen/deuterium (H/D) exchange kinetics and 19F NMR spectra, both being excellent methods for analyzing local environments. Human MnSOD was prepared in which all nine tyrosine residues in each subunit are replaced with 3-fluorotyrosine. The 19F NMR spectrum of this enzyme showed five sharp resonances that have been assigned by site-specific mutagenesis by replacing each 3-fluorotyrosine with phenylalanine; four 19F resonances not observed are near the paramagnetic manganese and extensively broadened. The temperature dependence of the line widths and chemical shifts of the 19F resonances were used to estimate conformational mobility. 3-Fluorotyrosine 169 at the dimeric interface showed little conformational mobility and 3-fluorotyrosine 45 at the tetrameric interface showed much greater mobility by these measures. In complementary studies, H/D exchange mass spectrometry was used to measure backbone dynamics in human MnSOD. Using this approach, amide hydrogen exchange kinetics were measured for regions comprising 78% of the MnSOD backbone. Peptides containing Tyr45 at the tetrameric interface displayed rapid exchange of hydrogen with deuterium while peptides containing Tyr169 in the dimeric interface only displayed moderate exchange. Taken together, these studies show that residues at the dimeric interface, such as Tyr169, have significantly less conformational freedom or mobility than do residues at the tetrameric interface, such as Tyr45. This is discussed in terms of the role in catalysis of residues at the dimeric interface.     

Polymorphism in the manganese superoxide dismutase gene

Oxidative stress and mitochondrial damage occur in sepsis. Manganese superoxide dismutase (MnSOD) provides the main defence against oxidative stress within mitochondria. Ala9Val is a single nucleotide polymorphism (SNP) in the MnSOD gene, predicted to affect intra-mitochondrial transport of the enzyme. We found a significant difference in the genotype frequency between healthy subjects (n = 100) and patients with sepsis (n = 40, p = 0.009). For assessment of functionality ten healthy subjects of each homozygous genotype (A/A or V/V) were studied. Peripheral blood mononuclear cells were separated and incubated for 18 h with lipopolysaccharide (LPS), followed by analysis of mitochondrial and cytosolic fractions. There was no difference between genotypes in MnSOD activity and cytochrome c concentration, and minor differences in total antioxidant capacity (TAC) and mitochondrial membrane potential, which did not affect response to LPS. Despite predictions from structural enzyme studies that mitochondrial trafficking would be affected by the Ala9Val polymorphism of the MnSOD gene had little functional effect.     

Polymorphism in the manganese superoxide dismutase gene

Oxidative stress and mitochondrial damage occur in sepsis. Manganese superoxide dismutase (MnSOD) provides the main defence against oxidative stress within mitochondria. Ala9Val is a single nucleotide polymorphism (SNP) in the MnSOD gene, predicted to affect intra-mitochondrial transport of the enzyme. We found a significant difference in the genotype frequency between healthy subjects (n = 100) and patients with sepsis (n = 40, p = 0.009). For assessment of functionality ten healthy subjects of each homozygous genotype (A/A or V/V) were studied. Peripheral blood mononuclear cells were separated and incubated for 18 h with lipopolysaccharide (LPS), followed by analysis of mitochondrial and cytosolic fractions. There was no difference between genotypes in MnSOD activity and cytochrome c concentration, and minor differences in total antioxidant capacity (TAC) and mitochondrial membrane potential, which did not affect response to LPS. Despite predictions from structural enzyme studies that mitochondrial trafficking would be affected by the Ala9Val polymorphism of the MnSOD gene had little functional effect.     

Metal uptake by manganese superoxide dismutase

Manganese superoxide dismutase is an important antioxidant defense metalloenzyme that protects cells from damage by the toxic oxygen metabolite, superoxide free radical, formed as an unavoidable by-product of aerobic metabolism. Many years of research have gone into understanding how the metal cofactor interacts with small molecules in its catalytic role. In contrast, very little is presently known about how the protein acquires its metal cofactor, an important step in the maturation of the protein and one that is absolutely required for its biological function. Recent work is beginning to provide insight into the mechanisms of metal delivery to manganese superoxide dismutase in vivo and in vitro.     

