Nickel superoxide dismutase: structural and functional roles of Cys2 and Cys6

Nickel superoxide dismutase (NiSOD) is unique among the family of superoxide dismutase enzymes in that it coordinates Cys residues (Cys2 and Cys6) to the redox-active metal center and exhibits a hexameric quaternary structure. To assess the role of the Cys residues with respect to the activity of NiSOD, mutations of Cys2 and Cys6 to Ser (C2S-NiSOD, C6S-NiSOD, and C2S/C6S-NiSOD) were carried out. The resulting mutants do not catalyze the disproportionation of superoxide, but retain the hexameric structure found for wild-type NiSOD and bind Ni(II) ions in a 1:1 stoichiometry. X-ray absorption spectroscopic studies of the Cys mutants revealed that the nickel active-site structure for each mutant resembles that of C2S/C6S-NiSOD and demonstrate that mutation of either Cys2 or Cys6 inhibits coordination of the remaining Cys residue. Mutation of one or both Cys residue(s) in NiSOD induces the conversion of the low-spin Ni(II) site in the native enzyme to a high-spin Ni(II) center in the mutants. This result indicates that coordination of both Cys residues is required to generate the native low-spin configurations and maintain catalytic activity. Analysis of the quaternary structure of the Cys mutants by differential scanning calorimetry, mass spectrometry, and size-exclusion chromatography revealed that the Cys ligands, particularly Cys2, are also important for stabilizing the hexameric quaternary structure of the native enzyme.     

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.     

Adriamycin-mediated nitration of manganese superoxide dismutase in the central nervous system: insight into the mechanism of chemobrain

Adriamycin (ADR), a potent anti-tumor agent, produces reactive oxygen species (ROS) in cardiac tissue. Treatment with ADR is dose-limited by cardiotoxicity. However, the effect of ADR in the other tissues, including the brain, is unclear because ADR does not pass the blood-brain barrier. Some cancer patients receiving ADR treatment develop a transient memory loss, inability to handle complex tasks etc., often referred to by patients as chemobrain. We previously demonstrated that ADR causes CNS toxicity, in part, via systemic release of cytokines and subsequent generation of reactive oxygen and nitrogen species (RONS) in the brain. Here, we demonstrate that treatment with ADR led to an increased circulating level of tumor necrosis factor-alpha in wild-type mice and in mice deficient in the inducible form of nitric oxide (iNOSKO). However, the decline in mitochondrial respiration and mitochondrial protein nitration after ADR treatment was observed only in wild-type mice, not in the iNOSKO mice. Importantly, the activity of a major mitochondrial antioxidant enzyme, manganese superoxide dismutase (MnSOD), was reduced and the protein was nitrated. Together, these results suggest that NO is an important mediator, coupling the effect of ADR with cytokine production and subsequent activation of iNOS expression. We also identified the mitochondrion as an important target of ADR-induced NO-mediated CNS injury.     

Selective 15N labeling and direct observation by NMR of the active-site glutamine of Fe-containing superoxide dismutase

The glutamine in position 69 is one of only three conserved active-site amino acid differencesbetween Fe- and Mn-containing superoxide dismutases (SODs). We have refined theconditions for extremely selective labeling of the side chains of glutamine with 15N, and thusobtained dramatically simplified spectra, despite the large size of Fe-SOD. The improvedresolution afforded by such highly specific labeling permits the use of direct 15N detectionto observe and assign Gln 69, even though its distance to the paramagnetic Fe2+ is only 5Å. Selective glutamine side-chain labeling is inexpensive and has general utility forlarge (and paramagnet-containing) proteins.     

