The oxidation of 3-hydroxyanthranilic acid by Cu,Zn superoxide dismutase: mechanism and possible consequences

Cu,Zn SOD, but not Mn SOD, catalyzes the oxidation of 3-hydroxyanthranilic acid (3-HA) under aerobic conditions. In the absence of O2, the Cu(II) of the enzyme is reduced by 3-HA. One plausible mechanism involves the reduction of the active site Cu(II) to Cu(I), which is then reoxidized by the O2- generated by autoxidation of the anthranilyl or other radicals on the pathway to cinnabarinate. We may call this the superoxide reductase, or SOR, mechanism. Another possibility invokes direct reoxidation of the active site Cu(I) by the anthranilyl, or other organic radicals, or by the peroxyl radicals generated by addition of O2 to these organic radicals. Such oxidations catalyzed by Cu,Zn SOD could account for the deleterious effects of the mutant Cu,Zn SODs associated with familial amyotrophic lateral sclerosis and of the overproduction or overadministration of wild-type Cu,Zn SOD.     

Effect of superoxide dismutase on the autoxidation of substituted hydro- and semi-naphthoquinones

The effect of superoxide dismutase on the autoxidation of hydro- and semi-1,4-naphthoquinones with different substitution pattern and covering a one-electron reduction potential range from -95 to -415 mV was examined. The naphthoquinone derivatives were reduced via one or two electrons by purified NADPH-cytochrome P-450 reductase or DT-diaphorase, respectively. Superoxide dismutase did not alter or slightly enhance the initial rates of enzymic reduction, whereas it affected in a different manner the following autoxidation of the semi- and hydroquinones formed. Autoxidation was assessed as NADPH oxidation in excess to the amounts required to reduce the quinone present, H2O2 formation, and the redox state of the quinones. Superoxide dismutase enhanced 2--8-fold the autoxidation of 1,4-naphthosemiquinones, following the reduction of the oxidized counterpart by NADPH-cytochrome P-450 reductase, except for the glutathionyl-substituted naphthosemiquinones, whose autoxidation was not affected by superoxide dismutase. Superoxide dismutase exerted two distinct effects on the autoxidation of naphthohydroquinones formed during DT-diaphorase catalysis: on the one hand, it enhanced slightly the autoxidation of 1,4-naphthohydroquinones with a hydroxyl substituent in the benzene ring: 5-hydroxy-1,4-naphthoquinone and the corresponding derivatives with methyl- and/or glutathionyl substituents at C2 and C3, respectively. On the other hand, superoxide dismutase inhibited the autoxidation of naphthohydroquinones that were either unsubstituted or with glutathionyl-, methyl-, methoxyl-, hydroxyl substituents (the latter in the quinoid ring). The inhibition of hydroquinone autoxidation was reflected as a decrease of NADPH oxidation, suppression of H2O2 production, and accumulation of the reduced form of the quinone. The enhancement of autoxidation of 1,4-naphthosemiquinones by superoxide dismutase has been previously rationalized in terms of the rapid removal of O2-. by the enzyme from the equilibrium of the autoxidation reaction (Q2-. + O2----Q + O2-.), thus displacing it towards the right. The superoxide dismutase-dependent inhibition of H2O2 formation as well as NADPH oxidation during the autoxidation of naphthohydroquinones--except those with a hydroxyl substituent in the benzene ring--seems to apply to those organic substrates which can break down with simultaneous formation of a semiquinone and O2-.. Inhibition of hydroquinone autoxidation by superoxide dismutase can be interpreted in terms of suppression by the enzyme of O2-.- dependent chain reactions or a direct catalytic interaction with the enzyme that might involve reduction of the semiquinone at expense of O2(-.).(ABSTRACT TRUNCATED AT 400 WORDS)     

Dismutation of superoxide radicals by ceruloplasmin--details of the mechanism

Like superoxide dismutase (SOD), human ceruloplasmin (Cp) scavenges superoxide anion radicals injected into the solution with the aid a high-voltage generator, hydrogen peroxide being the product of reaction. The O2-/H2O2 ratio is close to 2:1. The dismutase activity of Cp is about 1500 times lower than that of Cu, Zn-SOD isolated from human erythrocytes. The dismutation of O2- accomplished by SOD, "free" copper ions, native Cp or partly copper-depleted Cp, is inhibited with equal efficiency by cyanide. All the copper ions of the multicopper catalytic center of Cp are not essentially required for the dismutation of O2-, since the enzyme depleted of all type 2 Cu2+ and partly of type 1 Cu2+ lost none of its dismutase activity. Type 1 copper ions of Cp seem to play the leading role in the one-electron transfer occurring upon dismutation of O2-.     

