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• W2006365527 abstract "DNA is damaged in vivo by the Fenton reaction mediated by Fe2+ and cellular reductants such as NADH, which reduce Fe3+ to Fe2+ and allow the recycling of iron. To study the response of Escherichia coli to such cycling, the activities of several enzymes involved in nicotinamide nucleotide metabolism were measured following an H2O2 challenge. NADPH-dependent peroxidase, NADH/NADP+ transhydrogenase, and glucose-6-phosphate dehydrogenase were most strongly induced, increasing 2.5-3-fold. In addition, the cellular ratios of NADPH to NADH increased 6- or 92-fold 15 min after exposure to 0.5 or 5 mm H2O2, respectively. In vitro, NADH was oxidized by Fe3+ up to 16-fold faster than NADPH, despite their identical reduction potentials. To understand this rate difference, the interactions of Fe3+ and Ga3+ with NAD(P)H were examined by 1H, 13C, and 31P NMR spectroscopy. Association with NADH occurred primarily with adenine at N7 and the amino group, but for NADPH, strong metal interactions also occurred at the 2′-phosphate group. Interaction of M3+ (Fe3+ or Ga3+) with the adenine ring would bring it into close proximity to the redox-active nicotinamide ring in the folded form of NAD(P)H, but interaction of M3+ with the 2′-phosphate group would avoid this close contact. In addition, as determined by absorbance spectroscopy, the energy of the charge-transfer species was significantly higher for the Fe3+·NADPH complex than for the Fe3+·NADH complex. We therefore suggest that upon exposure to H2O2 the NADH pool is depleted, and NADPH, which is less reactive with Fe3+, functions as the major nicotinamide nucleotide reductant. DNA is damaged in vivo by the Fenton reaction mediated by Fe2+ and cellular reductants such as NADH, which reduce Fe3+ to Fe2+ and allow the recycling of iron. To study the response of Escherichia coli to such cycling, the activities of several enzymes involved in nicotinamide nucleotide metabolism were measured following an H2O2 challenge. NADPH-dependent peroxidase, NADH/NADP+ transhydrogenase, and glucose-6-phosphate dehydrogenase were most strongly induced, increasing 2.5-3-fold. In addition, the cellular ratios of NADPH to NADH increased 6- or 92-fold 15 min after exposure to 0.5 or 5 mm H2O2, respectively. In vitro, NADH was oxidized by Fe3+ up to 16-fold faster than NADPH, despite their identical reduction potentials. To understand this rate difference, the interactions of Fe3+ and Ga3+ with NAD(P)H were examined by 1H, 13C, and 31P NMR spectroscopy. Association with NADH occurred primarily with adenine at N7 and the amino group, but for NADPH, strong metal interactions also occurred at the 2′-phosphate group. Interaction of M3+ (Fe3+ or Ga3+) with the adenine ring would bring it into close proximity to the redox-active nicotinamide ring in the folded form of NAD(P)H, but interaction of M3+ with the 2′-phosphate group would avoid this close contact. In addition, as determined by absorbance spectroscopy, the energy of the charge-transfer species was significantly higher for the Fe3+·NADPH complex than for the Fe3+·NADH complex. We therefore suggest that upon exposure to H2O2 the NADH pool is depleted, and NADPH, which is less reactive with Fe3+, functions as the major nicotinamide nucleotide reductant. The utilization of oxygen as the terminal electron acceptor for aerobic respiration results in exposure to toxic reactive oxygen species that arise as incompletely reduced byproducts of respiratory electron transport (1Chance B. Sies H. Boveris A. Physiol. Rev. 1979; 59: 527-605Crossref PubMed Scopus (4782) Google Scholar) or as side products of enzymes such as xanthine oxidase, NADH/NADPH oxidase, monooxygenases, and cyclooxygenases (2Fridovich I. Science. 1978; 201: 875-880Crossref PubMed Scopus (2748) Google Scholar, 3Cerutti P.A. Science. 1985; 227: 375-381Crossref PubMed Scopus (2317) Google Scholar). In addition, environmental agents such as ionizing or near-UV radiation (4Tyrrell R.M. Pidoux M. Photochem. Photobiol. 1989; 49: 407-412Crossref PubMed Scopus (152) Google Scholar) and chemicals such as paraquat, plumbagin, and menadione can generate O2˙- and H2O2 in the cell (5Prieto-Alamo M.J. Abril N. Puyeo C. Carcinogenesis. 1993; 14: 237-244Crossref PubMed Scopus (29) Google Scholar). Reactive oxygen species cause damage to DNA, proteins, and lipids and are implicated in a variety of human pathologies including Alzheimer's disease, cancer, arteriosclerosis, and aging (6Barja G. Ageing Res. Rev. 2002; 1: 397-411Crossref PubMed Scopus (178) Google Scholar, 7Halliwell B. Drugs Aging. 