Irwin Fridovich

Duke University Medical Center | DUMC · Department of Biochemistry

Not applicable.

Highlights ► MnSOD does not act as a peroxidase of Amplex red. ► An artifact reported previously by the authors is involved. ► A role for MnSOD in cell signaling by acting as a peroxidase is unsupported.

The electronic structure of ground state oxygen, which is essential for the life of all aerobic organisms, makes it potentially dangerous for those organisms. Atmospheric oxygen contains two unpaired electrons with parallel spin states, which predisposes it to reduction by a univalent pathway. As a consequence, normal aerobic metabolism generates d...

Chemotaxis, both positive and negative, has been extensively studied in flagellated bacteria, such as Escherichia coli and Salmonella typhimurium, but not in gliding bacteria. The rapidly motile gliding bacterium Cytophaga johnsonae has been seen to be repelled by H2O2, OCl−, and N-chlorotaurine, as well as by low pH. Its response to H2O2 was elimi...

The family of superoxide dismutases (SODs) are well known for their antioxidant actions exerted by catalyzing the conversion of O(2)(·-) into H(2)O(2) plus oxygen. The importance of this action is revealed by the multiple phenotypic deficits exhibited by a variety of organisms that have been made to lack one or more of the SODs. Never the less ther...

In addition to its very efficient catalysis of the dismutation of superoxide ( O(2)(-) ) into O(2) plus H(2)O(2), Cu, Zn SOD acts less efficiently as a non-specific peroxidase. This peroxidase activity is CO(2) dependent although very slow peroxidation of some substrates occurs in the absence of CO2. The mechanism of that CO(2) dependence is explai...

Respiring organisms derive multiple benefits from molecular oxygen. However, some fraction of the oxygen used is unavoidably converted to dangerous substances such as the superoxide radical (O 2 ⁻ ), hydrogen peroxide (H 2 O 2 ) and the hydroxyl radical (HO). These intermediates of oxygen reduction threaten the integrity of diverse biological molec...

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Abstract In plants, as in other aerobic organisms, O2− is a commonly encountered intermediate of oxygen reduction and superoxide dismutases provide a defense against the potential cytotoxicity of this radical. The superoxide dismutases found in plants resemble those encountered in other organisms. Within chloroplasts one finds the CuZn enzyme, whil...

The Cu,Zn-superoxide dismutase (SOD1) has been reported to exert an S-nitrosylated glutathione (GSNO) denitrosylase activity that was augmented by a familial amyotrophic lateral sclerosis (FALS)-associated mutation in this enzyme. This putative enzymatic activity as well as the spontaneous decomposition of GSNO has been reexamined. The spontaneous...

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.

Human Cu,Zn-superoxide dismutase (hSOD1) has 4 cysteines per subunit. Cys57 and Cys148 are involved in an intrasubunit disulfide bond, while Cys6 and Cys111 are free. Cys6 is buried within the protein while Cys111 is on the surface, near the dimer interface. We examined by liquid chromatography-mass spectrometry the commercially purchased hSOD1 iso...

Cu,Zn SOD is known to be inactivated by HO(2)(-) and to be protected against that inactivation by a number of small molecules including formate, imidazole, and urate. This inactivation has been shown to be due to oxidation of a ligand field histidine residue by a bound oxidant formed by reaction of the active site Cu(II) with HO(2)(-). We now repor...

Introduction Assays Isoenzymes and Electromorphs Structure and Evolutionary Relationships Mechanisms Biosynthesis Conclusions

In a recent publication (Michel et al. Arch. Biochem. Biophys. 439:234-240; 2005) the authors argued that the catalytic rate constant, k(cat), for wild-type Cu,Zn-superoxide dismutase (Cu,Zn-SOD), determined previously by pulse radiolysis, was overestimated due to contamination with excess copper. They reported that addition of 0.1 mM EDTA to a sam...

Transition metals, such as Cu(+2), Mn(+2), and Co(+2), have been seen to catalyze the bicarbonate enhanced oxidation of a variety of substrates by H(2)O(2). In several of these cases it has been demonstrated that CO(2), rather than bicarbonate, is the enhancing species. Mechanisms that are in accord with the data involve a hypervalent state that ma...

Two new tri(ethyleneglycol)-derivatized Mn(III) porphyrins were synthesized with the aim of increasing their bioavailability, and blood-circulating half-life. These are Mn(III) tetrakis(N-(1-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)pyridinium-2-yl)porphyrin, MnTTEG-2-PyP5+ and Mn(III) tetrakis(N,N'-di(1-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)imidazolium-2-...