The role of superoxide radical in the autoxidation of cytochrome c.

The net rate of autoxidation of ferrocytochrome c was decreased by ferricytochrome c. Superoxide dismutase accelerated this autoxidation to a limit and overcame the inhibitory effect of ferricytochrome c. This was the case whether the autoxidationwas observed in the presence or in the absence of denaturants, such as alcohols orurea, and whether the superoxide dismutase used was the Cu-2+-Zn-2+ enzyme from bovine erythrocytes or the Mn-3+-enzyme from Escherichia coli. It can be deduced that the autoxidation of ferrocytochrome c, under a variety of conditions, geenerates O2 minus which can then dismute to H202 + O2 or can reduce ferricytochrome c back to ferrocytochrome c. Superoxide dismutase, by accelerating the dismutation of O2 minus, prevents the back reaction and thus exposes the true rate of reaction of ferrocytochrome c with molecular oxygen.     

Multiple actions of superoxide dismutase: why can it both inhibit and stimulate reduction of oxygen by hydroquinones?

Superoxide dismutase can either inhibit or stimulate autoxidation of different hydroquinones, suggesting multiple roles for O2.-. Inhibitory actions of superoxide dismutase include termination of O2.(-)-propagated reaction chains and metal chelation by the apoprotein. Together, chelation of metals and termination of O2.(-)-propagated chains can effectively prevent reduction of oxygen. Chain termination by superoxide dismutase can thus account for negligible accumulation of H2O2 without invoking a superoxide:semiquinone oxidoreductase activity for this enzyme. One stimulatory action of superoxide dismutase is to decrease thermodynamic limitations to reduction of oxygen. Whether superoxide dismutase inhibits or accelerates an autoxidation depends on the reduction potentials of the quinone and the availability of metal coordination for inner sphere electron transfers.     

Effect of superoxide dismutase on the autoxidation of various hydroquinones--a possible role of superoxide dismutase as a superoxide:semiquinone oxidoreductase

The autoxidation of DT-diaphorase-reduced 1,4-naphthoquinone, 2-OH-1,4-naphthoquinone, and 2-OH-p-benzoquinone is efficiently prevented by superoxide dismutase. This effect was assessed in terms of an inhibition of NADPH oxidation (over the amount required to reduce the available quinone), O2 consumption, and H2O2 formation. Superoxide dismutase also affects the distribution of molecular products -hydroquinone/quinone-involved in autoxidation, by favoring the accumulation of the reduced form of the above quinones. In contrast, the rate of autoxidation of DT-diaphorase-reduced 1,2-naphthoquinone is enhanced by superoxide dismutase, as shown by increased rates of NADPH oxidation, O2 consumption, and H2O2 formation and by an enhanced accumulation of the oxidized product, 1,2-naphthoquinone. These findings suggest that superoxide dismutase can either prevent or enhance hydroquinone autoxidation. The former process would imply a possible new activity displayed by superoxide dismutase involving the reduction of a semiquinone by O2-.. This activity is probably restricted to the redox properties of the semiquinones under study, as indicated by the failure of superoxide dismutase to prevent autoxidation of 1,2-naphthohydroquinone.     

Reactions involving superoxide and normal and unstable haemoglobins.

