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P R Gardner

Publications and source records attributed to P R Gardner.

At least 19 recordsLinked to original sources
PubMed2001

Dioxygen-dependent metabolism of nitric oxide in mammalian cells.

Steady-state gradients of NO within tissues and cells are controlled by rates of NO synthesis, diffusion, and decomposition. Mammalian cells and tissues actively decompose NO. Of several cell lines examined, the human colon CaCo-2 cell produces the most robust NO consumption activity. Cellular NO metabolism is mostly O2-dependent, produces near stoichiometric NO3-, and is inhibited by the heme poisons CN-, CO (K(I) approximately 3 microM), phenylhydrazine, and NO and the flavoenzyme inhibitor diphenylene iodonium. NO consumption is saturable by O2 and NO and shows apparent K(M) values for O2 and NO of 17 and 0.2 microM, respectively. Mitochondrial respiration, O2*-, and H2O2 are neither sufficient nor necessary for O2-dependent NO metabolism by cells. The existence of an efficient mammalian heme and flavin-dependent NO dioxygenase is suggested. NO dioxygenation protects the NO-sensitive aconitases, cytochrome c oxidase, and cellular respiration from inhibition, and may serve a dual function in cells by limiting NO toxicity and by spatially coupling NO and O2 gradients.

Aconitate Hydratase↗
PubMed2000

Nitric-oxide dioxygenase activity and function of flavohemoglobins. sensitivity to nitric oxide and carbon monoxide inhibition.

Widely distributed flavohemoglobins (flavoHbs) function as NO dioxygenases and confer upon cells a resistance to NO toxicity. FlavoHbs from Saccharomyces cerevisiae, Alcaligenes eutrophus, and Escherichia coli share similar spectra, O(2), NO, and CO binding kinetics, and steady-state NO dioxygenation kinetics. Turnover numbers (V(max)) for S. cerevisiae, A. eutrophus, and E. coli flavoHbs are 112, 290, and 365 NO heme(-1) s(-1), respectively, at 37 degrees C with 200 microm O(2). The K(M) values for NO are low and range from 0.1 to 0.25 microm. V(max)/K(M)(NO) ratios of 900-2900 microm(-1) s(-1) indicate an extremely efficient dioxygenation mechanism. Approximate K(M) values for O(2) range from 60 to 90 microm. NO inhibits the dioxygenases at NO:O(2) ratios of > or =1:100 and makes true K(M)(O(2)) values difficult to determine. High and roughly equal second order rate constants for O(2) and NO association with the reduced flavoHbs (17-50 microm(-1) s(-1)) and small NO dissociation rate constants suggest that NO inhibits the dioxygenase reaction by forming inactive flavoHbNO complexes. Carbon monoxide also binds reduced flavoHbs with high affinity and competitively inhibits NO dioxygenases with respect to O(2) (K(I)(CO) = approximately 1 microm). These results suggest that flavoHbs and related hemoglobins evolved as NO detoxifying components of nitrogen metabolism capable of discriminating O(2) from inhibitory NO and CO.

Alcaligenes↗
PubMed2000

Steady-state and transient kinetics of Escherichia coli nitric-oxide dioxygenase (flavohemoglobin). The B10 tyrosine hydroxyl is essential for dioxygen binding and catalysis.

