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I Fridovich

Publications and source records attributed to I Fridovich.

At least 19 recordsLinked to original sources
PubMed1992

Fumarase C, the stable fumarase of Escherichia coli, is controlled by the soxRS regulon.

Fumarase C was strongly induced by paraquat in a parental strain of Escherichia coli but was not induced in a strain lacking the soxRS response. Moreover, a strain that constitutively expresses the soxRS regulon contained more fumarase C than did the parental strain. The Mn-containing superoxide dismutase and glucose-6-phosphate dehydrogenase, members of the soxRS regulon, were similarly induced by paraquat. Mutational defects in glucose-6-phosphate dehydrogenase increased the induction of fumarase C by paraquat. For Mn-containing superoxide dismutase, responsiveness to paraquat was also enhanced in the glucose-6-phosphate dehydrogenase-defective strains. Overproduction of the Mn-containing superoxide dismutase, elicited by isopropyl beta-D-thiogalactoside in a tac-sodA fusion strain, did not diminish induction of fumarase C or of glucose-6-phosphate dehydrogenase by paraquat, and induction of these enzymes was more sensitive to paraquat when the cells were growing on succinate rather than on LB medium. These results indicate that fumarase C is a member of the soxRS regulon and that this regulon does not respond to changes in O2- concentration but perhaps does respond to some consequence of a decrease in the ratio of NADPH to NADP+.

Enzyme Induction↗
PubMed1992

Inactivation-reactivation of aconitase in Escherichia coli. A sensitive measure of superoxide radical.

The rapid inactivation of aconitase by O2-, previously seen to occur in vitro, was explored in vivo. A fraction of the aconitase in growing, aerobic, Escherichia coli is inactive at any instant but can be activated by imposition of anaerobic conditions. This reactivation occurred in the absence of protein synthesis and was inhibited by the ferrous chelator alpha,alpha'-dipyridyl. This fraction of inactive, but activatable, aconitase was increased by augmenting O2- production with paraquat, decreased by elevation of superoxide dismutase, and increased by inhibiting reactivation with alpha,alpha'-dipyridyl. The balance between inactive and active aconitase thus represented a pseudoequilibrium between inactivation by O2- and reactivation by restoration of Fe(II), and it provided, for the first time, a measure of the steady-state concentration of O2- within E. coli. On this basis, [O2-] was estimated to be approximately 20-40 pM in aerobic log phase E. coli containing wild type levels of superoxide dismutase and approximately 300 pM in a mutant strain lacking superoxide dismutase.

2,2'-Dipyridyl↗
PubMed1992

Transcriptional and maturational effects of manganese and iron on the biosynthesis of manganese-superoxide dismutase in Escherichia coli.

Anaerobically grown Escherichia coli contain an enzymatically active iron superoxide dismutase (Fe2-FeSOD) and an inactive iron-substituted manganese superoxide dismutase (Fe2-MnSOD). The anaerobic electron sink, nitrate plus paraquat, enhanced biosynthesis of the MnSOD polypeptide, with accumulation of inactive Fe2-MnSOD. The oxidant, diamide, in contrast, allowed anaerobic production of the active forms of MnSOD, i.e. Mn2-MnSOD and Mn/Fe-MnSOD. Nutritional supplementation with Mn(II) favored occupancy of the MnSOD active site with manganese and allowed anaerobic accumulation of Mn2-MnSOD in the absence of diamide. Enrichment of the anaerobic growth medium with Fe(II) both suppressed biosynthesis of the MnSOD polypeptide and inhibited formation of the active manganese-containing forms. A tac-sodA operon fusion was used to examine the effects of chelating agents and metals on maturation of nascent MnSOD, independent from the transcriptional effects these agents impose. Isopropyl-1-thio-beta-D-galactopyranoside (IPTG) elicited anaerobic biosynthesis of MnSOD, which accumulated as the inactive Fe2-MnSOD. Diamide, with IPTG, allowed formation of active Mn/Fe-MnSOD while 1,10-phenanthroline with IPTG resulted in accumulation of Mn2-MnSOD. These results suggest that iron participates in the redox-sensitive control of the formation of active MnSOD at two levels, i.e. that of transcription as well as that of maturation. During maturation of the nascent MnSOD polypeptide, iron and manganese compete for the metal-binding site; anaerobic conditions favor iron-binding, whereas oxidants, such as dioxygen or diamide, favor binding of manganese.