Superoxide ions (O2-) oxidized oxyhaemoglobin to methaemoglobin and reduced methaemoglobin to oxyhaemoglobin. The reactions of superoxide and H2O2 with oxyhaemoglobin or methaemoglobin and their inhibition by superoxide dismutase or catalase were used to detect the formation of superoxide or H2O2 on autoxidation of oxyhaemoglobin. The rate of autoxidation was decreased at about 35% in the presence of both enzymes. The copper-catalysed autoxidation of Hb (haemoglobin) was also shown to involve superoxide production. Superoxide was released on autoxidation of three unstable haemoglobins and isolated alpha and beta chains, at rates faster than with Hb A. Reactions of superoxide with Hb Christchurch and Hb Belfast were identical with those with Hb A, and occurred at the same rate. Hb Koln contrasted with the other haemoglobins in that the thiol groups of residue beta-93 as well as the haem groups reacted with superoxide. Haemichrome formation from methaemoglobin occurred very rapidly with Hb Christchurch and Hb Belfast, as well as the isolated chains, compared with Hb A. The process did not involve superoxide production or utilization. The relative importance of autoxidation and superoxide production compared with haemichrome formation in the haemolytic process associated with these abnormal haemoglobins and thalassaemia is considered.     

Oxygen activation during oxidation of methoxyhydroquinones by laccase from Pleurotus eryngii

Oxygen activation during oxidation of the lignin-derived hydroquinones 2-methoxy-1,4-benzohydroquinone (MBQH(2)) and 2, 6-dimethoxy-1,4-benzohydroquinone (DBQH(2)) by laccase from Pleurotus eryngii was examined. Laccase oxidized DBQH(2) more efficiently than it oxidized MBQH(2); both the affinity and maximal velocity of oxidation were higher for DBQH(2) than for MBQH(2). Autoxidation of the semiquinones produced by laccase led to the activation of oxygen, producing superoxide anion radicals (Q(*-) + O(2) <--> Q + O(2)(*-)). As this reaction is reversible, its existence was first noted in studies of the effect of systems consuming and producing O(2)(*-) on quinone formation rates. Then, the production of H(2)O(2) in laccase reactions, as a consequence of O(2)(*-) dismutation, confirmed that semiquinones autoxidized. The highest H(2)O(2) levels were obtained with DBQH(2), indicating that DBQ(*-) autoxidized to a greater extent than did MBQ(*-). Besides undergoing autoxidation, semiquinones were found to be transformed into quinones via dismutation and laccase oxidation. Two ways of favoring semiquinone autoxidation over dismutation and laccase oxidation were increasing the rate of O(2)(*-) consumption with superoxide dismutase (SOD) and recycling of quinones with diaphorase (a reductase catalyzing the divalent reduction of quinones). These two strategies made the laccase reaction conditions more natural, since O(2)(*-), besides undergoing dismutation, reacts with Mn(2+), Fe(3+), and aromatic radicals. In addition, quinones are continuously reduced by the mycelium of white-rot fungi. The presence of SOD in laccase reactions increased the extent of autoxidation of 100 microM concentrations of MBQ(*-) and DBQ(*-) from 4.5 to 30.6% and from 19.6 to 40.0%, respectively. With diaphorase, the extent of MBQ(*-) autoxidation rose to 13.8% and that of DBQ(*-) increased to 39.9%.     

On the mechanism of the Mn3(+)-induced neurotoxicity of dopamine:prevention of quinone-derived oxygen toxicity by DT diaphorase and superoxide dismutase