Augmentation, by CO(2)/HCO(3)(-), of Co(II)-catalyzed peroxidations was explored to clarify whether the rate enhancement was due to CO(2) or to HCO(3)(-). The rate of oxidation of NADH by Co(II) plus H(2)O(2), in Tris or phosphate, was markedly enhanced by CO(2)/HCO(3)(-). Phosphate was seen to inhibit the Co(II)-catalyzed peroxidation, probably du...

The absence of SOD1 in yeast has been found to result in inactivation of Lys4p. This [4Fe-4S]-containing dehydratase is in the pathway of biosynthesis of lysine, hence the oxygen-dependent lysine auxotrophy seen in this case. O(2)(-) is known to oxidize and thus destabilize the [Fe-4S] clusters of dehydratases; hence, this would make perfect sense...

Mn(II) can catalyze the decomposition of H(2)O(2) and, in the presence of H(2)O(2), can catalyze the oxidation of NADH. Strikingly, these processes depend on the simultaneous presence of both CO(2) and HCO(3)(-). This explains the exponential dependence of the rates on [HCO(3)(-)], previously noted by other workers. These processes are inhibited by...

Mn(III) ortho-tetrakis(N-ethylpyridinium-2-yl)porphyrin (Mn(III)TE-2-PyP(5+)) effectively scavenges reactive oxygen and nitrogen species in vitro, and protects in vivo, in different rodent models of oxidative stress injuries. Further, Mn(III)TE-2-PyP(5+) was shown to be readily reduced by cellular reductants such as ascorbic acid and glutathione. W...

Cationic Mn(III) porphyrins substituted on the methine bridge carbons (meso positions) with N-alkylpyridinium or N,N'-diethylimidazolium groups have been prepared and characterized, both chemically and as SOD mimics. The ortho tetrakis N-methylpyridinium compound was substantially more active than the corresponding para isomer. This ortho compound...

Three new Mn(III) porphyrin catalysts of O2.-dismutation (superoxide dismutase mimics), bearing ether oxygen atoms within their side chains, were synthesized and characterized: Mn(III) 5,10,15,20-tetrakis[N-(2-methoxyethyl)pyridinium-2-yl]porphyrin (MnTMOE-2-PyP(5+)), Mn(III)5,10,15,20-tetrakis[N-methyl-N'-(2-methoxyethyl)imidazolium-2-yl]porphyrin...

At pH 7.4, CO2, rather than HCO3-, markedly enhances the oxidation of diverse substrates by SOD1 plus H2O2. Since the concentration of CO2 would fall with rising pH in HCO3- buffers, it was of interest to explore the effects of pH on the peroxidase activity of SOD1 in the presence and in the absence of HCO3-. The rate of NADPH peroxidation in the H...

The frequently quoted figure for the fractional univalent reduction of oxygen to superoxide in mitochondria is certainly too high by at least one order of magnitude. This is so because the higher number (2%) was derived from mitochondria whose cytochrome c oxidase was blocked with cyanide. Nevertheless, even the more correct number (0.1%) means tha...

The frequently quoted figure for the fractional univalent reduction of oxygen to superoxide in mitochondria is certainly too high by at least one order of magnitude. This is so because the higher number (2%) was derived from mitochondria whose cytochrome c oxidase was blocked with cyanide. Nevertheless, even the more correct number (0.1%) means tha...

The Cu,Zn SOD catalyzes the bicarbonate-dependent oxidation of a wide range of substrates by H2O2. A mechanism in accord with this activity has been described. It involves the generation of a strong oxidant (Cu(I)O, Cu(II)OH, or Cu(III)) by reaction of the active site Cu with H2O2, followed by oxidation of bicarbonate to CO3-* that in turn diffuses...

The Cu,Zn superoxide dismutase catalyzes [Formula] -dependent oxidations by H2O2. This activity has been shown to depend on the creation of a bound oxidant at the Cu(II) by interactions with H2O2. The bound oxidant was then thought to oxidize [Formula] to [Formula], which diffuses into the bulk solution and there oxidizes diverse substrates. We now...

FALS-associated missense mutations of SOD1 exhibit a toxic gain of function that leads to the death of motor neurons. The explanations for this toxicity fall into two broad categories. One involves a gain of some oxidative activity, while the second involves a gain of protein: protein interactions. Among the postulated oxidative activities are the...

