Effect of expression of manganese superoxide dismutase in baculovirus-infected insect cells

It has previously been demonstrated that baculovirus infection of the Spodoptera frugiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell lines leads to oxidative stress as measured by protein and membrane lipid oxidation and that this oxidative damage contributes to cell death. As a result of these findings, it was hypothesized that baculovirus infection stimulates superoxide radical (O ₂ ^·— synthesis in the mitochondria and that the resulting O ₂ ^·— accumulation overwhelms the cells’ antioxidant defenses. We investigated the ability of manganese superoxide dismutase (MnSOD) expression (which reduces O ₂ ^·— to H₂O₂) to overcome the oxidative damage caused by baculovirus infection. It was found that MnSOD expression significantly reduced oxidative damage in baculovirus-infected Tn-5B1-4 cells but had no significant effect on oxidative damage in baculovirus-infected Sf-9 cells. The results are consistent with the hypothesis that O ₂ ^·— accumulation in the mitochondria is at least partially responsible for the oxidative damage resulting from the baculovirus infection of insect cells.     

A polymer supported manganese catalyst useful as a superoxide dismutase mimic

Polymer supported manganese was synthesized via a template polymerization involving functional monomers to afford a catalyst with superoxide dismutase activity.     

Kinetic and structural characterization of human manganese superoxide dismutase containing 3-fluorotyrosines

Incorporation of 3-fluorotyrosine and site-specific mutagenesis have been used with stopped-flow spectrophotometry and pulse radiolysis to investigate the catalytic properties of human manganese superoxide dismutase (MnSOD). All of the nine tyrosine residues in each of the four subunits of the homotetramer of human MnSOD were replaced with 3-fluorotyrosine. Previous studies showed that the crystal structures of the unfluorinated and fluorinated human MnSOD are nearly superimposable with the root-mean-square deviation for 198 -carbon atoms at 0.3 Å. However, the catalytic activity k_cat/K_m of the fluorinated MnSOD at 30 μM⁻¹ s⁻¹ was less than unfluorinated wild type at 800 μM⁻¹ s⁻¹. Comparison of the values of k_cat/K_m for fluorinated and unfluorinated wild-type andY34F MnSOD showed that this decrease for the fluorinated enzyme was in significant part due to 3-fluorotyrosine residues distant (>7 Å) from the active-site metal, not to 3-fluorotyrosine at position 34 close (5 Å) to the metal. Although many rate constants for the catalysis are decreased by this fluorination, the rate of dissociation of the product-inhibited complex appears unchanged by the presence of fluorinated tyrosines. These results suggest that Tyr34 is not a proton donor in the release of the product-inhibited complex, which involves protonation of a peroxo complex of the metal with release of hydrogen peroxide.     

The influence of amine/amide versus bisamide coordination in nickel superoxide dismutase

Nickel superoxide dismutase (NiSOD) is a mononuclear nickel-containing metalloenzyme that catalyzes the disproportionation of superoxide by cycling between NiII and NiIII oxidation states. In the reduced NiII oxidation state, the metal center is ligated by two cysteinate sulfurs, one amide nitrogen, and one amine nitrogen (from the N-terminus), while in the oxidized NiIII state, an imidazole nitrogen coordinates to the metal center. Herein, we expand on a previous report in which we described a functional metallopeptide-based NiSOD model compound [NiII(SODM1)] (SODM1 = H2N-HCDLPCGVYDPA-COOH) by exploring how acylation of the N-terminus (producing [NiII(SODM1-Ac)]) influences the properties of the metallopeptide. Titration results, GPC data, and mass-spectrometry data demonstrate that NiII coordinates to SODM1-Ac in a 1:1 ratio, while variable pH studies show that NiII coordination is strong at a pH of 7.5 and above but not observed below a pH of 6.2. This is higher than [NiII(SODM1)] by approximately 1.0 pH unit consistent with bisamide ligation. Ni K-edge XAS demonstrates that the NiII center is coordinated in a square-planar NiN2S2 coordination environment with Ni-N distances of 1.846(4) A and Ni-S distances of 2.174(3) A. Comparison of the electronic absorption and CD spectrum of [NiII(SODM1)] versus [NiII(SODM1-Ac)] in conjunction with time-dependent DFT calculations suggests a decrease in Ni covalency in the acylated versus unacylated metallopeptide. This decrease in covalency was also supported by DFT calculations and Ni L-edge XAS. [NiII(SODM1-Ac)] has a quasireversible NiII/NiIII redox couple of 0.49(1) V vs Ag/AgCl, which represents a -0.2 V shift compared with [NiII(SODM1)], while the peak separation suggests a change in the coordination environment upon oxidation (i.e., axial imidazole ligation). Using the xanthine/xanthine oxidase assay, we determine that [NiII(SODM1-Ac)] is less active than [NiII(SODM1)] by over 2 orders of magnitude (IC50 = 3(1) x 10-5 vs 2(1) x 10-7 M). Possible reasons for the decrease in activity are discussed.     