Dopamine (DA) is rapidly oxidized by Mn3(+)-pyrophosphate to its cyclized o-quinone (cDAoQ), a reaction which can be prevented by NADH, reduced glutathione (GSH) or ascorbic acid. The oxidation of DA by Mn3+, which appears to be irreversible, results in a decrease in the level of DA, but not in a formation of reactive oxygen species, since oxygen is neither consumed nor required in this reaction. The formation of cDAoQ can initiate the generation of superoxide radicals (O2-.) by reduction-oxidation cycling, i.e. one-electron reduction of the quinone by various NADH- or NADPH-dependent flavoproteins to the semiquinone (QH.), which is readily reoxidized by O2 with the concomitant formation of O2-.. This mechanism is believed to underly the cytotoxicity of many quinones. Two-electron reduction of cDAoQ to the hydroquinone can be catalyzed by the flavoprotein DT diaphorase (NAD(P)H:quinone oxidoreductase). This enzyme efficiently maintains DA quinone in its fully reduced state, although some reoxidation of the hydroquinone (QH2) is observed (QH2 + O2----QH. + O2-. + H+; QH. + O2----Q + O2-.). In the presence of Mn3+, generated from Mn2+ by O2-. (Mn2+ + 2H+ + O2-.----Mn3+ + H2O2) formed during the autoxidation of DA hydroquinone, the rate of autoxidation is increased dramatically as is the formation of H2O2. Furthermore, cDAoQ is no longer fully reduced and the steady-state ratio between the hydroquinone and the quinone is dependent on the amount of DT diaphorase present. The generation of Mn3+ is inhibited by superoxide dismutase (SOD), which catalyzes the disproportionation of O2-. to H2O2 and O2. It is noteworthy that addition of SOD does not only result in a decrease in the amount of H2O2 formed during the regeneration of Mn3+, but, in fact, prevents H2O2 formation. Furthermore, in the presence of this enzyme the consumption of O2 is low, as is the oxidation of NADH, due to autoxidation of the hydroquinone, and the cyclized DA o-quinone is found to be fully reduced. These observations can be explained by the newly-discovered role of SOD as a superoxide:semiquinone (QH.) oxidoreductase catalyzing the following reaction: O2-. + QH. + 2H+----QH2 + O2. Thus, the combination of DT diaphorase and SOD is an efficient system for maintaining cDAoQ in its fully reduced state, a prerequisite for detoxication of the quinone by conjugation with sulfate or glucuronic acid. In addition, only minute amounts of reactive oxygen species will be formed, i.e. by the generation of O2-., which through disproportionation to H2O2 and further reduction by ferrous ions can be converted to the hydroxyl radical (OH.). Absence or low levels of these enzymes may create an oxidative stress on the cell and thereby initiate events leading to cell death.     

Reversal of the superoxide dismutase reaction revisited

Reversal of the superoxide dismutase (SOD) reaction was measured in terms of the reduction of tetranitromethane (TNM) by O2-. Cu,ZnSOD caused a biphasic reduction of TNM by H2O2. The rapid initial phase was stoichiometric with the enzyme and was followed by a slower catalytic phase that was oxygen dependent and was augmented by HCO3-. The reaction scheme explaining this behavior is presented and a rate constant for the reduction of O2 by the cuprous enzyme is estimated. This rate constant is so low that it precludes significant O2- production by the reduced enzyme under the conditions explored.     

Dual effects of superoxide dismutase on the autoxidation of 1,4-naphthohydroquinone

The autoxidation of 1,4-naphthohydroquinone, in a phosphate, EDTA buffer at pH 7.4, exhibits an autocatalysis whose lag phase becomes more pronounced in the presence of either the Cu,Zn- or the Mn-containing superoxide dismutases. In contrast, the autoxidation of a second aliquot of the hydroquinone, added after complete oxidation of the first, is linear and is accelerated by superoxide dismutase. Catalase or inactive superoxide dismutase were without effect in either situation. These results are explicable in terms of a free radical chain reaction which is initially propagated by O2- and then, as the quinone accumulates, by univalent reduction of the quinone by the hydroquinone. Reduction of the quinone by O2- diminishes the overall rate of oxidation. It is not necessary to postulate catalysis by superoxide dismutase of the reduction of the semiquinone by O2-.     

Hydrogen peroxide damages the zinc-binding site of zinc-deficient Cu,Zn superoxide dismutase