Escherichia coli expresses an inducible flavohemoglobin possessing robust NO dioxygenase activity. At 37 degrees C, the enzyme shows a maximal turnover number (V(max)) of 670 s(-1) and K(m) values for NADH, NO, and O(2) equal to 4.8, 0.28, and approximately 100 microM, respectively. Individual reduction, ligand binding, and NO dioxygenation reactions were examined at 20 degrees C, where V(max) is approximately 94 s(-1). Reduction by NADH occurs in two steps. NADH reduces bound FAD with a rate constant of approximately 15 microM(-1) s(-1), and heme iron is reduced by FADH(2) with a rate constant of 150 s(-1). Dioxygen binds tightly to reduced flavohemoglobin, with association and dissociation rate constants equal to 38 microM(-1) s(-1) and 0.44 s(-1), respectively, and the oxygenated flavohemoglobin dioxygenates NO to form nitrate. NO also binds reversibly to reduced flavohemoglobin in competition with O(2), dissociates slowly, and inhibits NO dioxygenase activity at [NO]/[O(2)] ratios of 1:100. Replacement of the heme pocket B10 tyrosine with phenylalanine increases the O(2) dissociation rate constant approximately 80-fold and reduces NO dioxygenase activity approximately 30-fold, demonstrating the importance of the tyrosine hydroxyl for O(2) affinity and NO scavenging activity. At 37 degrees C, V(max)/K(m)(NO) is 2,400 microM(-1) s(-1), demonstrating that the enzyme is extremely efficient at converting toxic NO into nitrate under physiological conditions.

Amino Acids↗
PubMed1998

Constitutive and adaptive detoxification of nitric oxide in Escherichia coli. Role of nitric-oxide dioxygenase in the protection of aconitase.

Nitric oxide (NO.) is a naturally occurring toxin that some organisms adaptively resist. In aerobic or anaerobic Escherichia coli, low levels of NO. exposure inactivated the NO.-sensitive citric acid cycle enzyme aconitase, and inactivation was more effective when the adaptive synthesis of NO.-defensive proteins was blocked with chloramphenicol. Protection of aconitase in aerobically grown E. coli was dependent upon O2, was potently inhibited by cyanide, and was correlated with an induced rate of cellular NO. consumption. Constitutive and adaptive cellular NO. consumption in aerobic cells was also dependent upon O2 and inhibited by cyanide. Exposure of aerobic cells to NO. accordingly elevated the activity of the O2-dependent and cyanide-sensitive NO. dioxygenase (NOD). Anaerobic E. coli exposed to NO. or nitrate induced a modest O2-independent and cyanide-resistant NO.-metabolizing activity and a more robust O2-stimulated cyanide-sensitive activity. The latter activity was attributed to NOD. The results support a role for NOD in the aerobic detoxification of NO. and suggest functions for NOD and a cyanide-resistant NO. scavenging activity in anaerobic cells.

Aconitate Hydratase↗
PubMed1998

Nitric oxide dioxygenase: an enzymic function for flavohemoglobin.

Nitric oxide (NO*) is a toxin, and various life forms appear to have evolved strategies for its detoxification. NO*-resistant mutants of Escherichia coli were isolated that rapidly consumed NO*. An NO*-converting activity was reconstituted in extracts that required NADPH, FAD, and O2, was cyanide-sensitive, and produced NO3-. This nitric oxide dioxygenase (NOD) contained 19 of 20 N-terminal amino acids identical to those of the E. coli flavohemoglobin. Furthermore, NOD activity was produced by the flavohemoglobin gene and was inducible by NO*. Flavohemoglobin/NOD-deficient mutants were also sensitive to growth inhibition by gaseous NO*. The results identify a function for the evolutionarily conserved flavohemoglobins and, moreover, suggest that NO* detoxification may be a more ancient function for the widely distributed hemoglobins, and associated methemoglobin reductases, than dioxygen transport and storage.

Amino Acid Sequence↗
PubMed1997

Nitric oxide sensitivity of the aconitases.