A manganese superoxide dismutase mimic based on cis,cis-1,3,5-triaminocyclohexane

A novel manganese(iii) complex with a water soluble cis,cis-1,3,5-triaminocyclohexane-based ligand was synthesised and shown to exhibit superoxide dismutase activity.     

Modified expression of plasma glutathione peroxidase and manganese superoxide dismutase in human renal cell carcinoma

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is a powerful tool to separate thousands of polypeptides and to highlight the modification of protein expression in malignant diseases. By applying 2-D PAGE to ten normal human kidney and ten homologous renal cell carcinoma (RCC) tissues, we found two peptides in all ten normal tissues but not in RCCs and, conversely, two peptides were detected in all RCCs but not in normal tissues. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and internal sequence analysis, the two first peptides were identified as two isoforms of plasma glutathione peroxidase (GPxP). The two other peptides isolated in all RCCs but not in normal tissues were identified by N-terminal sequence analysis as multimeric forms of manganese superoxide dismutase (Mn-SOD). No multimeric Mn-SODs and only two monomeric forms were detected in normal tissues. GPxP and Mn-SOD are metallo-enzymes encoded on chromosome 5q32 and on chromosome 6p25, respectively. Their regions are within the locus 5q21-->qter and 6q21-6q27 on which deletions and translocations are described in some cytogenetic studies of RCC transformation. Therefore, our results might suggest a correlation between the modified expression of GPxP and Mn-SOD in tumor tissues and chromosomal modifications, and that the two proteins may be putative markers for diagnosis of RCC.     

Quinol-containing ligands enable high superoxide dismutase activity by modulating coordination number,charge, oxidation states and stability of manganese complexes throughout redox cycling

Reactivity assays previously suggested that two quinol-containing MRI contrast agent sensors for H₂O₂, [Mn(H2qp1)(MeCN)]²⁺ and [Mn(H4qp2)Br₂], could also catalytically degrade superoxide. Subsequently, [Zn(H2qp1)(OTf)]⁺ was found to use the redox activity of the H2qp1 ligand to catalyze the conversion of O₂˙⁻ to O₂ and H₂O₂, raising the possibility that the organic ligand, rather than the metal, could serve as the redox partner for O₂˙⁻ in the manganese chemistry. Here, we use stopped-flow kinetics and cryospray-ionization mass spectrometry (CSI-MS) analysis of the direct reactions between the manganese-containing contrast agents and O₂˙⁻ to confirm the activity and elucidate the catalytic mechanism. The obtained data are consistent with the operation of multiple parallel catalytic cycles, with both the quinol groups and manganese cycling through different oxidation states during the reactions with superoxide. The choice of ligand impacts the overall charges of the intermediates and allows us to visualize complementary sets of intermediates within the catalytic cycles using CSI-MS. With the diquinolic H4qp2, we detect Mn(iii)-superoxo intermediates with both reduced and oxidized forms of the ligand, a Mn(iii)-hydroperoxo compound, and what is formally a Mn(iv)-oxo species with the monoquinolate/mono-para-quinone form of H4qp2. With the monoquinolic H2qp1, we observe a Mn(ii)-superoxo ↔ Mn(iii)-peroxo intermediate with the oxidized para-quinone form of the ligand. The observation of these species suggests inner-sphere mechanisms for O₂˙⁻ oxidation and reduction that include both the ligand and manganese as redox partners. The higher positive charges of the complexes with the reduced and oxidized forms of H2qp1 compared to those with related forms of H4qp2 result in higher catalytic activity (k_cat ∼ 10⁸ M⁻¹ s⁻¹ at pH 7.4) that rivals those of the most active superoxide dismutase (SOD) mimics. The manganese complex with H2qp1 is markedly more stable in water than other highly active non-porphyrin-based and even some Mn(ii) porphyrin-based SOD mimics.

