Fe-superoxide Dismutase Research Articles

Synthesis, X-ray crystallographic characterization, and electronic structure studies of a di-azide iron(III) complex: implications for the azide adducts of iron(III) superoxide dismutase.

We have synthesized and characterized, using X-ray crystallographic, spectroscopic, and computational techniques, a six-coordinate diazide Fe (3+) complex, LFe(N 3) 2 (where L is the tetradentate ligand 7-diisopropyl-1,4,7-triazacyclononane-1-acetic acid), that serves as a model of the azide adducts of Fe (3+) superoxide dismutase (Fe (3+)SOD). While previous spectroscopic studies revealed that two distinct azide-bound Fe (3+)SOD species can be obtained at cryogenic temperatures depending on protein and azide concentrations, the number of azide ligands coordinated to the Fe (3+) ion in each species has been the subject of some controversy. In the case of LFe(N 3) 2, the electronic absorption and magnetic circular dichroism spectra are dominated by two broad features centered at 21 500 cm (-1) (approximately 4000 M (-1) cm (-1)) and approximately 30 300 cm (-1) (approximately 7400 M (-1) cm (-1)) attributed to N3 (-) --> Fe (3+) charge transfer (CT) transitions. A normal coordinate analysis of resonance Raman (RR) data obtained for LFe(N 3) 2 indicates that the vibrational features at 363 and 403 cm (-1) correspond to the Fe-N 3 stretching modes (nu Fe-N3) associated with the two different azide ligands and yields Fe-N 3 force constants of 1.170 and 1.275 mdyne/A, respectively. RR excitation profile data obtained with laser excitation between 16,000 and 22,000 cm (-1) reveal that the nu Fe-N3 modes at 363 and 403 cm (-1) are preferentially enhanced upon excitation in resonance with the N 3 (-) --> Fe (3+) CT transitions at lower and higher energies, respectively. Consistent with this result, density functional theory electronic structure calculations predict a larger stabilization of the molecular orbitals of the more strongly bound azide due to increased sigma-symmetry orbital overlap with the Fe 3d orbitals, thus yielding higher N 3 (-) --> Fe (3+) CT transition energies. Comparison of our data obtained for LFe(N 3) 2 with those reported previously for the two azide adducts of Fe (3+)SOD provides compelling evidence that a single azide is coordinated to the Fe (3+) center in each protein species.

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.

How do plants respond to copper deficiency?

The transition metal copper is essential for all organisms yet excess copper is toxic because of production of free radicals via its free form. Therefore, the levels of copper are precisely regulated in a cell. Under copper depleted conditions, the expression of Cu/Zn superoxide dismutase (SOD) is downregulated and its function is compensated by Fe SOD in chloroplasts of higher plants. We presented evidence that a microRNA, miR398, is involved in this downregulation of Cu/Zn SOD genes in Arabidopsis thaliana when grown at low copper levels, corresponding to less than 1 µM Cu in tissue culture media. However, a green alga, Chlamydomonas reinhardtii, adjusts to copper depletion by modifying the photosynthetic apparatus from copper containing plastocyanin to iron containing cytochrome c6-. During evolution plants modified one of the main strategies to respond to copper deficiency probably to adapt to different metal environments.

Multistate folding of a hyperthermostable Fe-superoxide dismutase (TcSOD) in guanidinium hydrochloride: The importance of the quaternary structure

Superoxide dismutases (SODs), which are the first line of cellular defense against the toxic effects of reactive oxygen species, are metalloenzymes that catalyze the disproportionation of superoxide radicals to produce oxygen and hydrogen peroxide. Although much effort has been devoted to the folding mechanisms of Cu/Zn-SODs, little is known about the folding of Fe-SODs. In this research, the equilibrium unfolding and refolding of TcSOD, a tetrameric hyperthermostable Fe-SOD, were investigated by circular dichroism, intrinsic fluorescence, ANS fluorescence, size-exclusion chromatography and cross-linking experiments. The results herein suggested that the guanidine hydrochloride-induced unfolding of TcSOD involved a stable monomeric intermediate and a possible tetrameric intermediate. The Gibbs free energy of TcSOD dissociation was about 3-fold larger than that of the monomeric intermediate unfolding, which suggested that the quaternary structure plays a crucial role in TcSOD stability. A comparison of the thermodynamic parameters between TcSOD and other SODs also suggested that the stability of quaternary structure might be responsible for the hyperthemostability of TcSOD.