Mutations in Cu,Zn superoxide dismutase (Cu,Zn SOD) account for approximately 20% of cases of familial amyotrophic lateral sclerosis (ALS), a late-onset neurodegenerative disease affecting motor neurons. These mutations decrease protein stability and lower zinc affinity. Zinc-deficient SOD (Cu,E SOD) has altered redox activities and is toxic to motor neurons in vitro. Using bovine SOD, we studied the effects of hydrogen peroxide (H(2)O(2)) on Cu,E SOD and Cu,Zn SOD. Hydrogen peroxide treatment of Cu,E SOD inactivated zinc binding activity six times faster than superoxide dismutase activity, whereas inactivation of dismutase activity occurred at the same rate for both Cu,Zn SOD and Cu,E SOD. Zinc binding by Cu,E SOD was also damaged by simultaneous generation of superoxide and hydrogen peroxide by xanthine oxidase plus xanthine. Although urate, xanthine, and ascorbate can protect superoxide dismutase activity of Cu,Zn SOD from inactivation, they were not effective at protecting Cu,E SOD. Hydrogen peroxide induced subtle changes in the tertiary structure but not the secondary structure of Cu,E SOD as detected by near and far UV circular dichroism. Our results suggest that low levels of hydrogen peroxide could potentially enhance the toxicity of zinc deficient SOD to motor neurons in ALS by rendering zinc loss from SOD irreversible.     

Mitochondrial respiratory chain and thioredoxin reductase regulate intermembrane Cu,Zn-superoxide dismutase activity: implications for mitochondrial energy metabolism and apoptosis

IMS (intermembrane space) SOD1 (Cu/Zn-superoxide dismutase) is inactive in isolated intact rat liver mitochondria and is activated following oxidative modification of its critical thiol groups. The present study aimed to identify biochemical pathways implicated in the regulation of IMS SOD1 activity and to assess the impact of its functional state on key mitochondrial events. Exogenous H2O2 (5 microM) activated SOD1 in intact mitochondria. However, neither H2O2 alone nor H2O2 in the presence of mitochondrial peroxiredoxin III activated SOD1, which was purified from mitochondria and subsequently reduced by dithiothreitol to an inactive state. The reduced enzyme was activated following incubation with the superoxide generating system, xanthine and xanthine oxidase. In intact mitochondria, the extent and duration of SOD1 activation was inversely correlated with mitochondrial superoxide production. The presence of TxrR-1 (thioredoxin reductase-1) was demonstrated in the mitochondrial IMS by Western blotting. Inhibitors of TxrR-1, CDNB (1-chloro-2,4-dinitrobenzene) or auranofin, prolonged the duration of H2O2-induced SOD1 activity in intact mitochondria. TxrR-1 inactivated SOD1 purified from mitochondria in an active oxidized state. Activation of IMS SOD1 by exogenous H2O2 delayed CaCl2-induced loss of transmembrane potential, decreased cytochrome c release and markedly prevented superoxide-induced loss of aconitase activity in intact mitochondria respiring at state-3. These findings suggest that H2O2, superoxide and TxrR-1 regulate IMS SOD1 activity reversibly, and that the active enzyme is implicated in protecting vital mitochondrial functions.     

Effects of superoxide dismutase on the autoxidation of 1,4-hydroquinone

During autoxidation of 1,4-hydroquinone (H2Q, less than 1 mM) at pH 7.4 and 37 degrees C, stoichiometric amounts of 1,4-benzoquinone (Q) and hydrogen peroxide were formed during the initial reaction. The reaction kinetics showed a significant induction period which was abolished by minute amounts of Q. Hydrogen peroxide and catalase were without effect on the autoxidation process. Transition metals apparently were not involved, since chelators like EDTA, DETAPAC, and desferrioxamine or FeSO4 had no influence on the autoxidation kinetics. Superoxide dismutase (SOD) did not abolish the induction period but dramatically enhanced the autoxidation rate by more than two orders of magnitude. The stimulatory effect was first-order in SOD concentration but showed saturation kinetics. The dependence of Q and hydrogen peroxide formation rates on H2Q concentration shows a biphasic behaviour: dependence on the square at low H2Q, but on the square root at high H2Q concentration. As revealed by calculatory simulations the results can be adequately described by the known reaction rate constants. The reaction starts with the comproportionation of H2Q and Q to yield two semiquinone molecules which autoxidize to give two superoxide radicals and two molecules of Q which enter into a new cycle of comproportionation. Because of unfavourable equilibria the autocatalytic reaction soon comes to steady state, and the further reaction is governed by the rate of superoxide removal. At excess SOD, the comproportionation reaction is rate-limiting, thus explaining the saturation effects of SOD. The experiments do not allow a decision between the two functions of SOD; the conventional action as a superoxide:superoxide oxidoreductase or as a semiquinone:superoxide oxidoreductase. In the latter reaction SOD is thought to be reduced by semiquinone with Q formation. In the second step the reduced enzyme would be re-oxidized by a superoxide radical which is formed during autoxidation of the second semiquinone molecule generated in the comproportionation reaction. From thermodynamic considerations, the latter function of SOD appears to be plausible.     