Manganese complexes with polydentate quinol-containing ligands are found to catalyze the degradation of superoxide through inner-sphere mechanisms. The redox activity of the ligand stabilizes higher-valent manganese species.     

Structure of reduced and oxidized manganese superoxide dismutase: a combined computational and experimental approach

Manganese superoxide dismutases catalyze the disproportionation of the superoxide radical anion to molecular oxygen and hydrogen peroxide. Recently, atomic-resolution crystal structures of the reduced and oxidized enzymes have been reported. They show an active site with the manganese ion bound to one aspartate, three histidine residues, and a solvent molecule. In this paper, we combine crystallographic refinement with quantum mechanical methods to show that the solvent ligand is undoubtedly a water molecule in the reduced state. However, the putative oxidized structure is to a large extent reduced during data collection, so that it contains a mixture of the Mn2+ and Mn3+ structure. The crystal structures show that the Mn-bound solvent molecule accepts a hydrogen bond from the side chain of the conserved Gln-146 residue. If the solvent ligand is water, then this could lead to a steric clash, but it is avoided by the plane of water molecule forming an angle of 72 degrees to the Mn-O bond. Such a conformation is also found outside the enzyme, giving a minimal destabilization of the reduced state. We show by molecular dynamics simulations that the suggested Mn2+-H2O and Mn3+-OH- structures are stable. Moreover, we show that the superoxide substrate may bind both in the first coordination sphere of the Mn ion, opposite to the aspartate ligand, or in the second sphere, close to the conserved Tyr-34 and His-30 residues, approximately 5 A from Mn. However, the second-sphere structures are not stable in long molecular dynamics simulations. We see no difference in the coordination between the reduced and the oxidized states of the enzyme.     

Comparative superoxide dismutase activity studies

Our results indicate that the various methods for measurement of superoxide dismutase (SOD) activity differ considerably as regards sensitivity. The activity difference may be particularly striking in the determination of the SOD activities of crude homogenates, because of the large number of disturbing factors. We consider the epinephrine-adrenochrome method to be the most suitable for measurement of the SOD activities of crude homogenates and purified enzyme preparations.     

Seven-coordinate iron and manganese complexes with acyclic and rigid pentadentate chelates and their superoxide dismutase activity

The reactions of seven-coordinate [Fe(III)(dapsox)(H(2)O)(2)]ClO(4).H(2)O (1), [Fe(II)(H(2)dapsox)(H(2)O)(2)](NO(3))(2).H(2)O (2), and [Mn(II)(H(2)dapsox)(CH(3)OH)(H(2)O)](ClO4)2(H2O) (3) complexes of the acyclic and rigid pentadentate H(2)dapsox ligand [H2dapsox = 2,6-diacetylpyridinebis(semioxamazide)] with superoxide have been studied spectrophotometrically, electrochemically, and by a submillisecond mixing UV/vis stopped-flow in dimethyl sulfoxide (DMSO). The same studies were performed on the seven-coordinate [Mn(II)(Me(2)[15]pyridinaneN(5))(H(2)O)(2)]Cl(2).H(2)O (4) complex with the flexible macrocyclic Me(2)[15]pyridinaneN(5) ligand (Me(2)[15]pyridinaneN(5) = trans-2,13-dimethyl-3,6,9,12,18-pentaazabicyclo[12.3.1]octadeca-1(18),14,16-triene), which belongs to the class of proven superoxide dismutase (SOD) mimetics. The X-ray crystal structures of 2-4 were determined. All complexes possess pentagonal-bipyramidal geometry with the pentadentate ligand in the equatorial plane and solvent molecules in the axial positions. The stopped-flow experiments in DMSO (0.06% of water) reveal that all four metal complexes catalyze the fast disproportionation of superoxide under the applied experimental conditions, and the catalytic rate constants are found to be (3.7 +/- 0.5) x 10(6), (3.9 +/- 0.5) x 10(6), (1.2 +/- 0.3) x 10(7), and (5.3 +/- 0.8) x 10(6) M(-1) s(-1) for 1-4, respectively. The cytochrome c McCord-Fridovich (McCF) assay in an aqueous solution at pH = 7.8 resulted in the IC(50) values (and corresponding kMcCF constants) for 3 and 4, 0.013 +/- 0.001 microM (1.9 +/- 0.2 x 10(8) M(-1) s(-1)) and 0.024 +/- 0.001 microM (1.1 +/- 0.3 x 10(8) M(-1) s(-1)), respectively. IC(50) values from a nitroblue tetrazolium assay are found to be 6.45 +/- 0.02 and 1.36 +/- 0.03 microM for 1 and 4, respectively. The data have been compared with those obtained by direct stopped-flow measurements and discussed in terms of the side reactions that occur under the conditions of indirect assays.     

