Inactivation of the human CuZn superoxide dismutase during exposure to O-2 and H2O2

Human copper-zinc superoxide dismutase undergoes inactivation when exposed to O₂^? and H₂O₂ generated during the oxidation of acetaldehyde by xanthine oxidase at pH 7.4 and 37° C. In contrast, human manganese superoxide dismutase is not inactivated under the same conditions. Catalase and Mn-superoxide dismutase protect CuZn superoxide dismutase from inactivation. Similar protection is observed with hydroxyl radical (OH.) scavengers, such as formate and mannitol. In contrast, other OH. scavengers such as ethanol and tert-butyl alcohol, have no protective action. The latter results indicate that “free OH.” is not responsible for the inactivation. Furthermore, H₂O₂ generated during the oxidation of glucose by glucose oxidase, i.e., without production of O₂^?, does not induce CuZn superoxide dismutase inactivation. A mechanism accounting for this $\text{O}{}_2{}^{\mathrm{?}}\text{H}{}_2\text{O}{}_2$-dependent inactivation of CuZn superoxide dismutase is proposed.     

The effects of superoxide dismutase on H2O2 formation

Numerous reports of the effects of overproduction of SODs have been explained on the basis of increased H2O2 production by the catalyzed dismutation of O2-. In this review we consider the effects of increasing [SOD] on H2O2 formation and question this explanation.     

The respiratory burst response to Histoplasma capsulatum by human neutrophils. Evidence for intracellular trapping of superoxide anion

Human neutrophils (PMN) have received little attention as to the role they play in host defense against Histoplasma capsulatum (Hc). We have characterized the binding and phagocytosis of Hc yeasts by human PMN and quantified the PMN respiratory burst in response to this organism. mAb specific for CD11a, CD11b, and CD11c all partially blocked the attachment of unopsonized yeasts to PMN; a mAb to CD18 inhibited attachment by greater than 90%. Thus, human PMN recognize and bind Hc yeasts via CD18 adhesion receptors as has been found for human cultured macrophages and alveolar macrophages. Unopsonized yeasts were phagocytosed by PMN, but phagocytosis was increased markedly by heat-labile and heat-stable serum opsonins. These opsonins promoted enhanced phagocytosis of yeasts by increasing the attachment of Hc yeasts to the PMN membrane. Phagocytosis of viable or heat-killed Hc yeasts by PMN did not induce the secretion of superoxide anion (O2-) as quantified by the reduction of cytochrome c. O2- was not detected when yeasts were opsonized in normal serum or immune serum, or at a ratio of yeasts to PMN of up to a 100:1. However, phagocytosis of opsonized yeasts by PMN did not prevent them from subsequently releasing O2- after further incubation with opsonized zymosan or PMA. Opsonized Hc yeasts clearly stimulated the PMN respiratory burst as quantified by intracellular reduction of nitroblue tetrazolium, reduction of cytochrome c in the presence of cytochalasin D, oxygen consumption, luminol-enhanced and nonenhanced chemiluminescence, and H2O2 production. These data suggest that phagocytosis of Hc yeasts by PMN is associated with intracellular entrapment of O2- that is not detectable by reduction of extracellular cytochrome c.     

Differentiation of activity of a superoxide dismutase inhibitor in human cells exposed to radiation, chemical mutagens and radioadaptive response

The superoxide dismutase (SOD) inhibitor, TRIEN, which enhanced the formation of gamma-induced DNA breaks in cells of healthy donors and patients with Marfan syndrome and Bloom syndrome (repair-defective hereditary diseases), had virtually no effect on the formation of radioadaptive response (RAR) in these systems. Similar results were obtained in studies on cell survival: TRIEN facilitated mortality in cells irradiated with gamma-rays but did not affect RAR formation. TRIEN also increased the deleterious effect of CdCl2, which indicates that SOD apparently plays a certain role in cell defence against this mutagen.     