A new paradigm: manganese superoxide dismutase influences the production of H2O2 in cells and thereby their biological state

The principal source of hydrogen peroxide in mitochondria is thought to be from the dismutation of superoxide via the enzyme manganese superoxide dismutase (MnSOD). However, the nature of the effect of SOD on the cellular production of H(2)O(2) is not widely appreciated. The current paradigm is that the presence of SOD results in a lower level of H(2)O(2) because it would prevent the non-enzymatic reactions of superoxide that form H(2)O(2). The goal of this work was to: a) demonstrate that SOD can increase the flux of H(2)O(2), and b) use kinetic modelling to determine what kinetic and thermodynamic conditions result in SOD increasing the flux of H(2)O(2). We examined two biological sources of superoxide production (xanthine oxidase and coenzyme Q semiquinone, CoQ(*-) that have different thermodynamic and kinetic properties. We found that SOD could change the rate of formation of H(2)O(2) in cases where equilibrium-specific reactions form superoxide with an equilibrium constant (K) less than 1. An example is the formation of superoxide in the electron transport chain (ETC) of the mitochondria by the reaction of ubisemiquinone radical with dioxygen. We measured the rate of release of H(2)O(2) into culture medium from cells with differing levels of MnSOD. We found that the higher the level of SOD, the greater the rate of accumulation of H(2)O(2). Results with kinetic modelling were consistent with this observation; the steady-state level of H(2)O(2) increases if K<1, for example CoQ(*-)+O(2)-->CoQ+O(2)(*-). However, when K>1, e.g. xanthine oxidase forming O(2)(*-), SOD does not affect the steady state-level of H(2)O(2). Thus, the current paradigm that SOD will lower the flux of H(2)O(2) does not hold for the ETC. These observations indicate that MnSOD contributes to the flux of H(2)O(2) in cells and thereby is involved in establishing the cellular redox environment and thus the biological state of the cell.     

Generation of superoxide ion in human red blood cell lysates

The generation of superoxide ion in human red blood cell lysates was investigated by an experimental method employing Cu,Zn superoxide dismutase as O2- scavenger and EPR to probe the oxidation state of the enzyme. The average value of the O2- flux in the erythrocytes of 8 normal individuals was (2.02 +/- 0.97) X 10(-8) M S-1. A progressive saturation of the rate of O2- production was found increasing PO2, KM = 1.04 X 10(-4) M, while the autoxidation of oxyhemoglobin did not contribute significantly to the measured O2- production.     

Copper,zinc superoxide dismutase and H2O2. Effects of bicarbonate on inactivation and oxidations of NADPH and urate,and on consumption of H2O2

Copper,zinc superoxide dismutase (Cu,Zn-SOD) catalyzes the HCO(3)(-)-dependent oxidation of diverse substrates. The mechanism of these oxidations involves the generation of a strong oxidant, derived from H(2)O(2), at the active site copper. This bound oxidant then oxidizes HCO(3)(-) to a strong and diffusible oxidant, presumably the carbonate anion radical that leaves the active site and then oxidizes the diverse substrates. Cu,Zn-SOD is also subject to inactivation by H(2)O(2). It is now demonstrated that the rates of HCO(3)(-)-dependent oxidations of NADPH and urate exceed the rate of inactivation of the enzyme by approximately 100-fold. Cu,Zn-SOD is also seen to catalyze a HCO(3)(-)-dependent consumption of the H(2)O(2) and that HCO(3)(-) does not protect Cu,Zn-SOD against inactivation by H(2)O(2). A scheme of reactions is offered in explanation of these observations.     