Characterization of a hyperthermostable Fe-superoxide dismutase from hot spring

A new gene encoding a thermostable Fe-superoxide dismutase (tcSOD) was identified from a metagenomic library prepared from a hot spring sample. The open reading frame of tcSOD encoded a 211 amino acid protein. The recombinant protein was overexpressed in Escherichia coli and confirmed to be a Fe-SOD with a specific activity of 1,890 U/mg using the pyrogallol method. The enzyme was highly stable at 80 degrees C and retained 50% activity after heat treatment at 95 degrees C for 2 h. It showed striking stability across a wide pH span from 4 to 11. The native form of the enzyme was determined as a homotetramer by analytical ultracentrifugation and gradient native polyacrylamide gel electrophoresis. Fe(2+) was found to be important to SOD activity and to the stability of tcSOD dimer. Comparative modeling analyses of tcSOD tetramer indicate that its high thermostability is mainly due to the presence of a large number of intersubunit ion pairs and hydrogen bonds and to a decrease in solvent accessible hydrophobic surfaces.

Variation in copper tolerance in Arabidopsis thaliana accessions Columbia, Landsberg erecta and Wassilewskija

Among the Arabidopsis thaliana accessions Columbia (Col), Landsberg erecta (Ler), and Wassilewskija (Ws), Ler and Ws showed higher copper (Cu) tolerance than Col, while accumulating more Cu. Thus, Cu tolerance did not appear to be related to metal exclusion. Rather, the higher Cu tolerance of Ler and Ws may reflect less Cu‐induced nutrient deficiency as they maintained higher sulfur, iron (Fe), and manganese levels than Col under Cu stress. Reverse transcription–polymerase chain reaction was used to compare the leaf transcript levels of nine genes involved in Cu metabolism, oxidative stress resistance or sulfate transport (Sultr 4;1). Excess Cu led to an overall decrease of the transcript levels of plastocyanin and two plastidic Cu transporters, PAA1 and PAA2. The Fe superoxide dismutase (SOD) gene FSD1 was also downregulated in the three accessions, while the cytosolic Cu/Zinc SOD (CSD1) was upregulated compared with the control conditions. These results may be related to differences in Fe and Cu accumulation. The transcript abundance of the Cu chaperones ATX1, CCS and CpCCP showed differential regulation among the three accessions in response to Cu supply. The vacuolar Sultr 4;1 was upregulated by Cu, most likely due to the lower ability to accumulate S. Total non‐protein thiol levels were not correlated with Cu tolerance.

Superoxide dismutase B gene (sodB)-deficient mutants of Francisella tularensis demonstrate hypersensitivity to oxidative stress and attenuated virulence.

A Francisella tularensis live vaccine strain mutant (sodB(Ft)) with reduced Fe-superoxide dismutase gene expression was generated and found to exhibit decreased sodB activity and increased sensitivity to redox cycling compounds compared to wild-type bacteria. The sodB(Ft) mutant also was significantly attenuated for virulence in mice. Thus, this study has identified sodB as an important F. tularensis virulence factor.

High Light Response of the Thylakoid Proteome in Arabidopsis Wild Type and the Ascorbate-Deficient Mutant vtc2-2. A Comparative Proteomics Study

The thylakoid proteome of chloroplasts contains multiple proteins involved in antioxidative defense, protein folding, and repair. To understand this functional protein network, we analyzed the quantitative response of the thylakoid-associated proteome of Arabidopsis (Arabidopsis thaliana) wild type and the ascorbate-deficient mutant vtc2-2 after transition to high light (HL; 1,000 micromol photons m(-2) s(-1)). The soluble thylakoid proteomes of wild type and vtc2-2 were compared after 0, 1, 3, and 5 d of HL using two-dimensional gels with three independent experiments, followed by a multivariant statistical analysis and tandem mass spectrometry. After 5 d of HL, both wild-type and vtc2-2 plants accumulated anthocyanins, increased their total ascorbate content, and lost 10% of photosystem II efficiency, but showed no bleaching. Anthocyanin and total ascorbate concentrations in vtc2-2 were respectively 34% and 20% of wild type, potentially leading to enhanced oxidative stress in vtc2-2. Forty-five protein spots significantly changed as a consequence of genotype, light treatment, or both. Independent confirmation was obtained from western blots. The most significant response was the up-regulation of thylakoid YCF37 likely involved in photosystem I assembly, and specific fibrillins, a flavin reductase-like protein, and an aldolase, each located in thylakoid-associated plastoglobules. Fe-superoxide dismutase was down-regulated in vtc2-2, while Cu,Zn-superoxide dismutase was up-regulated. vtc2-2 also showed a systematic up-regulation of a steroid dehydrogenase-like protein. A number of other stress-related proteins, several thylakoid proteases, and lumenal isomerases did not change, while PsbS increased in wild type upon light stress. These findings are discussed in terms of plastid metabolism and oxidative stress defense, and emphasize that understanding of the chloroplast stress-response network must include the enzymatic role of plastoglobules.