Comparison of the effects of superoxide dismutase and cytochrome c on luminol chemiluminescence produced by xanthine oxidase-catalyzed reactions

Superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) (SOD) and ferricytochrome c are used to check the effects on luminol chemiluminescence induced by a xanthine or hypoxanthine/xanthine oxidase/oxygen system. Luminol chemiluminescence has been attributed to superoxide anion radical (O2.-) in this system. From kinetic studies on the light intensity vs. time curves it is demonstrated that addition of SOD into the system does not affect the mechanism of O2.- generation, whilst ferricytochrome c dramatically alters the time-course of the reaction. This is interpreted as the effect of cytochrome c redox cycling by reaction with H2O2, modifying oxy-radical generation in the reaction medium. Also, an alternative mechanism for luminol chemiexcitation is proposed under certain experimental conditions.     

Phosphate, not superoxide dismutase, facilitates electron transfer from ferrous salts to cytochrome c.

Peterson and Eaton (1989, Biochem. Biophys. Res. Commun. 165, 164-167) reported that the copper- and zinc-containing, but not the manganese-containing, superoxide dismutase catalyzes the reduction of cytochrome c by ferrous salts. This activity, erroneously attributed to the enzyme, is now shown to have been due to inorganic phosphate.     

Pancreatic phospholipase A2--mediated enhancement of the respiratory burst response of human neutrophils

The aim of this study was to investigate the effects of exogenously added pancreatic phospholipase A2 (pPLA2) on the production of reactive oxygen species by human polymorphonuclear leukocytes (PMNs). Pancreatic PLA2 was used because PMNs do not possess a receptor for that enzyme and, therefore, the receptor-mediated effects could be excluded. Respiratory burst activity of PMNs was monitored by luminol-amplified chemiluminescence and the lipid composition of neutrophils after treatment with pPLA2 was determined by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Our results show that the products of the pPLA2 digestion of the PMN membrane--lysophospholipids and the corresponding free fatty acids--significantly enhanced the respiratory burst response of human neutrophils.     

Assay of superoxide dismutase: cautions relevant to the use of cytochrome c, a sulfonated tetrazolium, and cyanide.

Two commonly used assays for superoxide dismutase (SOD) activity have been compared, one using cytochrome c and the other using XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) as the indicating scavenger of superoxide. The use of cyanide to selectively suppress Cu,Zn-SOD and thus to allow assay of both Cu,Zn-SOD and Mn-SOD in mixtures of the two was also explored, as was the influence of pH. The XTT assay became more sensitive at elevated pH, because the rate of the superoxide/XTT reaction declines with increasing pH. This was clearly seen with the Cu,Zn-SOD but barely with Mn-SOD because the former retains full activity from pH 5 to 10 while the latter does not. Cyanide reacted with cytochrome c, but not XTT, in a concentration- and time-dependent manner and thus diminished its reducibility by superoxide. Cytochromes endogenous to tissue fractions were reduced by the xanthine oxidase reaction and this caused a decrease in absorbance 470 nm which interfered with the XTT assay. The alkalinizing effect of cyanide salts and the problems encountered in neutralizing cyanide stock solutions are discussed.     

The superoxide dismutase inhibitor diethyldithiocarbamate has antagonistic effects on apoptosis by triggering both cytochrome c release and caspase inhibition

Tumor necrosis factor-alpha (TNF-alpha) and etoposide both trigger a large and rapid production of reactive oxygen species (ROS) in HeLa cells. This occurs before translocations of the proapoptotic Bax and cytochrome c proteins, the loss of mitochondrial membrane potential (DeltaPsim), and apoptosis. We have used diethyldithiocarbamate (DDC), a well-known inhibitor of Cu, Zn superoxide dismutase to study the role of ROS in this system. We report that DDC strongly inhibits caspase activation, loss of DeltaPsim, and cell death induced by TNF-alpha or etoposide. Surprisingly, DDC does not inhibit Bax and cytochrome c translocations. On the contrary, we have observed that DDC can trigger the translocations of these proteins by itself, without altering DeltaPsim. Here, we report that DDC has at least two antagonistic apoptosis regulation functions. First, DDC triggers ROS-dependent Bax and cytochrome c translocations, which are potentially proapoptotic, and second, DDC inhibits caspase activation and activity, loss of DeltaPsim, and cell death, in a ROS-independent manner. Our results suggest an interesting model in which ROS-dependent Bax and cytochrome c translocations can be studied without interference from later apoptotic events.     