ENDOR and ESEEM investigation of the Ni-containing superoxide dismutase

Superoxide dismutases (SODs) protect cells against oxidative stress by disproportionating O₂ ⁻ to H₂O₂ and O₂. The recent finding of a nickel-containing SOD (Ni-SOD) has widened the diversity of SODs in terms of metal contents and SOD catalytic mechanisms. The coordination and geometrical structure of the metal site and the related electronic structure are the keys to understanding the dismutase mechanism of the enzyme. We performed Q-band ¹⁴N,^1/2H continuous wave (CW) and pulsed electron–nuclear double resonance (ENDOR) and X-band ¹⁴N electron spin echo envelope modulation (ESEEM) on the resting-state Ni-SOD extracted from Streptomyces seoulensis. In-depth analysis of the data obtained from the multifrequency advanced electron paramagnetic resonance techniques detailed the electronic structure of the active site of Ni-SOD. The analysis of the field-dependent Q-band ¹⁴N CW ENDOR yielded the nuclear hyperfine and quadrupole coupling tensors of the axial N_δ of the His-1 imidazole ligand. The tensors are coaxial with the g-tensor frame, implying the g-tensor direction is modulated by the imidazole plane. X-band ¹⁴N ESEEM characterized the hyperfine coupling of N_ε of His-1 imidazole. The nuclear quadrupole coupling constant of the nitrogen suggests that the hydrogen-bonding between N_ε–H and O_Glu-17 present for the reduced-state Ni-SOD is weakened or broken upon oxidizing the enzyme. Q-band ¹H CW ENDOR and pulsed ²H Mims ENDOR showed a strong hyperfine coupling to the protons(s) of the equatorially coordinated His-1 amine and a weak hyperfine coupling to either the proton(s) of a water in the pocket at the side opposite the axial N_δ or the proton of a water hydrogen-bonded to the equatorial thiolate ligand.     

Structural insight into the altered substrate specificity of human cytochrome P450 2A6 mutants

Human P450 2A6 displays a small active site that is well adapted for the oxidation of small planar substrates. Mutagenesis of CYP2A6 resulted in an increased catalytic efficiency for indole biotransformation to pigments and conferred a capacity to oxidize substituted indoles (Wu, Z.-L., Podust, L.M., Guengerich, F.P. J. Biol. Chem. 49 (2005) 41090-41100.). Here, we describe the structural basis that underlies the altered metabolic profile of three mutant enzymes, P450 2A6 N297Q, L240C/N297Q and N297Q/I300V. The Asn297 substitution abolishes a potential hydrogen bonding interaction with substrates in the active site, and replaces a structural water molecule between the helix B'-C region and helix I while maintaining structural hydrogen bonding interactions. The structures of the P450 2A6 N297Q/L240C and N297Q/I300V mutants provide clues as to how the protein can adapt to fit the larger substituted indoles in the active site, and enable a comparison with other P450 family 2 enzymes for which the residue at the equivalent position was seen to function in isozyme specificity, structural integrity and protein flexibility.     

Catalase, superoxide dismutase, and the production of O2-sensitive mutants of Bacillus coagulans

A number of facultatively anaerobic members of the genus Bacillus were screened for their catalase, diaminobenzidine peroxidase, and superoxide dismutase activities. A strain of Bacillus coagulans (7050) lacking peroxidatic activity and containing single catalatic and superoxide dismutase activities was selected. Responses of the superoxide dismutase activity and catalase level to the partial pressure of oxygen, and Fe and Mn levels, as well as to aerobic and fermentative metabolism, were determined. There appeared to be a relationship between high endogenous catalase levels and the high H2O2 evolution and KCN insensitivity of B. coagulans respiration. Bacillus coagulans 7050 was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine and screened for the expression of oxygen intolerance. All of the 38 stable oxygen sensitive mutants obtained had very low or completely absent catalatic activity and catalase protein. No mutant lacked superoxide dismutase, although five showed significantly lowered levels of the enzyme. Exogenous bovine liver catalase restored aerotolerance and reduced cell pleomorphism in the mutants.     

Gaining insight into the chemistry of lipoxygenases: a computational investigation into the catalytic mechanism of (8R)-lipoxygenase

Lipoxygenases (LOXs) are ubiquitous in nature and catalyze a range of life-essential reactions within organisms. In particular they are critical to the formation of eicosanoids, which are critical for normal cell function. However, a number of important questions about the reactivity and mechanism of these enzymes still remain. Specifically, although the initial step in the mechanism of LOXs has been well studied, little is known of subsequent steps. Thus, with use of a quantum mechanical/molecular mechanical approach, the complete catalytic mechanism of (8R)-LOX was investigated. The results have provided a better understanding of the general chemistry of LOXs as a whole. In particular, from comparisons with soybean LOX-1, it appears that the initial proton-coupled electron transfer may be very similar among all LOXs. Furthermore, LOXs appear to undergo multistate reactivity where potential spin inversion of an electron may occur either in the attack of O₂ or in the regeneration of the active site. Lastly, it is shown that with the explicit modeling of the environment, the regeneration of the active center likely occurs via the rotation of the intermediate followed by an outer-sphere $\rm{H^\cdot}$ transfer as opposed to the formation of a “purple intermediate” complex.     

A preliminary investigation into the roles played by the rectal gland and kidneys in the osmoregulation of the striped dogfish Poroderma africanum.

