Superoxide dismutase activities in Rhizobium phaseoli bacteria and bacteroids

Superoxide dismutase activity in free-living Rhizobium phaseoli is due to the presence of two different enzymes containing manganese or iron. Under usual culture conditions, the manganese-enzyme appears largely predominant but the induction of the iron-superoxide dismutase can be obtained by addition of methyl viologen to the culture media. The corresponding bacteroid, extracted from French-bean nodules, contains only a manganese-superoxide dismutase whose characteristics are similar to those of the bacterial enzyme. However, the activity of the microsymbiont is slightly lower than that of free-living cells. The presence of an active superoxide dismutase in the bacteroids suggests a significant formation of superoxide anion by their metabolism; this can be correlated with the existence of a large oxygen demand by the microsymbionts within the nodule, as suggested by their important oxygen uptake in vitro.     

Induction of the manganese-containing superoxide dismutase in Escherichia coli is independent of the oxidative stress (oxyR-controlled) regulon

The synthesis of manganese-superoxide dismutase in response to hydrogen peroxide and to paraquat was examined in strains of Escherichia coli with different mutations in the oxyR gene. Hydrogen peroxide treatment did not induce manganese-superoxide dismutase, but did induce the oxyR regulon. Paraquat induced this enzyme in a strain compromised in its ability to induce the defense response against oxidative stress (oxyR deletion) as well as in a strain that is constitutive and overexpresses the oxyR regulon. Catalase (HPI), but not manganese-superoxide dismutase, was over-expressed under anaerobic conditions in a strain harboring a constitutive oxyR mutation. The data clearly demonstrate that the induction of manganese-superoxide dismutase is independent of the oxyR-controlled regulon.     

Redox control of calcineurin by targeting the binuclear Fe(2+)-Zn(2+) center at the enzyme active site.

The interaction of protein serine/threonine phosphatase calcineurin (CaN) with superoxide and hydrogen peroxide was investigated. Superoxide specifically inhibited phosphatase activity of CaN toward RII (DLDVPIPGRFDRRVSVAAE) phosphopeptide in tissue and cell homogenates as well as the activity of the enzyme purified under reducing conditions. Hydrogen peroxide was an effective inhibitor of CaN at concentrations several orders of magnitude higher than superoxide. Inhibition by superoxide was calcium/calmodulin-dependent. Nitric oxide (NO) antagonized superoxide action on CaN. We provide kinetic and spectroscopic evidence that native, catalytically active CaN has a Fe(2+)-Zn(2+) binuclear center in its active site that is oxidized to Fe(3+)-Zn(2+) by superoxide and hydrogen peroxide. This oxidation is accompanied by a gain of manganese dependence of enzyme activity. CaN isolated by a conventional purification procedure was found in the oxidized, ferric enzyme form, and it became increasingly dependent on divalent cations. These results point to a complex redox regulation of CaN phosphatase activity by superoxide, which is modified by calcium, NO, and superoxide dismutase.     

Cellularly Generated Active Oxygen Species and Hela Cell Proliferation

In HeLa cells evidence is provided that active oxygen species such as hydrogen peroxide and superoxide at low levels are important growth regulatory signals. They may constitute a novel regulatory redox system of control superimposed upon the established cell growth signal transduction pathways. Whilst for example hydrogen peroxide can be added exogenously to elicit growth responses in these cells, it is clear that cellularly generated superoxide and hydrogen peroxide are important. Experiments with superoxide dismutase, superoxide dismutase mimics and inhibitors of both superoxide dismutase and xanthine oxidase suggest that superoxide generated intracellularly and superoxide released extracellularly are both relevant to growth control in HeLa cells.     

Cellularly Generated Active Oxygen Species and Hela Cell Proliferation

In HeLa cells evidence is provided that active oxygen species such as hydrogen peroxide and superoxide at low levels are important growth regulatory signals. They may constitute a novel regulatory redox system of control superimposed upon the established cell growth signal transduction pathways. Whilst for example hydrogen peroxide can be added exogenously to elicit growth responses in these cells, it is clear that cellularly generated superoxide and hydrogen peroxide are important. Experiments with superoxide dismutase, superoxide dismutase mimics and inhibitors of both superoxide dismutase and xanthine oxidase suggest that superoxide generated intracellularly and superoxide released extracellularly are both relevant to growth control in HeLa cells.     