Blotting, Western↗
PubMed1992

Superoxide generated by glutathione reductase initiates a vanadate-dependent free radical chain oxidation of NADH.

Vanadate V(V) markedly stimulated the oxidation of NADPH by GSSG reductase and this oxidation was accompanied by the consumption of O2 and the accumulation of H2O2. Superoxide dismutases completely eliminated this effect of V(V), whereas catalase was without effect, as was exogenous H2O2 added to 0.1 mM. These effects could be seen equally well in phosphate- or in 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid-buffered solutions. Under anaerobic conditions there was no V(V)-stimulated oxidation of NADPH. Approximately 4% of the electrons flowing from NADPH to O2, through GSSG reductase, resulted in release of O2-. The average length of the free radical chains causing the oxidation of NADPH, initiated by O2- plus V(V), was calculated to be in the range 140-200 NADPH oxidized per O2- introduced. We conclude that GSSG reductase, and by extension other O2(-)-producing flavoprotein dehydrogenases such as lipoyl dehydrogenase and ferredoxin reductase, catalyze V(V)-stimulated oxidation of NAD(P)H because they release O2- and because O2- plus V(V) initiate a free radical chain oxidation of NAD(P)H. There is no reason to suppose that these enzymes can act as NAD(P)H:V(V) oxidoreductases.

Animals↗
PubMed1992

Effects of overproduction of superoxide dismutases in Escherichia coli on inhibition of growth and on induction of glucose-6-phosphate dehydrogenase by paraquat.

Stationary phase inocula were more susceptible to the growth inhibitory effect of paraquat than were log phase inocula and this difference was exacerbated in strains overproducing superoxide dismutases (SOD). Glucose-6-phosphate dehydrogenase (G-6-PD), a member of the soxR regulon, was induced by paraquat promptly in the case of log phase cells; but only after a lag in stationary phase cells and this difference was also exaggerated in strains overproducing SOD. The negative consequences of overproduction of SOD on the adaptation of stationary phase cells to paraquat may be attributed to competition for cellular resources with an attendant delay in biosynthesis of other components of soxR. Since overproduction of SOD did not prevent log phase cells from inducing G-6-PD in response to paraquat, it appears likely that soxR can respond to aspects of redox status other than O2-. This conclusion is in accord with data which is already in the literature.

Enzyme Induction↗
PubMed1992

Exogenous quinones directly inhibit the respiratory NADH dehydrogenase in Escherichia coli.

The ability of naphthoquinones to generate reactive oxygen species has been widely exploited in studies of oxidative stress. However, excess superoxide dismutase and catalase failed to protect Escherichia coli in rich medium against growth inhibition by plumbagin, indicating that its toxic effect was not due to the production of partially reduced oxygen species. Respiration failed immediately upon the addition of growth-inhibitory levels of plumbagin. Studies in vitro showed that plumbagin and other redox-active quinones intercept electrons from NADH dehydrogenase, the primary respiratory dehydrogenase in glucose-containing media. An excess of oxidative substrate, such as plumbagin, inactivates this enzyme, which appears to be redox-regulated. The resultant respiratory arrest is a cautionary example of metabolic dysfunction from redox-cycling drugs that cannot be attributed to superoxide or hydrogen peroxide.

Cell Membrane↗
PubMed1992

Aluminum(III) facilitates the oxidation of NADH by the superoxide anion.

Al(III) augments the oxidation of reduced nicotinamide adenine dinucleotide (NADH) by enzymatic or photochemical sources of O2-. Superoxide dismutase, but not catalase, inhibited this action of Al(III). It thus appears that Al(III) forms a complex with O2-, which is a stronger oxidant than is O2- itself and which may contribute to the adverse biological effects of Al(III).