Presented here are the results of a preliminary investigation into ionic and osmotic regulation by the kidneys and rectal gland of the striped dogfish, Poroderma africanum. Fish with ligated rectal glands showed an increase in blood concentration of sodium and chloride within a short time period, reaching a maximum after four days. The blood concentration of the two ions then decreased over the following ten days. Control animals showed relatively unchanged blood-sodium and chloride levels, over the entire 14-day period. After salt loading, both control animals and those with ligated rectal glands showed initial rise in blood sodium and chloride levels, but these returned towards initial values within seven hours of injection. Comparison of the two groups indicates that the rectal gland may control blood-chloride levels more so than -sodium, although its action as a salt regulator does not seem very pronounced in either case. Urine and rectal gland fluid, were collected as a compound fluid, from normal fish, and the estimated cloacal salt loss is discussed. Urine from normal fish was also collected separately and was analysed for its contribution to salt loss. Results are discussed and compared with previous relevant findings.     

Spectroscopic characterization of polyethyleneglycol modified superoxide dismutase: 1H NMR studies on its Cu2Co2 derivative

Spectroscopic methods have been employed in order to understand the molecular basis of the decrease in enzymatic activity of the antiinflammatory enzyme copper-zinc superoxide dismutase (SOD) following the covalent binding of polyethyleneglycol (PEG) chains to the protein amino-groups. The PEG modification is a general method recently proposed to improve the therapeutic index of enzymes. 1H NMR spectra on the cobalt substituted PEG-modified SOD, Cu2Co2-PEG-SOD, have been recorded. The signals are quite broad with respect to the unmodified enzyme. This has been interpreted on the basis of the effect of molecular weight on the linewidth. The analysis has shown that the histidine hydrogens involved in metal binding at the enzyme active site are the same in both native and PEG-modified SOD. Similarly, circular dichroism and absorption spectra indicate that the overall conformation of the metal clusters is not perturbed upon modification. On the other hand, azide titration shows that the affinity constant of N-3 for SOD is largely reduced upon PEG modification (K = 154 M-1 and 75 M-1 for the native and modified SOD, respectively). These results indicate that the decrease in enzymatic activity upon surface modification with PEG is not caused by a perturbation of the active site geometry, but to a decrease in the channeling of the O2- ion towards the enzyme active site.     

The mechanism of DNA strand breakage by vitamin C and superoxide and the protective roles of catalase and superoxide dismutase.

Vitamin C breaks DNA only in the presence of oxygen. Superoxide dismutase has no effect on the reaction but catalase suppresses it. Superoxide also gives rise to breaks in DNA suppressible by both superoxide dismutase and catalase. The hydroxyl radical seems to be the agent responsible for strand cleavage itself.     

Molecular dynamics studies on mutants of Cu,Zn superoxide dismutase: The functional role of charged residues in the electrostatic loop VII

Molecular dynamics (MD) calculations have been performed on mutants of superoxide dismutase (SOD) on some residues present in the electrostatic loop. These calculations have provided the solution structures for the mutants Thr-137 → IIe and Arg; Lys-136 → Ala; Glu-132 → Gln; Glu-133 → Gln; Glu-132, Glu-133 → Gln-132, Gln-133 and → Gln-132, Lys-133. The structural and dynamic properties of these mutants have been correlated with the catalytic properties and available spectroscopic data. The water molecule present in the active site close to the copper ion in wild type (WT) SOD is missing in the MD average structure of the Thr-137 → IIe mutant, while this molecule is present in the MD average structures of all the other mutants and of WT SOD. This agrees with the experimental data. This is an important result that shows the validity of our calculations and their ability to reproduce even subtle structural features. Addition of one or more positive charges on the 132 and/or 133 positions does not sizably perturb the structure of the active site channel, while the introduction of a positively charged residue (Arg) on position 137 has a large effect on the structure of the electrostatic loop. Analysis of the MD average structures of these mutants has pointed out that the simple electrostatic effects of charged residues in the channel are not the only factor relevant for enzymatic behavior but that the structure of the electrostatic loop and the location of the charged residues also contribute to the catalytic properties of SOD. © 1994 John Wiley & Sons, Inc.     

