Kinetic analysis of the metal binding mechanism of Escherichia coli manganese superoxide dismutase

The acquisition of a catalytic metal cofactor is an essential step in the maturation of every metalloenzyme, including manganese superoxide dismutase (MnSOD). In this study, we have taken advantage of the quenching of intrinsic protein fluorescence by bound metal ions to continuously monitor the metallation reaction of Escherichia coli MnSOD in vitro, permitting a detailed kinetic characterization of the uptake mechanism. Apo-MnSOD metallation kinetics are "gated", zero order in metal ion for both the native Mn2+ and a nonnative metal ion (Co2+) used as a spectroscopic probe to provide greater sensitivity to metal binding. Cobalt-binding time courses measured over a range of temperatures (35-50 degrees C) reveal two exponential kinetic processes (fast and slow phases) associated with metal binding. The amplitude of the fast phase increases rapidly as the temperature is raised, reflecting the fraction of Apo-MnSOD in an "open" conformation, and its temperature dependence allows thermodynamic parameters to be estimated for the "closed" to "open" conformational transition. The sensitivity of the metallated protein to exogenously added chelator decreases progressively with time, consistent with annealing of an initially formed metalloprotein complex (k anneal = 0.4 min(-1)). A domain-separation mechanism is proposed for metal uptake by apo-MnSOD.     

Calorimetric studies on the tight binding metal interactions of Escherichia coli manganese superoxide dismutase

Escherichia coli apomanganese superoxide dismutase, prepared by removing the native metal ion under denaturing conditions, exhibits thermally triggered metal uptake behavior previously observed for thermophilic and hyperthermophilic superoxide dismutases but over a lower temperature range. Differential scanning calorimetry of aposuperoxide dismutase and metalated superoxide dismutase unfolding transitions has provided quantitative estimates of the metal binding affinities for manganese superoxide dismutase. The binding constant for Mn(II) (K(Mn(II)) = 3.2 x 10(8) m(-1)) is surprisingly low in light of the essentially irreversible metal binding characteristic of this family of proteins and indicates that metal binding and release processes are dominated by kinetic, rather than thermodynamic, constraints. The kinetic stability of the metalloprotein complex can be traced to stabilization by elements of the protein that are independent of the presence or absence of the metal ion reflected in the thermally triggered metalation characteristic of these proteins. Binding constants for Mn(III), Fe(II), and Fe(III) complexes were estimated using quasireversible values for the unfolding enthalpy and DeltaC(p) for apo-Mn superoxide dismutase and the observed T(m) values for unfolding the metalated species in the absence of denaturants. For manganese and iron complexes, an oxidation state-dependent binding affinity reflects the protein perturbation of the metal redox potential.     

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.     

Characterization of the Bacillus stearothermophilus manganese superoxide dismutase gene and its ability to complement copper/zinc superoxide dismutase deficiency in Saccharomyces cerevisiae.

Recombinant clones containing the manganese superoxide dismutase (MnSOD) gene of Bacillus stearothermophilus were isolated with an oligonucleotide probe designed to match a part of the previously determined amino acid sequence. Complementation analyses, performed by introducing each plasmid into a superoxide dismutase-deficient mutant of Escherichia coli, allowed us to define the region of DNA which encodes the MnSOD structural gene and to identify a promoter region immediately upstream from the gene. These data were subsequently confirmed by DNA sequencing. Since MnSOD is normally restricted to the mitochondria in eucaryotes, we were interested (i) in determining whether B. stearothermophilus MnSOD could function in eucaryotic cytosol and (ii) in determining whether MnSOD could replace the structurally unrelated copper/zinc superoxide dismutase (Cu/ZnSOD) which is normally found there. To test this, the sequence encoding bacterial MnSOD was cloned into a yeast expression vector and subsequently introduced into a Cu/ZnSOD-deficient mutant of the yeast Saccharomyces cerevisiae. Functional expression of the protein was demonstrated, and complementation tests revealed that the protein was able to provide tolerance at wild-type levels to conditions which are normally restrictive for this mutant. Thus, in spite of the evolutionary unrelatedness of these two enzymes, Cu/ZnSOD can be functionally replaced by MnSOD in yeast cytosol.     

Characterization of the product-inhibited complex in catalysis by human manganese superoxide dismutase.