A likely molecular basis of the susceptibility of Giardia lambliaTowards oxygen

Giardia lamblia is an amitochondrial protozoan susceptible to oxygen, but the molecular basis for it remains unclear. A Giardia NAD(P)H:menadione oxidoreductase (DT-diaphorase) is known to catalyse a single electron transfer reaction with quinones as the likely two-electron acceptor when oxygen is absent. Here we overexpressed this enzyme in Giardia trophozoites and observed a significantly enhanced susceptibility of the cells towards oxygen. A knock-down of this enzyme resulted, however, in more oxygen-tolerant Giardia cells growing equally well under anaerobic and aerobic conditions. The function of DT-diaphorase could be thus a major, if not the only, cause for the oxygen susceptibility of Giardia. Overexpressed DT-diaphorase is accompanied by increased intracellular hydrogen peroxide. An overexpression of Fe-superoxide dismutase in Giardia led also to a similarly heightened sensitivity to oxygen. Thus, generation of H2O2 from superoxide anion likely produced from DT-diaphorase catalysed reaction using oxygen as electron acceptor may constitute the molecular basis for Giardia susceptibility to oxygen. A functional homologue of DT-diaphorase in Giardia, NADH oxidase, uses oxygen as the preferred electron acceptor and reduces it to water. Overexpression of this enzyme in Giardia resulted in significantly enhanced growth under aerobic conditions. Giardia NADH oxidase could be thus an instrumental enzyme for the organism to adapt to and to tolerate an aerobic living environment.

Differential responses of stress proteins, antioxidant enzymes, and photosynthetic efficiency to physiological stresses in the Florida red tide dinoflagellate, Karenia brevis

This study identifies stress proteins and antioxidant enzymes that may play a role in the survival strategies of the Florida red tide dinoflagellate, Karenia brevis. Heat shock protein 60 (Hsp 60), mitochondrial small heat shock protein (mitosHsp), chloroplastic small heat shock protein (chlsHsp), Mn superoxide dismutase (SOD), and Fe SOD were first identified by Western blotting. The induction of these proteins in laboratory cultures in response to elevated temperatures, hydrogen peroxide, lead, or elevated light intensities was next assessed. In parallel, F V/ F M, a measurement of photosynthetic efficiency and common proxy of cellular stress, was determined. Hsp 60, Fe SOD, and Mn SOD were induced following exposure to elevated temperatures, hydrogen peroxide, or lead. MitosHsp responded only to heat, whereas chlsHsp responded only to H 2O 2-induced stress. The expression of stress proteins and antioxidant enzymes appears to be a more sensitive indicator of heat or chemically induced stresses than F V/ F M. However, F V/ F M decreased significantly in response to elevated light intensities that did not induce the expression of stress proteins. These results identify for the first time stress proteins and antioxidant enzymes in K. brevis, provide evidence for differential sensitivity of cellular organelles to various sources of stress, and confirm the presence of conserved stress responses observed across phyla in a dinoflagellate.

Synechocystis Fe superoxide dismutase gene confers oxidative stress tolerance to Escherichia coli

The superoxide dismutase (SOD) gene ( slr 1516) from the cyanobacterium Synechocystis sp. PCC 6803 was cloned and overexpressed in Escherichia coli BL 21 (DE3) using the pET-20b(+) expression vector. E. coli cells transformed with pET-SOD overexpressed the protein in cytosol, upon induction by isopropyl β- d-thiogalactopyranoside (IPTG). The recombinant protein was purified to near homogeneity by gel filtration and ion-exchange chromatography. The SOD activity of the recombinant protein was sensitive to hydrogen peroxide and sodium azide, confirming it to be FeSOD. The pET-FeSOD transformed E. coli showed significantly higher SOD activity and tolerance to paraquat-mediated growth inhibition compared to the empty vector transformed cells. Based on these results it is suggested that overexpression of FeSOD gene from a heterologous source like Synechocystis sp. PCC 6803 may provide protection to E. coli against superoxide radical-mediated oxidative stress mediated by paraquat.