Reduction of exogenous cytochrome c by Neurospora crassa conidia: effects of superoxide dismutase and blue light.

The reduction of externally added cytochrome c by Neurospora crassa conidia was observed. The reduction was stimulated by antimycin A and suppressed partially by superoxide dismutase. When conidia were treated with diethyldithiocarbamate, which inactivated endogenous superoxide dismutase, the cytochrome c reduction was stimulated. Blue light also stimulated the cytochrome c reduction. Azide, which inhibits photochemical reactions mediated by flavins, suppressed the blue light effect. Superoxide dismutase partially suppressed the cytochrome c reduction in the light. The results suggest that O2(-) participates in the cytochrome c reduction by conidia and the flavins or flavoproteins are candidates for the receptor pigment of blue light to stimulate the cytochrome c reduction. It was also suggested that the redox component(s), which could directly transfer its reducing equivalents to exogenous cytochrome c, was present at the surface of conidia.     

Superoxide dismutase does not inhibit the oxidation of cytochrome c and cytochrome oxidase.

An electrometric system was used to measure Ca⁺⁺ uptake by sarcoplasmic reticulum vesicles (SR). The method permits continuous recording of Ca⁺⁺ uptake and thus the valuation of kinetic parameters. Furthermore, the ultrasensitivity of the method permits to follow changes in Ca⁺⁺ concentration below 10^?6 M.     

A comparative kinetic analysis of the reactivity of plant, horse, and human respiratory cytochrome c towards cytochrome c oxidase

Two synthetic genes coding for human and Arabidopsis cytochrome c, respectively, have been designed and constructed, and the recombinant proteins have been over-expressed in Escherichia coli cells. Thus a comparative analysis of the two heme proteins, including horse cytochrome c as a reference, has been performed. In addition to their physico-chemical properties, the redox behavior of the three proteins has been analyzed by following the kinetics of both their reduction by flavin semiquinones (lumiflavin, riboflavin, and FMN) and oxidation by cytochrome c oxidase. The resulting data indicate that the accessibility and electrostatic charge of the active site do not differ in a significant way among the three proteins, but human cytochrome c exhibits some intriguing differences when interacting with cytochrome c oxidase that could be related to the amino acid changes underwent by the latter along evolution.     

Overexpression of manganese superoxide dismutase promotes the survival of prostate cancer cells exposed to hyperthermia

It has been hypothesized that exposure of cells to hyperthermia results in an increased flux of reactive oxygen species (ROS), primarily superoxide anion radicals, and that increasing antioxidant enzyme levels will result in protection of cells from the toxicity of these ROS. In this study, the prostate cancer cell line, PC-3, and its manganese superoxide dismutase (MnSOD)-overexpressing clones were subjected to hyperthermia (43°C, 1 h). Increased expression of MnSOD increased the mitochondrial membrane potential (MMP). Hyperthermic exposure of PC-3 cells resulted in increased ROS production, as determined by aconitase inactivation, lipid peroxidation, and H₂O₂ formation with a reduction in cell survival. In contrast, PC-3 cells overexpressing MnSOD had less ROS production, less lipid peroxidation, and greater cell survival compared to PC-3 Wt cells. Since MnSOD removes superoxide, these results suggest that superoxide free radical or its reaction products are responsible for part of the cytotoxicity associated with hyperthermia and that MnSOD can reduce cellular injury and thereby enhance heat tolerance.     