The classical view of the aerobic decomposition of Angeli's salt is that it releases NO(2)(-) + NO(-)/HNO the latter then reacting with O(2) to yield ONOO(-). An alternative that has recently been proposed envisions electron transfer to O(2) followed by decomposition to NO(2)(-) + NO. The classical view is now strongly supported by the observation...

We have studied the reaction kinetics of ten manganese porphyrins, differing in their meso substituents, with peroxynitrite (ONOO-) and carbonate radical anion (CO3.) using stopped-flow and pulse radiolysis, respectively. Rate constants for the reactions of Mn(III) porphyrins with ONOO- ranged from 1 x 10(5) to 3.4 x 10(7) m(-1) s(-1) and correlate...

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...

The Mn(III)meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnIIITE-2-PyP5+) is a potent superoxide dismutase (SOD) mimic in vitro and was beneficial in rodent models of oxidative stress pathologies. Its high activity has been ascribed to both the favorable redox potential of its metal center and to the electrostatic facilitation assured by the four...

The Mn(III)meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnIIITE-2-PyP5+) is a potent superoxide dismutase (SOD) mimic in vitro and was beneficial in rodent models of oxidative stress pathologies. Its high activity has been ascribed to both the favorable redox potential of its metal center and to the electrostatic facilitation assured by the four...

A childhood fascination with animals, plants, and insects was aided and abetted by many giants, beginning with my parents. The Bronx High School of Science and the City College of New York (CCNY) made a solid and priceless grounding in chemistry and biology available free of charge. Abe Mazur at CCNY revealed the wonders of biochemistry and illustr...

The sulfonated tetrazolium 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2-tetrazolium 5-carboxanilide (XTT) is advantageous in that it yields a water-soluble formazan, unlike most other available tetrazoliums. XTT is reducible by superoxide, as are other tetrazoliums, but is not directly reduced by xanthine oxidase plus xanthine or by glucose oxidase...

Rotational (atropo-) isomers of Mn(III) meso-tetrakis(N-alkylpyridinium-2-yl)porphyrins and corresponding metal-free porphyrin ligands (where alkyl is methyl, ethyl, n-butyl, n-hexyl) and Zn(II) meso-tetrakis(N-methyl(ethyl,n-hexyl)pyridinium-2-yl)porphyrins were separated and isolated by reverse-phase HPLC. The identity of the rotational isomers o...

The ability of short-chain sugars to cause oxidative stress has been examined using glycolaldehyde as the simplest sugar. Short-chain sugars autoxidize in air, producing superoxide and alpha,beta-dicarbonyls. In Escherichia coli the soxRS regulon mediates an oxidative stress response, which protects the cell against both superoxide-generating agent...

Radiation therapy (RT) is an important therapeutic modality in the treatment of thoracic tumors. The maximum doses to these tumors are often limited by the radiation tolerance of lung tissues. Lung injury from ionizing radiation is believed to be a consequence of oxidative stress and a cascade of cytokine activity. Superoxide dismutase (SOD) is a k...

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...

Simple algebraic manipulations and steady state assumptions have been applied to the elucidation of the effects of superoxide dismutase (SOD) on [NO] and [O(2)(-)], and on the rates of production of H(2)O(2) and of ONOO(-). The deductions help explain seemingly discordant published results and predictions.

Missense mutations in Cu,Zn-superoxide dismutase (SOD1) account for approximately 20% of familial amyotrophic lateral sclerosis (FALS) through some, as yet undefined, toxic gain of function that leads to gradual death of motor neurons. Mitochondrial swelling and vacuolization are early signs of incipient motor neuron death in FALS. We previously re...

The ortho, meta, and para isomers of cationic N-alkylpyridylporphyrins and their Zn(II) complexes were compared in terms of their photodynamic properties. The ortho Zn(II) complex was found to be the most efficient in causing photooxidation of NADH in vitro. In Escherichia coli, however, the para and meta isomers were better photosensitizers than t...

The interactions of Cu, Zn superoxide dismutase (SOD) with nitroxyl (NO-) and nitric oxide (NO), both of which are thought to be biologically significant, have been studied but remain undefined. Having previously noted that NO- can reduce Cu (II), Zn SOD aerobically, we now report that it also can do so anaerobically and that Cu, Zn SOD can catalyz...