Copper deficiency induced expression of Fe-superoxide dismutase gene in Matteuccia struthiopteris

Iron-superoxide dismutase (Fe-SOD) activity was not detected in extracts from the leaves of ferns, Equisetum arvense and Matteuccia struthiopteris. To know why ferns lack Fe-SOD activity, the Fe-SOD like gene ( MsFeSOD1) was isolated from M. struthiopteris and its expression was investigated with a focus on the metals Fe and Cu using the prothalli of the fern. The expression of MsFeSOD1 mRNA was induced by a deficiency of Cu, but Fe-SOD activity was not detected. The recombinant protein of MsFeSOD1 produced in E. coli showed Fe-SOD activity. These findings suggest that the fern Fe-SOD like gene was transcriptionally regulated by Cu but an additional mechanism is involved in the formation of an active enzyme.

Iron Metabolism in Tomato and Watermelon Plants: Influence of Nitrogen Source

Tomato and watermelon plants were grown in nutrient solutions in which nitrogen (N) was supplied as NO3 − (6 mM‐N) or NH4 + (6 mM‐N). The experiments were conducted to evaluate the effect which different N sources exert on iron (Fe) uptake and accumulation, on the enzymatic activities of aconitase (Aco), chelate reductase (FeCH‐R), peroxidase (POD), catalase (CAT), and Fe‐superoxide dismutase (FeSOD), and on biomass production. For both species of plants, fertilization with NH4 + caused the total Fe concentration to be lower, in the roots and in the leaves in relation to the concentrations recorded in plants fertilized with NO3 −. The response of the enzymes related to Fe correlated with their concentration. The plants treated with N‒NO3 − registered the highest activities in Aco, FeCH‐R, POD, and CAT for both tomato and watermelon. On the other hand, only in the tomato plants was the superoxide dismutase (SOD) activity appreciably influenced primarily by NH4 +, due possibly to the toxic effect of this N source. Finally, in relation to biomass production, fertilization with NH4 + drastically reduced growth in the tomato plants, while in watermelon plants, no significant alteration was detected in dry‐matter production, regardless of the N form used. It was concluded that the response of the parameters analyzed to NH4 + fertilization, in tomato and watermelon, compared to fertilization with NO3 − was similar. By contrast, tomato plants, but not watermelon plants, were negatively influenced by NH4 +.

Proton-coupled electron transfer in Fe-superoxide dismutase and Mn-superoxide dismutase

Fe-containing superoxide dismutase (FeSOD) and MnSOD are widely assumed to employ the same catalytic mechanism. However this has not been completely tested. In 1985, Bull and Fee showed that FeSOD took up a proton upon reduction [J. Am. Chem. Soc. 107 (1985) 3295]. We now demonstrate that MnSOD incorporates the same crucial coupling between electron transfer and proton transfer. The redox-coupled H + acceptor has been presumed to be the coordinated solvent molecule, in both FeSOD and MnSOD, however this is very difficult to test experimentally. We have now examined the most plausible alternative: that Tyr34 accepts a proton upon SOD reduction. We report specific incorporation of 13C in the C ζ positions of Tyr residues, assignment of the C ζ signal of Tyr34 in each of oxidized FeSOD and MnSOD, and direct NMR observations showing that in both cases, Tyr34 is in the neutral protonated state. Thus Tyr34 cannot accept a proton upon SOD reduction, and coordinated solvent is concluded to be the redox-coupled H + acceptor instead, in both FeSOD and MnSOD. We have also confirmed by direct 13C observation that the p K of 8.5 of reduced FeSOD corresponds to deprotonation of Tyr34. This work thus provides experimental proof of important commonalities between the detailed mechanisms of FeSOD and MnSOD.

Influence of temperature on biomass, iron metabolism and some related bioindicators in tomato and watermelon plants