Manganese superoxide dismutase in disease

Manganese superoxide dismutase (MnSOD) is essential for life as dramatically illustrated by the neonatal lethality of mice that are deficient in MnSOD. In addition, mice expressing only 50% of the normal compliment of MnSOD demonstrate increased susceptibility to oxidative stress and severe mitochondrial dysfunction resulting from elevation of reactive oxygen species. Thus, it is important to know the status of both MnSOD protein levels and activity in order to assess its role as an important regulator of cell biology.

Numerous studies have shown that MnSOD can be induced to protect against pro-oxidant insults resulting from cytokine treatment, ultraviolet light, irradiation, certain tumors, amyotrophic lateral sclerosis, and ischemia/reperfusion. In addition, overexpression of MnSOD has been shown to protect against pro-apoptotic stimuli as well as ischemic damage. Conversely, several studies have reported declines in MnSOD activity during diseases including cancer, aging, progeria, asthma, and transplant rejection. The precise biochemical/molecular mechanisms involved with this loss in activity are not well understood. Certainly, MnSOD gene expression or other defects could play a role in such inactivation. However, based on recent findings regarding the susceptibility of MnSOD to oxidative inactivation, it is equally likely that post-translational modification of MnSOD may account for the loss of activity. Our laboratory has recently demonstrated that MnSOD is tyrosine nitrated and inactivated during human kidney allograft rejection and human pancreatic ductal adenocarcinoma. We have determined that peroxynitrite (ONOO^-) is the only known biological oxidant competent to inactivate enzymatic activity, to nitrate critical tyrosine residues, and to induce dityrosine formation in MnSOD. Tyrosine nitration and inactivation of MnSOD would lead to increased levels of superoxide and concomitant increases in ONOO^- within the mitochondria which, could lead to tyrosine nitration/oxidation of key mitochondrial proteins and ultimately mitochondrial dysfunction and cell death. This article assesses the important role of MnSOD activity in various pathological states in light of this potentially lethal positive feedback cycle involving oxidative inactivation.     

Hypercholesterolemia increases manganese superoxide dismutase immunoreactive macrophages in myocardium

The effect of hypercholesterolemia on manganese superoxide dismutase (MnSOD)-containing macrophages was investigated in male New Zealand white rabbits. Macrophages from control animals, which were marked with the RAM-11 antibody, demonstrated co-localization with MnSOD immunoreactivity, e.g. in the peri- and paravascular space within the myocardium, but not in the bone marrow. In rabbits fed a 0.5% cholesterol-enriched diet for 42 days, a significant increase (P<0.01) of MnSOD-immunoreactive macrophages within the myocardium was found concomitant to the drastic elevation of serum cholesterol level. In the bone marrow, MnSOD immunoreactivity did not change after cholesterol feeding. Thus in cholesterol-fed rabbits, the increase of MnSOD-containing macrophages seems to parallel that of lipoproteins. MnSOD is considered as being protective against the cytotoxic effects of those superoxide anions, possibly generated in macrophages, which are involved in the metabolism of modified lipoproteins.     

Comparison of the crystal structures of genetically engineered human manganese superoxide dismutase and manganese superoxide dismutase from Thermus thermophilus: differences in dimer-dimer interaction.

The three-dimensional X-ray structure of a recombinant human mitochondrial manganese superoxide dismutase (MnSOD) (chain length 198 residues) was determined by the method of molecular replacement using the related structure of MnSOD from Thermus thermophilus as a search model. This tetrameric human MnSOD crystallizes in space group P2(1)2(1)2 with a dimer in the asymmetric unit (Wagner, U.G., Werber, M.M., Beck, Y., Hartman, J.R., Frolow, F., & Sussman, J.L., 1989, J. Mol. Biol. 206, 787-788). Refinement of the protein structure (3,148 atoms with Mn and no solvents), with restraints maintaining noncrystallographic symmetry, converged at an R-factor of 0.207 using all data from 8.0 to 3.2 A resolution and group thermal parameters. The monomer-monomer interactions typical of bacterial Fe- and Mn-containing SODs are retained in the human enzyme, but the dimer-dimer interactions that form the tetramer are very different from those found in the structure of MnSOD from T. thermophilus. In human MnSOD one of the dimers is rotated by 84 degrees relative to its equivalent in the thermophile enzyme. As a result the monomers are arranged in an approximately tetrahedral array, the dimer-dimer packing is more intimate than observed in the bacterial MnSOD from T. thermophilus, and the dimers interdigitate. The metal-ligand interactions, determined by refinement and verified by computation of omit maps, are identical to those observed in T. thermophilus MnSOD.     