Reactivity of tetrahydrobiopterin bound to nitric-oxide synthase.

Levels of tetrahydrobiopterin (BH(4)) bound to nitric-oxide synthase (NOS) were examined during multiple turnovers of the enzyme in the presence of an NADPH-regenerating system. Our findings show that NOS-bound BH(4) does not remain in a static state but undergoes redox reactions. Under these experimental conditions, the redox state of BH(4) was determined by the balance between calcium/calmodulin (Ca(2+)/CaM)-dependent oxidation of BH(4) mediated by the uncoupled formation of superoxide/hydrogen peroxide on the one hand and by reductive regeneration of BH(4) on the other hand. BH(4) oxidation was appreciably increased in the presence of arginine. Levels of NOS-bound BH(4) were also examined under single turnover conditions in the absence of an NADPH-regenerating system and in the presence of added superoxide dismutase and catalase to suppress the accumulation of superoxide and hydrogen peroxide. BH(4) oxidation was again dependent on Ca(2+)/CaM. The insensitivity to superoxide dismutase and catalase suggested that the single turnover oxidation of BH(4) did not proceed through superoxide/peroxide, although the involvement of these oxidants could not be definitively excluded. The amount of BH(4) oxidized was highest in the presence of arginine, and this oxidation significantly exceeded that in the presence of N(G)-hydroxy-L-arginine. The findings that single turnover oxidation of BH(4) is stimulated by arginine in the presence of Ca(2+)/CaM and that BH(4) is regenerated are consistent with a role for the pterin as an electron donor in product formation; this role remains to be defined.     

Redox-dependent signal transduction

Reactive oxygen species (ROS) such as superoxide anions and hydrogen peroxide appear to be transiently produced in response to growth factor and cytokine stimulation. A variety of evidence suggests that this ligand-stimulated change in the cellular redox state participates in downstream signal transduction. This review will focus on the effects of ROS on signal transduction pathways, the molecules that regulate intracellular ROS production and the potential protein targets of oxidants.     

Modification of proteins by active oxygen and their degradation

A detailed analysis of literary data concerning the oxidative modification of proteins by active oxygen species was carried out. It was shown that intermediate products of molecular oxygen reduction, e.g., superoxide anion radical, hydrogen peroxide and hydroxyl radical, can induce the inactivation of enzymes in vitro as a result of oxidative modification of certain amino acid residues necessary for the maintenance of native properties of the enzyme. In some cases modification of enzymes results in their degradation by proteolytic enzymes. Besides, some enzymes catalyzing the interconversions of active oxygen species (catalase superoxide dismutase, cytochrome P-450) are also inactivated in the course of catalysis under the oxidative action of active oxygen species. It was assumed that the oxidative modification of proteins appears to be one of the mechanisms which control their degradation in the cell. The hydroxyl radical oxidizing the amino acid residues located in the vicinity of the site of its synthesis is a direct modifying species. The superoxide anion radical and hydrogen peroxide are hydroxyl radical precursors and are responsible for the transport of oxidizing equivalents in the cell.     

Human copper-zinc superoxide dismutase complements superoxide dismutase-deficient Escherichia coli mutants

An Escherichia coli double mutant, sodAsodB, that is deficient in both bacterial superoxide dismutases (Mn superoxide dismutase and iron superoxide dismutase) is unable to grow on minimal medium in the presence of oxygen and exhibits increased sensitivity to paraquat and hydrogen peroxide. Expression of the evolutionarily unrelated eukaryotic CuZn superoxide dismutase in the sodAsodB E. coli mutant results in a wild-type phenotype with respect to aerobic growth on minimal medium and in resistance to paraquat and hydrogen peroxide. This supports the hypothesis that superoxide dismutation is the in vivo function of these proteins. Analysis of the growth of sodAsodB cells containing plasmids encoding partially active CuZn superoxide dismutases, produced by in vitro mutagenesis, shows a correlation between cell growth and enzyme activity. Thus, the sodAsodB strain provides a controlled selection for varying levels of superoxide dismutase activity.     