Aluminum↗
PubMed1992

Suppression of oxidative envelope damage by pseudoreversion of a superoxide dismutase-deficient mutant of Escherichia coli.

Mutants of Escherichia coli that are devoid of superoxide dismutase (SOD) fail to grow in aerobic minimal medium. This is largely because of the O2- sensitivities of several amino acid biosynthetic pathways, since amino acid supplements can restore growth, albeit at a slow rate. We now report that growth in amino acid-supplemented medium can be further stimulated by the presence of extracellular osmolytes. Osmolytes also partially suppress the amino acid requirements of the SOD mutant. These data suggest that the combination of oxidative injury and turgor pressure permeabilizes the cell envelope and that critical metabolites, including the limiting products of damaged biosynthetic pathways, escape from the cell. External osmolytes may offer protection by countervailing the usual turgor pressure and thus stabilizing the damaged envelope. This model is consistent with the previous observation that deficiency of cell wall components is lethal to SOD mutants. A pseudorevertant that can grow at a moderate rate in normosmotic medium without amino acid supplementation has been obtained (J. A. Imlay and I. Fridovich, Mol. Gen. Genet. 228:410-416, 1991). Analysis suggests that the suppressor mutation allows the envelope either to resist or to tolerate oxidative lesions. Study of the pseudorevertant may illuminate the molecular basis of this oxidative envelope injury.

Amino Acids↗
PubMed1991

Superoxide sensitivity of the Escherichia coli aconitase.

Mutants of Escherichia coli lacking superoxide dismutase (SOD) activity were used to explore the sensitivity of aconitase toward O2 and O2-. The aconitase activity in SOD-free extracts was rapidly lost under aerobic conditions and exogenous SOD afforded a concentration-dependent protection. The rate of the inactivating reaction between O2- and aconitase was estimated to be of the order of 10(9) M-1 s-1. The competitive inhibitors fluorocitrate and tricarballylate provided some protection, and at saturating concentrations, they decreased the rate of the inactivating reaction by 100- and 10-fold, respectively. Aconitase was markedly less sensitive to O2 than it was to O2-. Aerobic growth on succinate involves a greater dependence upon aconitase than does growth on glucose and, as expected, the deleterious consequences of SOD deficiency were more pronounced on succinate than on glucose. Moreover, aconitase activity was lower in extracts of aerobically grown SOD mutants, than it was in the parental strain. We suppose that inactivation of aconitase by O2- involves oxidative attack on the prosthetic iron-sulfur cluster. The extreme sensitivity of aconitase to inactivation by O2- suggests that its inactivation will be an early event in the oxidative stress imposed by hyperoxia, ultraviolet irradiation or redox-cycling agents, such as viologens or quinones.

Aconitate Hydratase↗
PubMed1991

Desferal-Mn(III) in the therapy of diquat-induced cataract in rabbit.

In rabbit lenses subjected to oxidative stress, induced by 1 mM diquat in vitro, there were 7- to 10-fold increases (p less than 0.001) in malondialdehyde, conjugated dienes, and carbonyl dienes, indicating extensive peroxidation of cellular membrane lipids, and approximately a 60% decrease in reduced glutathione. In the presence of 0.1-5 mM Desferal-Mn(III) these changes were diminished by 50-70%. In an experimental group of 12 rabbits having diquat-induced cataract, Desferal-Mn(III) (5% w/v) applied topically as a 50-microliters eye drop four times per day and a single intraperitoneal dose of 64 mg/kg body wt daily for 5 weeks (including pretreatment for 1 week) retarded the progression of lens opacities, whereas, in a control group of 6 rabbits treated with the vehicle (0.15 M NaCl) cataract progressed to an advanced grade. Treatment with Desferal-Mn(III) also significantly diminished production of O2.- and OH. in the lens, aqueous humor, and vitreous humor, and of H2O2 in the aqueous humor and vitreous humor. It also suppressed lipid peroxidation and oxidation of protein-SH of the lens and restored lenticular glutathione and ascorbate to normal levels.

Animals↗
PubMed1991

Effects of overproduction of superoxide dismutase on the toxicity of paraquat toward Escherichia coli.