Radioprotection by short-term oxidative preconditioning: Role of manganese superoxide dismutase

Manganese superoxide dismutase (MnSOD) is vital to the protection of mitochondria and cells against oxidative stress. Earlier, we demonstrated that catalytically active homo-tetramer of MnSOD can be stabilized by oxidative cross-linking. Here we report that this effect may be translated into increased radioresistance of mouse embryonic cells (MECs) by pre-exposure to oxidative stress. Pre-treatment of MECs with antimycin A, rotenone or H₂O₂ increased their survival after irradiation. Using MnSOD siRNA, we show that MECs with decreased MnSOD levels displayed a lowered ability to preconditioning. Thus oxidative preconditioning may be used for targeted regulation of MnSOD.

Structured summary

MINT-7288408: MnSOD (uniprotkb:P04179) and MnSOD (uniprotkb:P04179) physically interact (MI:0915) by zymography (MI:0512)     

Expression of manganese superoxide dismutase is not altered in transgenic mice with elevated level of copper-zinc superoxide dismutase

In evaluating the relative expression of copper-zinc and manganese superoxide dismutase (CuZnSOD and MnSOD) in vivo in states like Down syndrome in which one dismutase is present at increased levels, we measured activities of both enzymes, in tissues of control and transgenic mice constitutively expressing increased levels of CuZnSOD, during exposure to normal and elevated oxygen tensions. Using SOD gel electrophoresis assay, CuZnSOD and MnSOD activities of brain, lung, heart, kidney, and liver from mice exposed to either normal (21%) or elevated (>99% oxygen, 630 torr) oxygen tensions for 120 h were compared. Whereas CuZnSOD activity was elevated in tissues of transgenic relative to control mice under both normoxic or hyperoxic conditions, MnSOD activities in organs of transgenic mice were remarkably similar to those of controls under both conditions. To confirm the accuracy of this method in quantitating MnSOD relative to CuZnSOD expression, two other methods were utilized. In lung, which is the organ exposed to the highest oxygen tension during ambient hyperoxia, a sensitive, specific ELISA for MnSOD was used. Again, MnSOD protein was not different in transgenic relative to control mice during exposure to air or hyperoxia. In addition, lung MnSOD protein was not changed significantly by exposure to hyperoxia in either group. In kidney, a mitochondrion-rich organ, SOD assay, before and after inactivation of CuZnSOD with diethyldithiocarbamate, was used. MnSOD activity was not different in organs from air-exposed transgenic relative to control mice. The data indicated that expression of MnSOD in vivo was not affected by overexpression of the CuZnSOD and, therefore, the two enzymes are probably regulated independently.     

Outer sphere mutations perturb metal reactivity in manganese superoxide dismutase

Tyrosine 34 and glutamine 146 are highly conserved outer sphere residues in the mononuclear manganese active site of Escherichia coli manganese superoxide dismutase. Biochemical and spectroscopic characterization of site-directed mutants has allowed functional characterization of these residues in the wild-type (wt) enzyme. X-ray crystallographic analysis of three mutants (Y34F, Q146L, and Q146H) reveal subtle changes in the protein structures. The Y34A mutant, as well as the previously reported Y34F mutant, retained essentially the full superoxide dismutase activity of the wild-type enzyme, and the X-ray crystal structure of Y34F manganese superoxide dismutase shows that mutation of this strictly conserved residue has only minor effects on the positions of active site residues and the organized water in the substrate access funnel. Mutation of the outer sphere solvent pocket residue Q146 has more dramatic effects. The Q146E mutant is isolated as an apoprotein lacking dismutase activity. Q146L and Q146H mutants retain only 5-10% of the dismutase activity of the wild-type enzyme. The absorption and circular dichroism spectra of the Q146H mutant resemble corresponding data for the superoxide dismutase from a hyperthermophilic archaeon, Pyrobaculum aerophilum, which is active in both Mn and Fe forms. Interestingly, the iron-substituted Q146H protein also exhibits low dismutase activity, which increases at lower pH. Mutation of glutamine 146 disrupts the hydrogen-bonding network in the active site and has a greater effect on protein structure than does the Y34F mutant, with rearrangement of the tyrosine 34 and tryptophan 128 side chains.     