The reduction with excess H(2)O(2) of human Mn(III) superoxide dismutase (SOD) and the active-site mutant Y34F Mn(III)SOD was measured by scanning stopped-flow spectrophotometry and revealed the presence of an intermediate in the reduction of the manganese. The visible absorption spectrum of this intermediate closely resembled that of the enzyme in the inhibited, zero-order phase of the catalyzed disproportionation of superoxide. The decay of the visible spectrum of this intermediate was 2-fold faster for the wild-type compared with the mutant Y34F Mn-SOD. This correlates with the enhanced product inhibition of Y34F during the catalysis of O-(2) dismutation. The visible spectrum of the product-inhibited complex resembles that of the azide-Mn-SOD complex, suggesting that the inhibited complex has expanded geometry about the metal to octahedral. This study shows that the inhibited complex responsible for the zero-order phase in the catalysis by Mn-SOD of superoxide dismutation can be reached through both the forward (O-(2)) and reverse (H(2)O(2)) reactions, supporting a mechanism in which the zero-order phase results from product inhibition.     

Characterization of heparin binding of human extracellular superoxide dismutase

The C-terminal domain of human extracellular superoxide dismutase (hEC-SOD) plays a crucial role in the protein's interaction with heparin. Here we investigated this interaction in more detail by comparing the heparin-binding characteristics of two variants of hEC-SOD: the two fusion proteins containing the hEC-SOD C-terminal domain and a synthetic peptide homologous to the C-terminal. The interaction studies were performed using a surface plasmon resonance based technique on a BIAcore system. It should be emphasized that this is a model system. However, the kinetic constants, as measured, are valid in a comparative sense. Comparison of affinities for size-fractionated heparins revealed that octa- or decasaccharides are the smallest heparin fragments that can efficiently interact with the C-terminal domain of hEC-SOD. At physiological salt concentration, and pH 7.4, the hEC-SOD/heparin interaction was found to be of a high-affinity type, with an equilibrium dissociation constant, K(d), of 0.12 microM, which is 700 and 10-20 times lower than the K(d) values for the synthetic peptide and the fusion proteins, respectively. However, when an alpha-helical structure was induced in the synthetic peptide, by addition of 10% trifluoroethanol, the K(d) decreased to 0.64 microM. The differences in the K(d) values were mainly governed by differences in the association rate constants (k(ass)). The hEC-SOD/heparin interaction itself was found to have a fairly high dissociation rate constant (0.1 s(-)(1)), and a very high association rate constant (8 x 10(5) M(-)(1) s(-)(1)), suggesting that the interaction is mainly controlled by the association. These results together with circular dichroism spectra of the synthetic peptide suggest that an alpha-helical structure in the C-terminal is essential for optimal binding to heparin and that other parts of hEC-SOD moderate the affinity. Our data also demonstrate that the tetramerization itself does not substantially increase the affinity.     

Unfolding and inactivation of monomeric superoxide dismutase from E. coli by SDS

The inactivation and the unfolding of the naturally monomeric Cu, Zn, superoxide dismutase from E. coli upon addition of sodium dodecylsulphate have been studied. In contrast to the bovine enzyme, CD, EPR, NMR spectroscopy and pulsed low resolution NMR measurements found an unfolding transition followed by inactivation of the enzyme. During this transition the active site becomes accessible to the bulk water. The unfolding is reversible and both, the tridimensional structure of the protein and the active site, can be restored upon dialysis. In addition, unfolding occurs without loss of metals in the solution.     

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.     

Subunit dissociation and metal binding by Escherichia coli apo-manganese superoxide dismutase

Metal binding by apo-manganese superoxide dismutase (apo-MnSOD) is essential for functional maturation of the enzyme. Previous studies have demonstrated that metal binding by apo-MnSOD is conformationally gated, requiring protein reorganization for the metal to bind. We have now solved the X-ray crystal structure of apo-MnSOD at 1.9 Å resolution. The organization of active site residues is independent of the presence of the metal cofactor, demonstrating that protein itself templates the unusual metal coordination geometry. Electrophoretic analysis of mixtures of apo- and (Mn₂)-MnSOD, dye-conjugated protein, or C-terminal Strep-tag II fusion protein reveals a dynamic subunit exchange process associated with cooperative metal binding by the two subunits of the dimeric protein. In contrast, (S126C) (SS) apo-MnSOD, which contains an inter-subunit covalent disulfide-crosslink, exhibits anti-cooperative metal binding. The protein concentration dependence of metal uptake kinetics implies that protein dissociation is involved in metal binding by the wild type apo-protein, although other processes may also contribute to gating metal uptake. Protein concentration dependent small-zone size exclusion chromatography is consistent with apo-MnSOD dimer dissociation at low protein concentration (K_D = 1 × 10⁻⁶ M). Studies on metal uptake by apo-MnSOD in Escherichia coli cells show that the protein exhibits similar behavior in vivo and in vitro.     