Effect of cytochrome c on the generation and elimination of O2*- and H2O2 in mitochondria

The primary recognized function of cytochrome c is to act as an electron carrier transferring electrons from complex III to complex IV in the respiratory chain of mitochondria. Recent studies on cell apoptosis reveal that cytochrome c is responsible for the programmed cell death when it is released from mitochondria to cytoplasm. In this study we present evidence showing that cytochrome c plays an antioxidative role by acting on the generation and elimination of O(2)(*) and H(2)O(2) in mitochondria. The O(2)(*) and H(2)O(2) generation in cytochrome c-depleted Keilin-Hartree heart muscle preparation (HMP) is 7-8 times higher than that in normal HMP. The reconstitution of cytochrome c to the cytochrome c-depleted HMP causes the O(2)(*) and H(2)O(2) generation to exponentially decrease. An alternative electron-leak pathway of the respiratory chain is suggested to explain how cytochrome c affects on the generation and elimination of O(2)(*) and H(2)O(2) in mitochondria. Enough cytochrome c in the respiratory chain is needed for keeping O(2)(*) and H(2)O(2) at a lower physiological level. A dramatic increase of O(2)(*) and H(2)O(2) generation occurs when cytochrome c is released from the respiratory chain. The burst of O(2)(*) and H(2)O(2), which happens at the same time as cytochrome c release from the respiratory chain, should have some role in the early stage of cell apoptosis.     

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.     

Effects of iron and manganese availability on growth and production of superoxide dismutase by Streptococcus suis

A complex medium supported good growth of the type strain of Streptococcus suis irrespective of the presence or absence of a high concentration (1 microM) of the iron chelating agent, ethylenediamine di-o-hydroxyphenylacetic acid. Good growth was also obtained using a complex medium that had been treated with Chelex-100 to reduce the iron content, but only if this medium was supplemented with manganese; supplementation with iron had little effect. Collectively, these results indicate that S. suis requires manganese, but not iron, for growth. Polyacrylamide gel electrophoresis of cell extracts followed by activity staining revealed the presence of a single manganese-cofactored superoxide dismutase; activity staining and enzyme assays revealed that manganese availability during growth affected the activity of the superoxide dismutase in cell extracts. The results are discussed with respect to the capacity of S. suis to avoid damage by reactive oxygen species.     

Kinetic evidence for the re-definition of electron transfer pathways from cytochrome c to O2 within cytochrome oxidase

The reaction with O2 of equimolar mixtures of cytochrome c and cytochrome c oxidase in high and low ionic strength buffers has been examined by flow-flash spectrophotometry at room temperature. In low ionic strength media where cytochrome c and the oxidase are bound in an electrostatic, 1:1 complex some of the cytochrome c is oxidised at a faster rate than a metal centre of the oxidase. In contrast, when cytochrome c and cytochrome c oxidase are predominantly dissociated at high ionic strength cytochrome c oxidation occurs only slowly (t1/2 = 5 s) following the complete oxidation of the oxidase. These results demonstrate that maximal rates of electron transfer from cytochrome c to O2 occur when both substrates are present on the enzyme. The heterogeneous oxidation of cytochrome c observed in the complex implies more than one route for electron transfer within the enzyme. Possibilities for new electron transfer pathways from cytochrome c to O2 are proposed.     

The reduction of cytochrome b558 and the activity of the respiratory burst oxidase from human neutrophils.

It is known that in respiratory burst oxidase preparations engaged in O2- production, cytochrome b558, a characteristic oxidase component, is partly reduced. This result has been interpreted in terms of a mechanism in which cytochrome b558 functions as an electron-carrying component of the respiratory burst oxidase, its level of reduction reflecting a steady-state partitioning of the cytochrome between reduced and oxidized forms as it ferries electrons from NADPH to oxygen. Kinetic arguments based on this interpretation have supported the proposal that the cytochrome is reduced at a rate sufficient to account for the rate of O2- production by activated neutrophils. We have confirmed the partial reduction of cytochrome b558 in neutrophil cytoplasts and in oxidase preparations exposed to NADPH, but have found that the reduction of the cytochrome bears no apparent relation to the activity of the oxidase, and can occur when NADPH is added to neutrophil membrane preparations that are unable to manufacture O2-. We therefore conclude that the NADPH-dependent reduction of cytochrome b558 seen in these preparations is unlikely to be a reflection of a catalysis-related steady state and that inferences drawn from such observations regarding the kinetic competence of the cytochrome may need to be reconsidered.