A role for manganese superoxide dismutase in apoptosis after photosensitization

The oxidative stress triggered by photodynamic therapy (PDT) involves generation of cytotoxic reactive oxygen species, including superoxide radical, accumulation of de novo-generated ceramide, and induction of apoptosis. Since PDT with the photosensitizer phthalocyanine Pc 4 induces mitochondrial damage and the superoxide scavenger manganese superoxide dismutase (MnSOD) is localized to mitochondria, here we tested genetically the role of MnSOD in apoptosis and ceramide accumulation after photosensitization with Pc 4. Jurkat cells overexpressing wild-type MnSOD were protected from Pc 4-PDT-initiated apoptosis, but not from increased ceramide response to Pc 4-PDT. In Jurkat cells overexpressing mutant MnSOD, however, DEVDase activation and ceramide formation were promoted post-Pc 4-PDT. Similarly, in MnSOD-null cells, Pc 4-PDT-induced apoptosis, as well as ceramide accumulation, were enhanced compared to their normal counterparts. The data show that MnSOD affects sensitivity of cells to Pc 4-PDT-initiated apoptosis, and partly ceramide accumulation, suggesting that the processes are superoxide-mediated.     

Hydrogen bonding in human manganese superoxide dismutase containing 3-fluorotyrosine

Incorporation of 3-fluorotyrosine and site-specific mutagenesis has been utilized with Fourier transform infrared (FTIR) spectroscopy and x-ray crystallography to elucidate active-site structure and the role of an active-site residue Tyr34 in human manganese superoxide dismutase (MnSOD). Calculated harmonic frequencies at the B3LYP/6-31G** level of theory for L-tyrosine and its 3-fluorine substituted analog are compared to experimental frequencies for vibrational mode assignments. Each of the nine tyrosine residues in each of the four subunits of the homotetramer of human MnSOD was replaced with 3-fluorotyrosine. The crystal structures of the unfluorinated and fluorinated wild-type MnSOD are nearly superimposable with the root mean-square deviation for 198 alpha-carbon atoms at 0.3 A. The FTIR data show distinct vibrational modes arising from 3-fluorotyrosine in MnSOD. Comparison of spectra for wild-type and Y34F MnSOD showed that the phenolic hydroxyl of Tyr34 is hydrogen bonded, acting as a proton donor in the active site. Comparison with crystal structures demonstrates that the hydroxyl of Tyr34 is a hydrogen bond donor to an adjacent water molecule; this confirms the participation of Tyr34 in a network of residues and water molecules that extends from the active site to the adjacent subunit.     

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.     

Magnetization of manganese superoxide dismutase from Thermus thermophilus.

The ground state magnetic properties of manganese superoxide dismutase from Thermus thermophilus in its native and reduced forms have been determined using saturation magnetization data. Parallel EPR measurements were used to verify that commonly encountered paramagnetic impurities were at low concentration relative to the metalloprotein. The native enzyme contains high spin Mn(III) (S = 2) with D = +2.44(5) cm-1 and E/D = 0. The reduced enzyme contains high spin Mn(II) (S = 5/2) with D = +0.50(5) cm-1 and E/D = 0.027. These results are in keeping with the suggestions of several previous groups of workers concerning the permissible oxidation and spin states of the manganese, but the zero field splitting parameters are unlike those of known manganese model compounds. In addition, the extinction coefficient for the visible region absorption maximum of the native enzyme and the corresponding difference extinction coefficient (native minus reduced) have been measured using saturation magnetization data to quantitate Mn(III) present. The result, epsilon 480 = 950(80) M-1 cm-1 (delta epsilon 480 = 740(60) M-1 cm-1) agrees with the previously reported value of epsilon 480 = 910 M-1 cm-1 found by total manganese determination (Sato, S. and Nakazawa, K. (1978) J. Biochem. 83, 1165-1171). The wide variation in the reported visible region extinction coefficients of manganese superoxide dismutases from different sources is discussed.     

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.     

大肠埃希菌Mn-SOD在乳酸乳球菌中的表达

本文根据GenBank中报道的大肠埃希菌MG1655全基因组DNA序列中SOD的编码基因序列设计引物,PCR扩增大肠埃希菌锰超氧化物歧化酶(Mn-SOD)基因,并将其克隆入原核高效表达质粒载体pBV220中构建重组质粒pBV220-sod,并将其电转入乳酸乳球菌MG1363中获得了成功表达,为SOD发酵奶的研制奠定了基础。