Manganese-mediated oxidative damage of cellular and isolated DNA by isoniazid and related hydrazines: non-Fenton-type hydroxyl radical formation.

The mechanism by which hydrazines induce damage to cellular and isolated DNA in the presence of metal ions has been investigated by pulsed-field gel electrophoresis (PFGE), DNA sequencing methods, and the ESR spin-trapping technique. For the detection of single-strand breaks by PFGE, an experimental procedure with alkali treatment has been designed. Isoniazid, hydrazine, and phenylhydrazine induced DNA single- and double-strand breaks in cells pretreated with Mn(II), whereas iproniazid did not. With isolated 32P-DNA, isoniazid produced DNA damage in the presence of Cu(II), Mn(II), or Mn(III). Iproniazid damage isolated DNA only in the presence of Cu(II). The Cu(II)-mediated DNA damage by isoniazid or iproniazid is due to active oxygen species other than hydroxyl free radical (.OH), presumably the Cu(I)-peroxide complex. Cleavage of isolated DNA by isoniazid plus Mn(II) occurred without marked site specificity. The DNA damage was inhibited by .OH scavengers and superoxide dismutase (SOD) but not by catalase, suggesting the involvement of .OH formed via O2- but not via H2O2. Consistently, in ESR experiments .OH formation was observed during Mn(II)-catalyzed autoxidation of isoniazid, and the .OH formation was inhibited by SOD, but not by catalase. Iproniazid plus Mn(II) produced no or little .OH. We propose a reaction mechanism for the .OH formation without a H2O2 intermediate during manganese-catalyzed autoxidation of hydrazine. The present and previous data raise the possibility that hydrazines plus Mn(II)-induced cellular DNA damage may occur, at least in part, through the non-Fenton-type reaction.     

Copper complexes of 1,10-phenanthroline and related compounds as superoxide dismutase mimetics

In a preliminary study we tested CuSO4.5H2O, (Cu(II]2[3,5-diisopropylsalicylate]4.2H2O and a number of copper complexes of substituted 1,10-phenanthrolines for superoxide anion dismutase activity. It appeared that this activity depends on the ligands involved and might be governed by the redox potential of the Cu(I) complex/Cu(II) complex couple. The strong superoxide anion dismutase activity of Cu(II)[DMP]2 complex can be expected considering its high redox potential. Rather surprisingly is the superoxide anion dismutase activity of the Cu(I)[DMP]2 complex since it involves oxidation to Cu(II)[DMP]2 complex. From regression analysis it was established that steric and field effects of the substituents of the investigated phenanthrolines play an important role in SOD activity and therefore it is concluded that complex formation is important for the superoxide dismutase-like activity.     

Metal-catalyzed oxidation reactions and mass spectrometry: the roles of ascorbate and different oxidizing agents in determining Cu-protein-binding sites

Further study has been made of metal-catalyzed oxidation (MCO) reactions and mass spectrometry as a method to determine the binding site of copper in metalloproteins. The role of ascorbate and a variety of oxidizing agents, including O2, H2O2, and S2O8(2-), have been investigated using Cu/Zn superoxide dismutase (SOD) as a model system. Ascorbate is found to play two competing roles in the MCO reactions. It reduces Cu(II), which initiates and maintains the generation of reactive oxygen species, and it scavenges radicals, which helps to localize oxidation products to amino acids near the metal center. An ascorbate concentration of 100 mM is found to be optimal with regard to localizing oxidation products to only the Cu-binding residues (His44, His46, His61, and His118) of Cu/Zn SOD. This concentration of ascorbate is very similar to the optimum concentration found in our previous studies of different Cu-binding proteins. Another notable result from this study is the observation that S2O8(2-) is more effective as an oxidant than O2 or H2O2 in the MCO reactions. Because S2O8(2-) is more stable in solution than H2O2, using it as an oxidizing agent results in much less nonspecific oxidation to the protein. The overall results of this study suggest that general MCO reaction conditions may exist for determining the metal-binding site of a wide range of Cu-binding proteins.