Enzymatic defenses against the toxicity of oxygen and of streptonigrin in Escherichia coli.

Anaerobically grown Escherichia coli K-12 contain only one superoxide dismutase and that is the iron-containing isozyme found in the periplasmic space. Exposure to oxygen caused the induction of a manganese-containing superoxide dismutase and of another, previously undescribed, superoxide dismutase, as well as of catalase and peroxidase. These inductions differed in their responsiveness towards oxygen. Thus the very low levels of oxygen present in deep, static, aerobic cultures were enough for nearly maximal induction of the manganese-superoxide dismutase. In contrast, induction of the new superoxide dismutase, catalase, and peroxidase required the much higher levels of oxygen achieved in vigorously agitated aerobic cultures. Anaerobically grown cells showed a much greater oxygen enhancement of the lethality of streptonigrin than did aerobically grown cells, in accord with the proposal that streptonigrin can serve as an intracellular source of superoxide. Anaerobically grown cells in which enzyme inductions were prevented by puromycin were damaged by exposure to air. This damage was evidenced both as a decline in viable cell count and as structural abnormalities evident under an electron microscope.     

Regulation of glutamine synthetase by metal-catalyzed oxidative modification in the marine oxyphotobacterium Prochlorococcus.

The inactivation of glutamine synthetase (GS; EC 6.3.1.2) by metal-catalyzed oxidation (MCO) systems was studied in several Prochlorococcus strains, including the axenic PCC 9511. GS was inactivated in the presence of various oxidative systems, either enzymatic (as NAD(P)H+NAD(P)H-oxidase+Fe(3+)+O(2)) or non-enzymatic (as ascorbate+Fe(3+)+O(2)). This process required the presence of oxygen and a metal cation, and is prevented under anaerobic conditions. Catalase and peroxidase, but not superoxide dismutase, effectively protected the enzyme against inactivation, suggesting that hydrogen peroxide mediates this mechanism, although it is not directly responsible for the reaction. Addition of azide (an inhibitor of both catalase and peroxidase) to the MCO systems enhanced the inactivation. Different thiols induced the inactivation of the enzyme, even in the absence of added metals. However, this inactivation could not be reverted by addition of strong oxidants, as hydrogen peroxide or oxidized glutathione. After studying the effect of addition of the physiological substrates and products of GS on the inactivation mechanism, we could detect a protective effect in the case of inorganic phosphate and glutamine. Immunochemical determinations showed that the concentration of GS protein significantly decreased by effect of the MCO systems, indicating that inactivation precedes the degradation of the enzyme.     

Role of oxidative stress in Drosophila aging.

We review the role that oxidative damage plays in regulating the lifespan of the fruit fly, Drosophila melanogaster. Results from our laboratory show that the lifespan of Drosophila is inversely correlated to its metabolic rate. The consumption of oxygen by adult insects is related to the rate of damage induced by oxygen radicals, which are purported to be generated as by-products of respiration. Moreover, products of activated oxygen species such as hydrogen peroxide and lipofuscin are higher in animals kept under conditions of increased metabolic rate. In order to understand the in vivo relationship between oxidative damage and the production of the superoxide radical, we generated transgenic strains of Drosophila melanogaster that synthesize excess levels of enzymatically active superoxide dismutase. This was accomplished by P-element transformation of Drosophila melanogaster with the bovine cDNA for CuZn superoxide dismutase, an enzyme that catalyzes the dismutation of the superoxide radical to hydrogen peroxide and water. Adult flies that express the bovine SOD in addition to native Drosophila SOD are more resistant to oxidative stresses and have a slight but significant increase in their mean lifespan. Thus, resistance to oxidative stress and lifespan of Drosophila can be manipulated by molecular genetic intervention. In addition, we have examined the ability of adult flies to respond to various environmental stresses during senescence. Resistance to oxidative stress, such as that induced by heat shock, is drastically reduced in senescent flies. This loss of resistance is correlated with the increase in protein damage generated in old flies by thermal stress and by the insufficient protection from cellular defense systems which includes the heat shock proteins as well as the oxygen radical scavenging enzymes. Collectively, results from our laboratory demonstrate that oxidative damage plays a role in governing the lifespan of Drosophila during normal metabolism and under conditions of environmental stress.     