Escherichia coli, lacking cytoplasmic superoxide dismutases, exhibits a variety of oxygen-dependent phenotypic deficits. Enrichment of the growth medium with Mn(II) relieved those deficits. Extracts of cells grown on Mn(II)-rich medium exhibited superoxide dismutase-like activity that was due partially to low-molecular-weight and partially to high-...

A series of ortho isomers of meso-tetrakis(N-alkylpyridyl)porphyrins (alkyl being methyl, ethyl, n-propyl, n-butyl, n-hexyl, and n-octyl) and their Mn(III) complexes were synthesized and characterized by elemental analysis, uv/vis spectroscopy, electrospray ionization mass spectrometry and electrochemistry. An increase in the number of carbon atoms...

In a recent review published in this journal,(1) Koppenol traced the history of the Fenton reaction and of the catalytic decomposition of H(2)O(2) by iron salts. If his purpose was to shed light on current understanding of related chemistry in biological systems, he failed. Moreover, he managed to sow confusion by inaccurate reporting of the work o...

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...

Rat liver was homogenized in isotonic buffer, fractionated by differential centrifugation, and then subfractionated by equilibrium sedimentation in Nycodenz gradients. Fractions were assayed for both Cu,Zn-superoxide dismutase (SOD) and Mn-SOD by exploiting the cyanide sensitivity of the former activity and by the use of specific antibodies. As exp...

Nitroxyl (NO−) may be produced by nitric-oxide synthase and by the reduction of NO by reduced Cu,Zn-SOD. The ability of NO− to cause oxidations and of SOD to inhibit such oxidations was therefore explored. The decomposition of Angeli's salt (AS) produces NO− and that in turn caused the aerobic oxidation of NADPH, directly or indirectly. O⨪2 was pro...

It has been reported in the literature that the endogenous estrogen metabolite 2-methoxyestradiol (2-ME) inhibits both manganese and copper,zinc superoxide dismutases (Mn and Cu,Zn SODs) and that this mechanism is responsible for 2-ME's ability to kill cancer cells. In fact, as demonstrated using several SOD assays including pulse radiolysis, 2-ME...

The evanescent nature of reactive oxygen and nitrogen species, the multiple cellular mechanisms evolved to maintain these substances at low (submicromolar) concentrations within the vascular system, and the often multifaceted nature of their reactivities have made measurement of these compounds within the vasculature problematic. This review attemp...

Each of my published papers ignites a flood of memories of the students and postdoctoral fellows I worked with, of the competitors we contended with, and of the pleasures of achieving an understanding of something formerly hidden from view. I have here recorded only a small fraction of those memories, but that is enough to give the reader a feeling...

Reactive oxygen species contribute to ischemic brain injury. This study examined whether the porphyrin catalytic antioxidant manganese (III) meso-tetrakis (N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP(5+)) reduces oxidative stress and improves outcome from experimental cerebral ischemia. Rats that were subjected to 90 min focal ischemia and 7 d rec...

We have developed and tested several synthetic superoxide dismutase (SOD) mimetic metalloporphyrin compounds to determine their ability to protect/ameliorate radiation-induced (RT) normal tissue injury, and, at the same time, to produce significant anti-tumor activity. In rats with R3230 AC mammary adenocarcinoma tumors, a significant inhibition of...

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 oth...

A manganese(III) complex of biliverdin IX dimethyl ester, (MnIIIBVDME)2, was prepared and characterized by elemental analysis, UV/vis spectroscopy, cyclic voltammetry, chronocoulometry, electrospray mass spectrometry, freezing-point depression, magnetic susceptibility, and catalytic dismuting of superoxide anion (O2.-). In a dimeric conformation ea...

The copper- and zinc-containing superoxide dismutase can catalyze the oxidation of ferrocyanide by O⨪2 as well as the reduction of ferricyanide by O⨪2. Thus, it can act as a superoxide dismutase (SOD), a superoxide reductase (SOR), and a superoxide oxidase (SOO). The human manganese-containing SOD does not exert SOR or SOO activities with ferrocyan...

Abstract—The evanescent nature of reactive oxygen and nitrogen species, the multiple cellular mechanisms evolved to

The extent to which sugars serve as targets for superoxide was examined using glycolaldehyde as the simplest sugar and using superoxide dismutase (SOD)-replete and SOD-null strains growing under aerobic and anaerobic conditions. Glycolaldehyde was more toxic to the SOD-null strain than to its SOD-replete parent, and this differential effect was oxy...