Gross overproduction of the manganese-containing superoxide dismutase in Escherichia coli, by virtue of a multicopy plasmid bearing the sodA gene, decreases enumeration on paraquat-containing agar plates. This reflects growth inhibition, not lethality, since cells on these plates can be rescued by exclusion of dioxygen. Growth in liquid medium revealed that the control strain adapted to growth in the presence of paraquat more rapidly than did the overproducer. Glucose-6-phosphate dehydrogenase, taken as a representative of the superoxide-inducible soxR regulon, was induced during exposure to paraquat to a much greater extent in the control than in the superoxide dismutase-over-producing strain. These results support the view that overproduction of superoxide dismutase interferes with induction of the soxR regulon and thus prevents a balanced adaptation to the multiple aspects of the toxicity of aerobic paraquat.

Anaerobiosis↗
PubMed1991

Assay of metabolic superoxide production in Escherichia coli.

Superoxide production has been measured in subcellular fractions of SOD-deficient Escherichia coli provided with physiological reductants. Although cytosolic enzyme(s) do generate O2-., the larger portion is produced by autoxidation of components of the respiratory electron-transport chain. At 37 degrees C and with pO2, NADH, and NAD+ levels matching those in vivo, respiring membrane vesicles generate 3 O2-./10,000 electrons transferred. This corresponds to intracellular O2-. production, in glucose-fed cells, of 5 microM/s. The high SOD content of normal cells restricts O2-. accumulation to 2.10(-10) M, with a moderate gradient from the membrane to the center of the cell. SOD-deficient mutants achieve a much higher steady-state content of O2-.. Rates of superoxide-mediated inactivation of certain enzymes are sufficiently rapid that even 10(-10) M O2-. imposes a significant oxidative stress.

Cell Membrane↗
PubMed1991

Superoxide sensitivity of the Escherichia coli 6-phosphogluconate dehydratase.

The activity of 6-phosphogluconate dehydratase was significantly lower in extracts of aerobically grown Escherichia coli deficient in superoxide dismutase (sodAsodB) and in mutants lacking the inducible manganese-containing superoxide dismutase (sodA), exposed to the redox-cycling agent paraquat, than in the parental strain. Growth of these strains on a gluconate minimal medium was also impaired under these conditions. The enzyme was most susceptible to dioxygen in superoxide dismutase (SOD)-free extracts, and exogenous SOD afforded a concentration-dependent protection against inactivation. The amount of SOD necessary for full protection was comparable to the amount normally present in extracts of aerobic E. coli (7-36 units/mg protein), and the rate of reaction of O2- with the dehydratase was estimated to be approximately 2.0 x 10(8) M-1 s-1. The dehydratase was much less sensitive to O2 or H2O2 than to O2-. The virtual substrate, alpha-glycerophosphate, provided partial protection. Iron chelators, thiol-reactive reagents, and oxidants, including nitrite and diamide, inactivated the enzyme. Fluoride ions stabilized the dehydratase and blocked the effect of oxidants. The O2(-)-sensitive target site is proposed to be an iron-sulfur cluster which is readily destroyed by oxidation.

Bacterial Proteins↗
PubMed1991

In vivo competition between iron and manganese for occupancy of the active site region of the manganese-superoxide dismutase of Escherichia coli.

Three forms of the dimeric manganese superoxide dismutase (MnSOD) were isolated from aerobically grown Escherichia coli which contained 2 Mn, 1 Mn and 1 Fe, or 2 Fe, respectively. These are designated Mn2-MnSOD, Mn,Fe-MnSOD, and Fe2-MnSOD. Substitution of iron in place of manganese, eliminated catalytic activity, decreased the isoelectric point, and increased the native electrophoretic anodic mobility, although circular dichroism, high performance liquid chromatography gel exclusion chromatography, and sedimentation equilibrium revealed no gross changes in conformation. Moreover, replacement of iron by manganese restored enzymatic activity. Fe2-MnSOD and the iron-superoxide (FeSOD) of E. coli exhibit distinct optical absorption spectra. These data indicate that the active site environments of E. coli MnSOD and FeSOD must differ. They also indicate that competition between iron and manganese for nascent MnSOD polypeptide chains occurs in vivo, and copurification of these variably substituted MnSODs can explain the substoichiometric manganese contents and the variable specific activities which have been reported for this enzyme.