Differential roles played by the native cysteine residues of the yeast glutathione transporter, Hgt1p

Hgt1p, a high-affinity glutathione transporter from the yeast Saccharomyces cerevisiae , belongs to the structurally uncharacterized oligopeptide transporter (OPT) family. To initiate structural studies on Hgt1p, a cysteine-free (cys-free) Hgt1p was generated. This cys-free Hgt1p was nonfunctional and pointed to a critical role being played by the native cysteine residues of Hgt1p. To investigate their role, genetic and biochemical approaches were undertaken. Functional suppressors of the cys-free Hgt1p were isolated, and yielded double revertants bearing C622 and C632. Subsequent biochemical characterization of the individual C622S/A or C632S/A mutations revealed that both these cysteine residues were, in fact, individually indispensable for Hgt1p function and were required for trafficking to the plasma membrane. However, despite their essentiality, the presence of only these two native cysteines in Hgt1p generated a very weak glutathione transporter with minimal functional activity. Hence, the remaining 10 cysteines were also contributing towards Hgt1p activity, although they were not found to be singly responsible or crucial for Hgt1p functional activity. These residues, however, contributed cumulatively towards the stability and the functionality of Hgt1p, without affecting the trafficking to the cell surface. The study reveals differential roles for the cysteines of Hgt1p and provides first insights into the structural features of an OPT family member.     

Analysis of the roles of amino acid residues in the flavoprotein tryptophan 2-monooxygenase modified by 2-oxo-3-pentynoate: characterization of His338, Cys339, and Cys511 mutant enzymes

The flavoprotein tryptophan 2-monooxygenase catalyzes the oxidative decarboxylation of tryptophan to indoleacetamide. His338, Cys339, and Cys511 of the Pseudomonas savastanoi enzyme were previously identified as possible active-site residues by modification with 2-oxo-3-pentynoate ([G. Gadda, L.J. Dangott, W.H. Johnson Jr., C.P. Whitman, P.F. Fitzpatrick, Biochemistry 38 (1999) 5822-5828]). The H338N, C339A, and C511S enzymes have been characterized to determine the roles of these residues in catalysis. The steady-state kinetic parameters with both tryptophan and methionine decrease only slightly in the case of the H338N and C339A enzymes; the decrease in activity is greater for the C511S enzyme. Only in the case of the C511S enzyme do deuterium kinetic isotope effects on kinetic parameters indicate a significant change in catalytic rates. The structural bases for the effects of the mutations can be interpreted by identification of L-amino acid oxidase and tryptophan monooxygenase as homologous proteins.     

Examination of the nickel site structure and reaction mechanism in Streptomyces seoulensis superoxide dismutase

Superoxide dismutases are metalloenzymes involved in protecting cells from oxidative damage arising from superoxide radical or reactive oxygen species produced from superoxide. Examples of enzymes containing Cu, Mn, and Fe as the redox-active metal have been characterized. Recently, a SOD containing one Ni atom per subunit was reported. The amino acid sequence of the NiSOD deduced from the nucleotide sequence of the structural gene sodN from Streptomyces seoulensis is reported and has no homology with other SODs. X-ray absorption spectroscopic studies coupled with EPR of the Ni center show that the Ni in the oxidized (as isolated) enzyme is in a five-coordinate site composed of three S-donor ligands, one N-donor, and one other O- or N-donor. This unique coordination environment is modified by the loss of one N- (or O-) donor ligand in the dithionite-reduced enzyme. The NiSOD activity was determined by pulse radiolysis, and a value of kcat = 1.3 x 10(9) M-1 s-1 per Ni was obtained. The rate is pH sensitive and drops off rapidly above pH 8. The results characterize a novel class of metal center active in catalyzing the redox chemistry of superoxide and, when placed in context with other nickel enzymes, suggest that thiolate ligation is a prerequisite for redox-active nickel sites in metalloenzymes.     

HSP25 overexpression attenuates oxidative stress-induced apoptosis: roles of ERK1/2 signaling and manganese superoxide dismutase