Aconitases are important cellular targets of nitric oxide (NO.) toxicity, and NO.-derived species, rather than NO. per se, have been proposed to mediate their inactivation. NO.-mediated inactivation of the Escherichia coli aconitase and the porcine mitochondrial aconitase was investigated. In E. coli, aconitase activity decreased by approximately 70% during a 2-h exposure to an atmosphere containing 120 ppm NO. in N2. The NO.-inactivated aconitase reactivated poorly in E. coli under anaerobic or aerobic conditions. Elevated superoxide dismutase activity did not affect the aerobic inactivation of aconitase by NO., thus indicating a limited role of the NO.- and superoxide-derived species peroxynitrite. Glutathione-deficient and glutathione-containing E. coli were comparably sensitive to NO.-mediated aconitase inactivation, thus excluding the participation of S-nitrosoglutathione or more oxidizing NO.-derived species. NO. progressively decreased aconitase activity in extracts in the presence of substrates, and inactivation was greatest at an acidic pH with cis-aconitate. The porcine mitochondrial aconitase was sensitive to NO. when exposed at pH 6.5, but not at pH 7.5, and irreversible inactivation occurred during catalysis. The requirement of an acidic pH or substrates for sensitivity may explain the reported resistance of aconitases to NO. in vitro (Castro, L., Rodriguez, M., and Radi, R. (1994) J. Biol. Chem. 269, 29409-29415; Hausladen, A., and Fridovich, I. (1994) J. Biol. Chem. 269, 29405-29408). An S-nitrosation of the aconitase [4Fe-4S] center catalyzed by the solvent-exposed electron withdrawing iron atom (Fea) is proposed.

Aconitate Hydratase↗
PubMed1997

Superoxide-driven aconitase FE-S center cycling.

O2- produced by the autoxidation of respiratory chain electron carriers, and other cellular reductants, inactivates bacterial and mammalian iron-sulfur-containing (de)hydratases including the citric acid cycle enzyme aconitase. Release of the solvent-exposed iron atom and oxidation of the [4Fe-4S]2+ cluster accompanies loss of catalytic activity. Rapid reactivation is achieved by iron-sulfur cluster reduction and Fe2+ insertion. Inactivation-reactivation is a dynamic and cyclical process which modulates aconitase and (de)hydratase activities in Escherichia coli and mammalian cells. The balance of inactive and active aconitase provides a sensitive measure of the changes in steady-state O2- levels occurring in living cells and mitochondria under stress conditions. Aconitases are also inactivated by other oxidants including O2, H2O2, NO, and ONOO- which are associated with inflammation, hyperoxia and other pathophysiological conditions. Loss of aconitase activity during oxidant stress may impair energy production, and the liberation of reactive iron may further enhance oxidative damage. Iron-sulfur center cycling may also serve adaptive functions by modulating gene expression or by signaling metabolic quiescence.

Aconitate Hydratase↗
PubMed1996

Failure of tumor necrosis factor and interleukin-1 to elicit superoxide production in the mitochondrial matrices of mammalian cells.

Subversion of mitochondrial electron transport to the production of O2.- has been proposed as a mechanism of tumor necrosis factor (TNF)-mediated cell killing and to a lesser extent interleukin-1 (IL-1) and lipopolysaccharide (LPS) cytotoxicity. We utilized the O2.- -sensitive aconitases to measure changes in steady-state 02.- levels in the mitochondrial matrix and cytoplasm of cultured mammalian cells in response to these inflammatory mediators. TNF alpha did not measurably affect aconitase activity, and thus mitochondrial 02.- production, in either cultured human A549 cells or murine L929 cells while TNF alpha clearly caused cytotoxicity as revealed by impaired mitochondrial respiration. IL-1 alpha and Escherichia coli LPS also failed to affect the aconitase activity in A549 cells. Neither the O2.- scavenger Mn(III) TMPyP nor the H2O2 scavenger catalase protected L929 cells against the cytotoxicity of TNF alpha. In conclusion, TNF, IL-1, and LPS do not appear to exert cytotoxicity, or MnSOD gene induction effects, by eliciting mitochondrial O2.- production.

Aconitate Hydratase↗
PubMed1996

Superoxide production by the mycobacterial and pseudomonad quinoid pigments phthiocol and pyocyanine in human lung cells.