Tomato, Lycopersicon esculentum L. cv. RX-335, and watermelon plants, Citrullus lanatus [Thom.] Mansf. cv. F-90 were grown under controlled conditions at three different temperatures (10 degrees, 25 degrees and 35 degrees C) for 30 days. The aim of the experiment was to analyse the effect of the different temperatures on Fe uptake and distribution, as well as the behaviour of the main bioindicators of this element. Thus, we analysed the total and free Fe concentrations and H2O2 concentrations, as well as enzymatic activities of Fe-chelate reductase (FeCH-R), aconitase (Aco), guaiacol peroxidase (GPX), catalase (CAT), and Fe-superoxide dismutase (FeSOD), and the dry weight of the plants. The effect caused by each temperature varied according to the species of plant. Our results indicate that heat stress appears in tomato plants when grown at 35 degrees C (above the optimal temperature for growth), while in watermelon plants, which need more heat than do tomatoes, cold stress appears at 10 degrees C (below the optimal temperature for growth). Despite these differences between the two species, the results under conditions of thermal stress were the same: 1) decreased shoot weight, 2) reduced Fe uptake, 3) depressed activities of FeCH-R, Aco, GPX, CAT and 4) boosted SOD activity. In short, our results appear to indicate that, whether heat in tomato plants or cold stress in watermelon plants, Fe uptake was diminished, as were the enzymatic activities related to the levels of this micronutrient in the plant. The high FeSOD activity in these plants could be explained by a defensive response to heat or cold stress.

Mutants of Cyanobacterium Synechocystis sp. PCC 6803 with Insertional Inactivation of the sodB Gene Encoding Fe-Superoxide Dismutase

The sodB gene encoding the only superoxide dismutase (Fe-SOD) in cells of the cyanobacterium Synechocystis sp. PCC 6803 was inactivated with gentamycin resistance aacC1 marker insertions located in the direct or reverse direction in the sodB gene. The corresponding ΔsodB12 and ΔsodB22 mutants are characterized by the complete absence of superoxide dismutase activity and the loss of viability upon standard photoautotrophic cultivation. Mutant cells can grow under conditions of a decreased illumination intensity and upon addition of NaHCO3 with catalase or bovine serum albumin in the growth medium. The ΔsodB22 mutant is auxotrophic for leucine due to the polar effect of insertion into the sodB gene on the downstream leuB gene controlling leucine biosynthesis. These data suggest that Fe-SOD is very important for providing resistance of Synechocystis 6803 cells to oxidative stress and thatsodB and leuB genes are organized into a single operon.

Antioxidative enzymes in cultivars of pepper plants with different sensitivity to cadmium

The effect of growing five different cultivars of pepper plants ( Capsicum annuum L.) with CdCl 2 concentrations ranging from 0.125 to 0.5 mM on different physiological parameters, and antioxidative enzyme activities of leaves was studied. On the basis of growth parameters, pepper plants were relatively tolerant to cadmium, although metal concentrations higher than 0.125 mM produced a significant inhibition of growth, net photosynthesis, and water use efficiency. Different sensitivities to Cd ++ ions were observed among cultivars, Abdera being the most resistant to cadmium stress, while Mondo and Herminio were the most sensitive cultivars. Cadmium concentrations of 0.5 mM produced an increase in the activity of glutathione reductase, and guaiacol peroxidase in most cultivars, while catalase and superoxide dismutase (SOD) were slightly depressed. The analysis of the SOD activity pattern by native-PAGE showed the presence in most cultivars of four SODs which were identified as Mn–SOD, Fe–SOD, CuZn–SOD I and CuZn–SOD II. However, the two CuZn–SODs were absent in the Cd-sensitive cv. Herminio. The growth of pepper plants with 0.5 mM cadmium inhibited the activity of CuZn–SODs in all cultivars, while the activity of Mn- and Fe–SOD was enhanced. The activity of NADPH-dehydrogenases (glucose-6- P-dehydrogenase, 6-phosphogluconate dehydrogenase, NADP–isocitrate dehydrogenase and malic enzyme) showed a Cd-dependent enhancement in most cultivars, the highest increase being observed in the tolerant cv. Abdera. These results suggest that in pepper plants the tolerance to Cd toxicity is more dependent on the availability of NADPH than on its antioxidant capacity.

Differential expression and localization of Mn and Fe superoxide dismutases in the heterocystous cyanobacterium Anabaena sp. strain PCC 7120.