HSP25 has been shown to induce resistance to radiation and oxidative stress; however, its exact mechanisms remain unclear. In the present study, a high concentration of H2O2 was found to induce DNA fragmentation in L929 mouse fibroblast cells, and HSP25 overexpression attenuated this phenomenon. To elucidate the mechanisms of H2O2-mediated cell death, ERK1/2, p38 MAPK, and JNK1/2 phosphorylation in the cells after treatment with H2O2 were examined. ERK1/2 and JNK1/2 were activated by H2O2; ERK1/2 activation was inhibited in HSP25-overexpressed cells, while JNK1/2 was indifferent. Inhibition of ERK1/2 activation by treatment of the cells with PD98059 or dominant-negative ERK2 transfection blocked H2O2-induced cell death; similarly treated HSP25-overexpressed cells were not at all affected. Moreover, inhibition of JNK1/2 by dominant-negative JNK1 or JNK2 transfection did not affect H2O2-mediated cell death in control cells. Dominant-negative Ras or Raf transfection inhibited H2O2-mediated ERK1/2 activation and cell death in control cells. On the contrary, HSP25-overexpressed cells did not show any differences. Upstream pathways of H2O2-mediated ERK1/2 activation and cell death involved both tyrosine kinase (PDGFbeta receptor and Src) and PKCdelta, while in HSP25-overexpressed cells these kinases did not respond to H2O2 treatment. Since HSP25 overexpression reduced reactive oxygen species (ROS), increased manganese superoxide dismutase (MnSOD) gene expression, and increased enzyme activity, involvement of MnSOD in HSP25-mediated attenuation of H2O2-mediated ERK1/2 activation and cell death was examined. Blockage of MnSOD with antisense oligonucleotides prevented DNA fragmentation and returned the ERK1/2 activation to the control level. Indeed, when MnSOD was overexpressed in L929 cells, similar to in HSP25-overexpressed cells, DNA fragmentation and ERK1/2 activation were reduced. From the above results, we suggest for the first time that reduced oxidative damage by HSP25 was due to MnSOD-mediated downregulation of ERK1/2.     

An investigation of Cu2Zn2 superoxide dismutase and its Ile-137 mutant at high pH

The activity profile of the CU₂Zn₂HSOD Ile-137 mutant has a pK_a of 9.6, i. e. one unit lower than the wild type (WT). This property has allowed us to investigate the inactive high pH form of the enzyme before denaturation occurs. The electronic and EPR spectra do not change with the above pK_a. The ¹H NMR spectrum of the CU₂CO₂-analog reveals slight decreases in the hyperfine shifts of the protons of His-48 at high pH, which are consistent with a water molecule becoming closer to the copper ion, as detected through water ¹H T ₁ ^–1 NMR measurements. The affinity of azide at high pH is lower than at low pH, though still sizeable. The WT follows the same pattern up to pH pK_a. It appears that the drop in activity is not related to any major change involving the metal coordination sphere, but is related to changes in the electrostatic potential due to the deprotonation process. Offprint requests to: I. Bertini     

Structural, kinetic and computational investigation of Vitis vinifera DHDPS reveals new insight into the mechanism of lysine-mediated allosteric inhibition

Lysine is one of the most limiting amino acids in plants and its biosynthesis is carefully regulated through inhibition of the first committed step in the pathway catalyzed by dihydrodipicolinate synthase (DHDPS). This is mediated via a feedback mechanism involving the binding of lysine to the allosteric cleft of DHDPS. However, the precise allosteric mechanism is yet to be defined. We present a thorough enzyme kinetic and thermodynamic analysis of lysine inhibition of DHDPS from the common grapevine, Vitis vinifera (Vv). Our studies demonstrate that lysine binding is both tight (relative to bacterial DHDPS orthologs) and cooperative. The crystal structure of the enzyme bound to lysine (2.4 Å) identifies the allosteric binding site and clearly shows a conformational change of several residues within the allosteric and active sites. Molecular dynamics simulations comparing the lysine-bound (PDB ID 4HNN) and lysine free (PDB ID 3TUU) structures show that Tyr132, a key catalytic site residue, undergoes significant rotational motion upon lysine binding. This suggests proton relay through the catalytic triad is attenuated in the presence of lysine. Our study reveals for the first time the structural mechanism for allosteric inhibition of DHDPS from the common grapevine.     

Insights into the mechanism of activation of the phosphorylation-independent response regulator NblR. Role of residues Cys69 and Cys96

Cyanobacteria respond to environmental stress conditions by adjusting their photosynthesis machinery. In Synechococcus sp. PCC 7942, phycobilisome degradation and other acclimation responses after nutrient or high light stress require activation by the phosphorylation-independent response regulator NblR. Structural modelling of its receiver domain suggested a role for Cys69 and Cys96 on activation of NblR. Here, we investigate this hypothesis by engineering Cys to Ala substitutions. In vivo and in vitro analyses indicated that mutations Cys69Ala and/or Cys96Ala have a minor impact on NblR function, structure, size, or oligomerization state of the protein, and that Cys69 and Cys96 do not seem to form disulphide bridges. Our results argue against the predicted involvement of Cys69 and Cys96 on NblR activation by redox sensing.