The quinoid pigments pthiocol, produced by Mycobacterium tuberculosis, and pyocyanine, produced by Pseudomonas aeruginosa, were examined for their effects on O2.- production in cultured human lung epithelial-like A549 cells. Intracellular O2.- levels were measured using the O2.-sensitive aconitase(s), and rates of O2.- generation were assessed from rates of antimycin-resistant respiration. Elevated O2.- was detected in cells exposed to < 25 microM phthiocol and < 2 microM pyocyanine in neutral pH medium, and both agents impaired cell growth. The O2.- scavenging manganoporphyrin, Mn(III)TMPyP, partially protected cells against pyocyanine and phthiocol-mediated growth inhibition. O2.- production by phthiocol and pyocyanine was enhanced by acidification of the growth medium. Surprisingly, the dicumarol-inhibitable quinoid detoxification enzyme DT-diaphorase was a significant source of phthiocol and pyocyanine-mediated O2.- generation in cells. O2.- production in macrophages by the phthiocol analog, menadione, was shown to impair macrophage mitochondrial respiration and bactericidal activity toward Escherichia coli. Phthiocol and pyocyanine, by producing O2.-/H2O2, and inhibiting host cell aconitase activity, energetics, and other host cell functions, may contribute to the pathogenicity of M. tuberculosis and P. aeruginosa.

Aconitate Hydratase↗
PubMed1996

Superoxide scavenging by Mn(II/III) tetrakis (1-methyl-4-pyridyl) porphyrin in mammalian cells.

The superoxide dismutase mimic Mn(II/III) tetrakis (1-methyl-4-pyridyl) porphyrin (Mn(II/III)TMPyP) was examined for its superoxide radical (O2.-)-scavenging ability in cultured mammalian cells. Mn(III)TMPyP (< 5 microM) added to culture media relieved growth inhibition and decreased the inactivation of the O2(.-)-sensitive enzyme aconitase in cells exposed to the O2(.-)-generating phenazine pyocyanine. Treatment of cells with Mn(III)TMPyP did not measurably affect cellular O2.- production as revealed by rates of cyanide-resistant respiration with or without added pyocyanine. In contrast, Mn(II/III)TMPyP enhanced O2.- production in cells when the redox-active naphthoquinone menadione was present as measured by both increased cyanide-resistant respiration rates and aconitase inactivation. In vitro, Mn(II/III)TMPyP catalyzed the oxidation of ascorbate, and menadione enhanced this effect. Mn(III)TMPyP did not protect aconitase when O2.- production was elicited in mitochondria by antimycin A and the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The results support a reductant-O2.-:oxidoreductase mechanism for O2.- scavenging by Mn(II/III)TMPyP in the mammalian cytosol as proposed for its action in Escherichia coli, but also indicate that Mn(II/III)TMPyP can either scavenge or produce O2.- in cells depending upon the prevailing pathways of Mn(II/III)TMPyP oxidation-reduction.

Aconitate Hydratase↗
PubMed1995

Superoxide radical and iron modulate aconitase activity in mammalian cells.

Aconitase is a member of a family of iron-sulfur-containing (de)hydratases whose activities are modulated in bacteria by superoxide radical (O2-.)-mediated inactivation and iron-dependent reactivation. The inactivation-reactivation of aconitase(s) in cultured mammalian cells was explored since these reactions may impact important and diverse aconitase functions in the cytoplasm and mitochondria. Conditions which increase O2-. production including exposure to the redox-cycling agent phenazine methosulfate (PMS), inhibitors of mitochondrial ubiquinol-cytochrome c oxidoreductase, or hyperoxia inactivated aconitase in mammalian cells. Overproduction of mitochondrial Mn-superoxide dismutase protected aconitase from inactivation by PMS or inhibitors of ubiquinol-cytochrome c oxidoreductase, but not from normobaric hyperoxia. Aconitase activity was reactivated (t1/2 of 12 +/- 3 min) upon removal of PMS. The iron chelator deferoxamine impaired reactivation and increased net inactivation of aconitase by O2-.. The ability of ubiquinol-cytochrome c oxidoreductase-generated O2-. to inactivate aconitase in several cell types correlated with the fraction of the aconitase activity localized in mitochondria. Extracellular O2-. generated with xanthine oxidase did not affect aconitase activity nor did exogenous superoxide dismutase decrease aconitase inactivation by PMS. The results demonstrate a dynamic and cyclical O2-.-mediated inactivation and iron-dependent reactivation of the mammalian [4Fe-4S] aconitases under normal and stress conditions and provide further evidence for the membrane compartmentalization of O2-..