Superoxide dismutases (Sods) play very important roles in preventing oxidative damages in aerobic organisms. The nitrogen-fixing heterocystous cyanobacterium Anabaena sp. strain PCC 7120 has two Sod-encoding genes: a sodB, encoding a soluble iron-containing Sod (FeSod), and a sodA, encoding a manganese-containing Sod (MnSod). The FeSod was purified and characterized. A recombinant FeSod was also obtained by overproduction in Escherichia coli. Immunoblot study of the FeSod during induction of heterocyst differentiation showed that the cells produced six- to eightfold more FeSod 8 h after a shift from a nitrogen-replete condition to a nitrogen-depleted condition. However, the amount of FeSod protein in filaments with mature heterocysts was the same as that in filaments grown with combined nitrogen. Superoxide anion-generating chemicals such as methyl viologen did not induce upregulation of the sodB gene expression. The predicted preprotein of the sodA gene has a leader peptide and a motif for membrane attachment at the N terminus of the mature protein. Activity staining after gel electrophoresis of the purified thylakoid membranes showed that most of the MnSod in Anabaena sp. strain PCC 7120 was located on thylakoids toward the lumenal side. Expression of the sodA gene in E. coli shows that the leader peptide was required for its activity and the membrane localization of the MnSod. Northern hybridization detected one 0.82-kb transcript of sodA. The sodA gene was upregulated by methyl viologen, whereas its amount was unchanged during heterocyst differentiation. Immunoblotting and activity staining showed that isolated heterocysts contained a lower but still significant amount of FeSod, suggesting that its function is required in heterocysts. No MnSod was observed in isolated heterocysts. These results show that the two different Sod proteins have differentiated roles in Anabaena sp. strain PCC 7120.

Thermal-induced Changes in the Secondary Conformation of Superoxide Dismutase Containing Different Metal Ions

The thermal stability of three superoxide dismutases (SODs) with different metal ions (Mn, Cu/Zn, Fe) in the solid state was studied by a Fourier transform infrared (FT-IR) microspec-troscopy combined with thermal analyzer. The IR spectra showed a maximum peak at 1652 cm−1 for all the native SODs in the amide I band, suggesting a predominant random coil with less α-helix structures. By heating each sample, a shoulder at 1631 cm−1 in the amide I band gradually appeared from 45° C for Fe SOD and from 50° C for Mn SOD but another shoulder at 1639 cm−1 appeared from 50° C for Cu/Zn SOD. The peak at 1631 cm−1 is due to the intermolecular β-sheet structure, but the peak at 1639 cm−1 corresponds to the major intramolecular β-sheet with less random coil structure. This reveals that in the first heating process the transformation from random coil/a-helix structure to β-sheet structure initiated from around 45–50° C. There was about 16–22% compositional change resulting from that transformation. However, both additional shoulders stood there and did not restore to their original spectra even with cooling to room temperature, suggesting the denaturation and irreversible properties of the solid SODs after heating. The thermal-dependent denaturation and irreversibility of Mn SOD, Cu/Zn SOD and Fe SOD were clearly evidenced by the increase in intramolecular and intermolecular β-sheet structure.

Changes in antioxidant activity in sub-cellular fractions of tolerant and susceptible wheat genotypes in response to long term salt stress

Effects of long term, medium level (electrical conductivity of extract (ECe)=6.85 dS m −1) sodium chloride (NaCl) salinity were studied in tolerant (Kharchia 65) and susceptible (HD 2687) wheat genotypes. NaCl salinity caused decrease in relative water content (RWC), chlorophyll (CHL), membrane stability index (MSI) and ascorbic acid (AA) content, and increased the contents of hydrogen peroxide (H 2O 2), thiobarbituric acid reactive substances (TBARS) (measure of lipid peroxidation) and activities of superoxide dismutase (SOD), its various isozymes, ascorbate peroxidase (APOX) and glutathione reductase (GR) in wheat genotypes Kharchia 65 (tolerant) and HD 2687 (susceptible). Salinity tolerant wheat cv. Kharchia 65 showed less decline in RWC, CHL, MSI estimated in whole tissue than salt sensitive HD 2687. Kharchia 65 also exhibited less decrease in AA content, less increase in H 2O 2, TBARS contents and higher increase in SOD and its isozymes, APOX and GR in all sub-cellular fractions than salt sensitive HD 2687. H 2O 2, TBARS contents and AA contents were higher in chloroplastic fraction. Chloroplastic fraction showed higher total SOD, APOX and GR activity, followed by mitochondrial fraction in case of total SOD and GR, while cytosolic fraction was in second place in case of APOX activity. Though Mn–SOD activity was highest in mitochondrial fraction, but residual activity was also observed in cytosolic fraction. Cu/Zn–SOD and Fe–SOD were observed in all the sub-cellular fractions, however, the activities were higher in chloroplastic fraction for both the isoforms. Total Cu/Zn–SOD activity, sum of activity observed in all the fractions, was higher than other SOD isoforms. Susceptibility of HD 2687 to long-term salinity stress seems to be due to relatively less induction of SOD isozymes, no induction in chloroplastic and mitochondrial APOX and cytosolic GR and decrease in chloroplastic GR under salt stress resulting in higher oxidative stress in the form of H 2O 2 and TBARS contents and decrease in MSI and CHL.