Amino Acids↗
PubMed1991

Isolation and genetic analysis of a mutation that suppresses the auxotrophies of superoxide dismutase-deficient Escherichia coli K12.

The most striking phenotype associated with superoxide dismutase (SOD) deficiency in Escherichia coli is the inability to grow in aerobic minimal medium, which is due to the sensitivity of several amino acid biosynthetic pathways to superoxide. We have isolated two classes of pseudorevertants that grow on minimal medium at modest rates. Of these, the class that exhibited the faster growth carries mutations at a single locus, denoted ssa, which was mapped to 4 min on the E. coli chromosome. This class constituted the majority of the spontaneous pseudorevertants that were selected by the transfer of independent SOD-deficient cultures in minimal medium from anaerobic to aerobic growth conditions. Pseudoreversion at ssa suppressed requirements for a variety of unrelated amino acid supplements. Further, the SOD-deficient strains were unable to assimilate diaminopimelic acid from the growth medium, whereas the ssa pseudorevertants did so. The viability of these pseudorevertants indicates that superoxide-sensitive biosynthetic enzymes do retain some function in SOD-deficient cells during aerobic growth.

Alleles↗
PubMed1991

The roles of O2-, HO(.), and secondarily derived radicals in oxidation reactions catalyzed by vanadium salts.

V(IV) decomposed H2O2, with evolution of O2, in a free radical chain process involving O2- and HO(.). When V(IV) was limiting, the presence of V(V) augmented O2 evolution because it allowed production of additional V(IV) from the reduction of V(V) by O2-. Gradual addition of V(IV) increased the yield of O2 evolved, per V(IV) added, to greater than 1--a clear indication of a free radical chain reaction. Reductants such as ethanol, Hepes, and NADH imposed a phase of O2 consumption because of HO.-initiated oxidation reactions. The radical produced from the reaction of HO. with ethanol was unable to directly oxidize NADH, whereas that produced from Hepes was able to do so. Ethanol consequently inhibited the oxidation of NADH by anaerobic V(IV) + H2O2, whereas Hepes did not. These results, and others reported herein, are explained on the basis of a coherent set of reactions. Data already in the literature are also clarified on the basis of these reactions.

Free Radical Scavengers↗
PubMed1991

Quinolinate synthetase: the oxygen-sensitive site of de novo NAD(P)+ biosynthesis.

The ability of niacin to relieve the growth-inhibiting effect of hyperoxia on Escherichia coli can be attributed to the dioxygen sensitivity of quinolinate synthetase. The activity of this enzyme within E. coli was diminished by exposure of the cells to 4.2 atm O2, while the activity in extracts was rapidly decreased by 0.2 atm O2. Neither catalase nor superoxide dismutase afforded detectable protection against the inactivating effect of O2, indicating that H2O2 and O2- were not significant intermediates in this process. Nevertheless, H2O2 at 1.0 mM did inactivate quinolinate synthetase, even under anaerobic conditions and in the absence of catalatic activity which might have generated O2. Addition of paraquat to aerobic cultures of E. coli caused an inactivation of quinolinate synthetase, which may be explained in terms of an increase in the production of H2O2. The O2-dependent inactivation of quinolinate synthetase in extracts was gradually reversed during anaerobic incubation and this reactivation was blocked by alpha, alpha'-dipyridyl or by 1,10-phenanthroline. The sequence of the quinolinate synthetase "A" protein contains a--cys-w-x-cys-y-z-cys--sequence, which is characteristic of (Fe-S)4-containing proteins. This sequence, together with the effect of the Fe(II)-chelating agents, suggests that the O2-sensitive site of quinolinate synthetase is an iron-sulfur cluster which is essential for the dehydration reaction catalyzed by the A protein.

2,2'-Dipyridyl↗