Aconitate Hydratase↗
PubMed1994

Aconitase is a sensitive and critical target of oxygen poisoning in cultured mammalian cells and in rat lungs.

The effect of hyperoxia on activity of the superoxide-sensitive citric acid cycle enzyme aconitase was measured in cultured human epithelial-like A549 cells and in rat lungs. Rapid and progressive loss of > 80% of the aconitase activity in A549 cells was seen during a 24-hr exposure to a PO2 of 600 mmHg (1 mmHg = 133 Pa). Inhibition of mitochondrial respiratory capacity correlated with loss of aconitase activity in A549 cells exposed to hyperoxia, and this effect could be mimicked by fluoroacetate (or fluorocitrate), a metabolic poison of aconitase. Exposure of rats to an atmospheric PO2 of 760 mmHg or 635 mmHg for 24 hr caused respective 73% and 61% decreases in total lung aconitase activity. We propose that early inactivation of aconitase and inhibition of the energy-producing and biosynthetic reactions of the citric acid cycle contribute to the sequelae of lung damage and edema seen during exposure to hyperoxia.

Aconitate Hydratase↗
PubMed1993

NADPH inhibits transcription of the Escherichia coli manganese superoxide dismutase gene (sodA) in vitro.

We have previously reported that the thiols glutathione, dithiothreitol, and beta-mercaptoethanol suppress transcription of the Escherichia coli manganese-containing superoxide dismutase gene (sodA) in an in vitro coupled transcription plus translation system (Gardner, P. R., and Fridovich, I. (1987) J. Biol. Chem. 262, 17591-17595). We now report that NADPH, but not NADH, selectively decreases transcription of sodA in vitro and that an NADPH generating system utilizing glucose 6-phosphate and the corresponding dehydrogenase markedly augments this suppressive effect. A redox buffer containing various ratios of oxidized and reduced glutathione also modulated transcription of sodA thus demonstrating the existence of a redox-sensitive mechanism controlling sodA transcription. Fusion of a 120-base pair fragment, containing 90 base pairs of DNA upstream of the sodA transcription initiation site, to a promoterless galactokinase gene (galK) conferred redox-sensitivity to GalK synthesis. We propose that these redox effects act through a redox-sensitive regulator of sodA and that the anabolic reduction charge, [NADPH]/([NADPH]+[NADP+]), is one cellular signal controlling sodA transcription.

Base Sequence↗
PubMed1993

Effect of glutathione on aconitase in Escherichia coli.

The effect of glutathione (GSH) on the superoxide-sensitive [4Fe-4S]-containing aconitase of Escherichia coli was explored. A mutant deficient in GSH biosynthesis, designated gshA, grew slower in a defined medium than did the parental strain and this effect was more pronounced when succinate was supplied as the carbon source in place of glucose. This suggested that the citric acid cycle was compromised in the gshA strain. Aconitase activity was approximately 25% lower in GSH-deficient cells growing on either glucose or succinate, and was lower still in strains producing less superoxide dismutase. Addition of GSH to the medium stimulated growth of the gshA strain on succinate. It also elevated the aconitase activity in the presence of chloramphenicol, which was added to block protein synthesis. Dithiothreitol and 2-mercaptoethanol were much less effective in this regard than was GSH. Exposure of cultures to 4.2 atm O2 caused a rapid decline in aconitase activity and this was the case in both GSH-proficient and GSH-deficient E. coli; however, the reactivation which was seen when the hyperoxic exposure was terminated was significantly impaired in the gshA strain. There is a dynamic balance between inactivation of aconitase by superoxide and reactivation by Fe(II) and this balance is altered in GSH-deficient E. coli. GSH may facilitate reactivation of aconitase, and of other [4Fe-4S]-containing dehydratases, by increasing the rate of transfer of Fe(II) to the [3Fe-4S] site.

Aconitate Hydratase↗
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↗
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

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↗