Reaction between NO• and manganese tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnIIITE-2-PyP5+) was investigated at 25°C. At high excess of NO• (1.5 mM) the reaction with the oxidized, air-stable form MnIIITE-2-PyP5+ (5 μM), proceeds very slowly (t1/2 ≅ 60 min). The presence of excess ascorbate (1 mM) produces the reduced form, MnIITE-2-PyP4+, which...

Manganese(III) complexes of isomeric tetrakis(N-alkylpyridyl)porphyrins (N-alkyl = N-methyl, M or N-ethyl, E), MnTM(E)-2(3,4)-PyP5+, are being developed as superoxide dismutase (SOD) mimics. Simultaneously, techniques for their purification, identification and characterization are being pursued. Electrospray mass spectrometry (ESMS) proved to be an...

Manganese(III) complexes of isomeric tetrakis(N-alkylpyridyl)porphyrins (N-alkyl = N-methyl, M or N-ethyl, E), MnTM(E)-2(3,4)-PyP5+, are being developed as superoxide dismutase (SOD) mimics. Simultaneously, techniques for their purification, identification and characterization are being pursued. Electrospray mass spectrometry (ESMS) proved to be an...

The use of lucigenin luminescence as a measure of ¿O-(2) has been questioned because lucigenin has been shown to be capable of mediating the production of O-(2). This being the case, lucigenin can signal the presence of O-(2) even in systems not producing it in the absence of lucigenin. The reduction potential of lucigenin should be in accord with...

The interaction of Cu,ZnSOD with H2O2 generates an oxidant at the active site that can then cause either the inactivation of this enzyme or the oxidation of a variety of exogenous substrates. We show that the rate of inactivation, imposed by 10-mM H2O2 at 25°C and pH 7.2, is not influenced by 10-mM HCO3−; whereas the oxidation of 2,2′-azino-bis-[3-...

The soxRS regulon orchestrates a multifaceted defense against oxidative stress, by inducing the transcription of 15 genes. The induction of this regulon by redox agents, known to mediate O 2. production, led to the view that O 2. is one signal to which it responds. However, redox cycling agents deplete cellular reduc-tants while producing O 2. , an...

Superoxide (O2-) poses multiple threats, which are diminished by a family of metalloenzymes, the superoxide dismutases. Among the damaging effects of O2- are direct oxidation of low-molecular-weight reductants; inactivation of a select group of enzymes; and reaction with NO to yield the strong oxidant, peroxynitrite. Of even greater import is the a...

The log kcat values for the dismutation of O2•- by a series of monohydroxoiron(III) and aquamanganese(III) porphyrins, including ortho, meta, and para isomers of 5,10,15,20-tetrakis(N-alkylpyridiniumyl)porphyrins, were found to vary linearly with the metal-centered redox potentials (E1/2) for the M(III)/M(II) couple. Each 120 mV increase in E1/2 im...

Three isomers of manganese(III) 5,10,15, 20-tetrakis(N-methylpyridyl)porphyrin (MnTMPyP) were evaluated for their reaction with peroxynitrite. The Mn(III) complexes reacted with peroxynitrite anion with rate constants of 1.85 x 10(7), 3.82 x 10(6), and 4.33 x 10(6) M(-1) s(-1) at 37 degrees C for MnTM-2-PyP, MnTM-3-PyP, and MnTM-4-PyP, respectively...

The soxRS regulon orchestrates a multifaceted defense against oxidative stress, by inducing the transcription of approximately 15 genes. The induction of this regulon by redox agents, known to mediate O-2 production, led to the view that O-2 is one signal to which it responds. However, redox cycling agents deplete cellular reductants while producin...

Nitroreductase A catalyzes the divalent reduction of nitro compounds, quinones, and dyes by NADPH. In this paper, nitroreductase A is induced in Escherichia coli by exposure to paraquat in a manner that depends on the expression of soxR. Nitroreductase activity was only slightly induced by paraquat in a strain bearing a mutational defect in the gen...

The lack of superoxide dismutase and the consequent elevation of [O2-] imposes, on Escherichia coli, auxotrophies for branched chain, sulfur-containing, and aromatic amino acids. The former two classes of auxotrophies have already been explained, whereas the third is explained herein. Thus O2- is shown to interfere with the production of erythrose-...

A byproduct of normal aerobic metabolism is the generation of dangerously reactive intermediates of the reduction of O2. These include O2-, H2O2, and HO. and arise because of the predisposition of O2 for univalent reductions. These reactive oxygen species (ROS), and others that they can engender, threaten all cellular macromolecules, and defenses a...