Spectroscopic and computational investigation of second-sphere contributions to redox tuning in Escherichia coli iron superoxide dismutase

In Fe- and Mn-dependent superoxide dismutases (SODs), second-sphere residues have been implicated in precisely tuning the metal ion reduction potential to maximize catalytic activity (Vance, C. K.; Miller, A.-F. J. Am. Chem. Soc. 1998, 120, 461-467). In the present study, spectroscopic and computational methods were used to characterize three distinct Fe-bound SOD species that possess different second-coordination spheres and, consequently, Fe(3+/2+)reduction potentials that vary by approximately 1 V, namely, FeSOD, Fe-substituted MnSOD (Fe(Mn)SOD), and the Q69E FeSOD mutant. Despite having markedly different metal ion reduction potentials, FeSOD, Fe(Mn)SOD, and Q69E FeSOD exhibit virtually identical electronic absorption, circular dichroism, and magnetic circular dichroism (MCD) spectra in both their oxidized and reduced states. Likewise, variable-temperature, variable-field MCD data obtained for the oxidized and reduced species do not reveal any significant electronic, and thus geometric, variations within the Fe ligand environment. To gain insight into the mechanism of metal ion redox tuning, complete enzyme models for the oxidized and reduced states of all three Fe-bound SOD species were generated using combined quantum mechanics/molecular mechanics (QM/MM) geometry optimizations. Consistent with our spectroscopic data, density functional theory computations performed on the corresponding active-site models predict that the three SOD species share similar active-site electronic structures in both their oxidized and reduced states. By using the QM/MM-optimized active-site models in conjunction with the conductor-like screening model to calculate the proton-coupled Fe(3+/2+) reduction potentials, we found that different hydrogen-bonding interactions with the conserved second-sphere Gln (changed to Glu in Q69E FeSOD) greatly perturb the p K of the Fe-bound solvent ligand and, thus, drastically affect the proton-coupled metal ion reduction potential.     

Temperature-dependent coordination of phosphine to five-coordinate alkenylruthenium complexes

The red, five-coordinate complexes Ru(CO)Cl(PPh(3))2(CH=CHPh) and [Ru(CO)Cl(PPh(3))2]2(mu-CH=CHC(6)H(4)CH=CH) undergo reversible coordination of PPh(3) at low temperature to produce the pale yellow, six-coordinate complexes Ru(CO)Cl(PPh(3))3(CH=CHPh) and [Ru(CO)Cl(PPh(3))3]2(mu-CH=CHC(6)H(4)CH=CH). X-ray crystal structures of the latter complex and of the hydride complex RuH(CO)Cl(PPh(3))3 were obtained. 1H and 31P NMR spectra between 20 and -70 degrees C exhibit large changes in both equilibrium constants and dynamic effects. Thermodynamic parameters, DeltaH = -17.5 +/- 2.0 kcal/mol and DeltaS = -57.5 +/- 7.6 eu, were obtained for PPh(3) coordination to the monoruthenium complex, and activation parameters, DeltaH = 20.6 +/- 0.7 kcal/mol and DeltaS = 41.6 +/- 2.0 eu, were obtained for the reverse decoordination. Coordination of PPh(3) was not observed upon cooling of the shorter bridged complex, [Ru(CO)Cl(PPh(3))2]2(mu-CH=CHCH=CH).