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.     

Complementation of SOD-deficient Escherichia coli by manganese porphyrin mimics of superoxide dismutase activity

Cationic Mn(III) porphyrins substituted on the methine bridge carbons (meso positions) with N-alkylpyridinium or N,N'-diethylimidazolium groups have been prepared and characterized, both chemically and as SOD mimics. The ortho tetrakis N-methylpyridinium compound was substantially more active than the corresponding para isomer. This ortho compound also exhibited a more positive redox potential and greater ability to facilitate the aerobic growth of a SOD-deficient Escherichia coli. Analogs with longer alkyl side chains and with methoxyethyl side chains, as well as with N,N'-diethylimidazolium and N,N'-dimethoxyethylimidazolium groups on the meso positions, have been prepared in anticipation of greater penetration of the cells due to greater lipophilicity. We now report that the more lipophilic compounds were effective at complementing the SOD-deficient E. coli at lower concentrations than were needed with the less lipophilic compounds. The greater efficacy of the more lipophilic compounds was achieved at the cost of greater toxicity that became apparent when these compounds were applied at higher concentrations.     

Specific and cooperative binding of E. coli single-stranded DNA binding protein to mRNA.

Fluorometric titration of E. coli single-stranded DNA binding protein with various RNAs showed that the protein specifically and cooperatively binds to its own mRNA. The binding inhibited in vitro expression of ssb and bla but not nusA. This inhibition takes place at a physiological concentration of SSB. The function of the protein in gene regulation is discussed.     

RNA interference mediated knockdown of apolipophorin-III leads to knockdown of manganese superoxide dismutase in Hyphantria cunea

Apolipophorin-III (apoLp-III), a hemolymph protein that facilitates lipid transport in aqueous media in insects was recently shown to play a role in insect immune activation. Here, we report another novel possible function of apoLp-III in insects. To identify genes affected by apoLp-III expression in larvae, we decreased endogenous apoLp-III mRNA in Hyphantria cunea (Hc) through RNA interference; subsequently, we observed lower levels of antioxidant enzymes, including manganese superoxide dismutase (MnSOD), glutathione S-transferase, and immune proteins. Knockdown of Hc apoLp-III led to decreased MnSOD expression in fat body tissues and elevated superoxide anion levels in Hc fat body cells, suggesting that Hc apoLp-III is involved in the action and/or expression of antioxidant enzymes, especially MnSOD.     

Characterization of the antioxidant status of the heterozygous manganese superoxide dismutase knockout mouse

The antioxidant status of several tissues (liver, kidney, lung, brain, heart, muscle, stomach, and spleen) from heterozygous manganese superoxide dismutase (MnSOD) mutant mice (Sod2-/+) was characterized. The activity of MnSOD was decreased (30 to 80%) in all tissues examined. The levels of mRNA coding for the major antioxidant enzymes (CuZnSOD, catalase, and glutathione peroxidase) were not significantly altered in liver, kidney, heart, lung, or brain in the Sod2-/+ mice. The activities of the enzymes were not altered in any of these tissues, with the exception of a decrease in glutathione peroxidase activity in muscle in the Sod2-/+ mice compared to the Sod2+/+ mice. Thus, there was no up-regulation of the activities of the major antioxidant enzymes to compensate for the decrease in MnSOD activity. Reduced glutathione levels were 30 to 50% lower in the lung, brain, and muscle of the Sod2-/+ mice compared to the wild-type Sod2+/+ mice. In addition, the ratio of GSH/GSSG was decreased approximately 50% in Sod2-/+ muscle, indicating that the decrease in MnSOD activity in the Sod2-/+ mice results in some degree of oxidative stress in this tissue.     

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.