Induction of manganese-superoxide dismutase by membrane-binding drugs in Escherichia coli.

Treatment of exponentially growing cells of Escherichia coli with membrane-binding drugs such as chlorpromazine (CPZ) and procaine resulted in an induction of manganese-superoxide dismutase (Mn-SOD). A slight decrease was observed in the amount of Fe-SOD. The induction of Mn-SOD required de novo synthesis of this enzyme, since it was suppressed by rifampin. The treatment did not cause the induction of Mn-SOD when performed under anaerobic conditions. In E. coli cells with a sodA-lacZ operon fusion, CPZ and procaine induced beta-galactosidase in the presence of oxygen, whereas it was not expressed and was not induced by CPZ and procaine under anaerobic conditions. Although CPZ reduced the ability of cell suspensions to take up oxygen, it increased the cyanide-resistant fraction of the total respiration. Therefore, it appeared likely that the induction of the sodA gene was a response to an increase in superoxide radical production mediated by these membrane-binding drugs in E. coli cells, possibly by disruption of the electron transport systems in the cell membranes.     

Structural Analysis of Peroxide-Soaked MnSOD Crystals Reveals Side-On Binding of Peroxide to Active-Site Manganese

The superoxide dismutase (SOD) enzymes are important antioxidant agents that protect cells from reactive oxygen species. The SOD family is responsible for catalyzing the disproportionation of superoxide radical to oxygen and hydrogen peroxide. Manganese- and iron-containing SOD exhibit product inhibition whereas Cu/ZnSOD does not. Here, we report the crystal structure of Escherichia coli MnSOD with hydrogen peroxide cryotrapped in the active site. Crystallographic refinement to 1.55 Å and close inspection revealed electron density for hydrogen peroxide in three of the four active sites in the asymmetric unit. The hydrogen peroxide molecules are in the position opposite His26 that is normally assumed by water in the trigonal bipyramidal resting state of the enzyme. Hydrogen peroxide is present in active sites B, C, and D and is side-on coordinated to the active-site manganese. In chains B and D, the peroxide is oriented in the plane formed by manganese and ligands Asp167 and His26. In chain C, the peroxide is bound, making a 70° angle to the plane. Comparison of the peroxide-bound active site with the hydroxide-bound octahedral form shows a shifting of residue Tyr34 towards the active site when peroxide is bound. Comparison with peroxide-soaked Cu/ZnSOD indicates end-on binding of peroxide when the SOD does not exhibit inhibition by peroxide and side-on binding of peroxide in the product-inhibited state of MnSOD.     

Production of superoxide anions and hydrogen peroxide in Ehrlich ascites tumour cell nuclei.

Nuclei isolated from Ehrlich-Lettré ascites tumour cells catalyze the co-oxidation of epinephrine to adrenochrome in the presence of NADPH. Adrenochrome formation is sensitive to superoxide dismutase but not to scavengers of hydroxyl radicals or singlet oxygen. Addition of NADPH also initiates the production of hydrogen peroxide. Moreover measurements of superoxide dismutase activity indicate the presence of this enzyme in the ascites cell nuclei, although the sensitivity of adrenochrome formation to externally added superoxide dismutase indicates that the endogenous enzyme is not sufficient for a complete protection from superoxide radicals.     

Production of superoxide anions and hydrogen peroxide in Ehrlich ascites tumour cell nuclei

Nuclei isolated from Ehrlich-Lettré ascites tumour cells catalyze the co-oxidation of epinphrine to adrenochrome in the presence of NADPH. Adrenochrome formation is senstive to superoxide dismutase but not to scavengers of hydroxyl radicals or singler oxygen. Addition of NADPH also initiates the production of hydrogen peroxide. Moreover measurements of superoxide dismutase activity indicate the presence of this enzyme in the ascites cell nuclei, although the sensitivity of adrenochrome formation to externally added superoxide dismutase indicates that the endogenous enzyme is not sufficient for a complete protection from superoxide radicals.