2001; 18: 635-716Crossref Scopus (1226) Google Scholar, 8Markesbery W.R. Carney J.M. Brain Pathol. 1999; 9: 133-146Crossref PubMed Scopus (742) Google Scholar, 9Olsinki R. Gackowski D. Foksinski M. Rozalski R. Rozalski K. Jaruga P. Free Radic. Biol. Med. 2002; 33: 192-200Crossref PubMed Scopus (254) Google Scholar).Exposure of Escherichia coli or mammalian cells to hydrogen peroxide results in two modes of killing: Mode I cytotoxicity peaks at <3 mm H2O2, whereas Mode II cytotoxicity occurs between 3 and 25 mm and is independent of the dose. These complex kinetics are also observed for DNA damage in vitro. This toxicity is attributable to DNA damage by reactive oxygen species generated via the Fenton reaction (10Imlay J.A. Chin S.M. Linn S. Science. 1988; 240: 640-642Crossref PubMed Scopus (1224) Google Scholar). H2O2+Fe2++H+→Fe3++[ -OH]+H2O Reaction 1REACTION 1 The hydroxyl radical is an extremely powerful oxidant that reacts with most organic substrates at nearly diffusion-limited rates (11Halliwell B. Gutteridge J.M.C. Methods Enzymol. 1990; 186: 1-85Crossref PubMed Scopus (4414) Google Scholar). Studies of oxidative DNA damage by the Fenton reaction have indicated that the species [ -OH] is not free hydroxyl radical, but is likely stabilized by coordination to the DNA-bound iron (12Qian S.Y. Buettner G.R. Free Radic. Biol. Med. 1999; 26: 1447-1456Crossref PubMed Scopus (224) Google Scholar, 13Lloyd R. Hanna P.M. Mason R.P. Free Radic. Biol. Med. 1997; 22: 885-888Crossref PubMed Scopus (313) Google Scholar).To continue DNA damage, a reductant must be present in vivo to regenerate Fe2+ from Fe3+. Genetic studies suggested that NADH might act as this reductant, and biochemical studies showed that NADH could drive DNA damage by iron and peroxide in vivo (10Imlay J.A. Chin S.M. Linn S. Science. 1988; 240: 640-642Crossref PubMed Scopus (1224) Google Scholar, 14Imlay J.A. Linn S. Science. 1988; 240: 1302-1309Crossref PubMed Scopus (1646) Google Scholar). The level of NAD(H) drops severely in glucose-starved E. coli cells, and these cells are remarkably resistant to H2O2 (14Imlay J.A. Linn S. Science. 1988; 240: 1302-1309Crossref PubMed Scopus (1646) Google Scholar). Alternatively, raising the NADH level in vivo by eliminating or negatively regulating NADH dehydrogenase activity either genetically or chemically by inhibiting electron transport with KCN, dramatically sensitizes E. coli to killing by H2O2. KCN does not further enhance the sensitivity of an NADH dehydrogenase mutant to H2O2 (14Imlay J.A. Linn S. Science. 1988; 240: 1302-1309Crossref PubMed Scopus (1646) Google Scholar). Acceleration of DNA damage in isolated nuclei also increases dramatically in the presence of NAD(P)H and Fe3+ bound to EDTA or diethylenetriaminepentaacetic acid (15Peskin A.V. Free Radic. Biol. Med. 1996; 20: 313-318Crossref PubMed Scopus (4) Google Scholar).NADPH is an essential cofactor for the catalytic activities of glutathione peroxidase (16Sinha B.K. Mimnaugh E.G. Rajagopalan S. Meyers C.E. Cancer Res. 1989; 49: 3844-3848PubMed Google Scholar, 17Mezzetti A. Di Ilio C. Calafione A.M. Aceto A. Marzio L. Frederici G. Cuccurello F. J. Mol. Cell Cardiol. 1990; 22: 935-938Abstract Full Text PDF PubMed Scopus (30) Google Scholar), catalase (18Kirkman H.N. Gaetani G.F. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 4343-4347Crossref PubMed Scopus (362) Google Scholar), and NADPH-dependent alkylhydroperoxidase (19McKie J.H. Douglas K.T. FEBS Lett. 1991; 279: 5-8Crossref PubMed Scopus (64) Google Scholar). Moreover, the majority of NADPH in mammalian cells is bound to catalase, which it reactivates after the enzyme is inactivated by H2O2 (18Kirkman H.N. Gaetani G.F. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 4343-4347Crossref PubMed Scopus (362) Google Scholar, 20Hillar A. Nicholls P. Switala J. Loewen P.C. Biochem. J. 1994; 300: 531-539Crossref PubMed Scopus (69) Google Scholar). It might be expected that, besides NAD(P)H, glutathione could facilitate redox cycling for the Fenton reaction. However, glutathione synthase and glutathione reductase mutants exhibit normal sensitivity to Mode I killing (21Greenberg J.T. Demple B. J. Bacteriol. 1986; 168: 1026-1029Crossref PubMed Google Scholar, 22Imlay J.A. Linn S. J. Bacteriol. 1987; 169: 2967-2976Crossref PubMed Scopus (292) Google Scholar). As a result of the induction of the soxRS regulon under oxidative stress, the expression of glucose-6-phosphate dehydrogenase (G6PD) 1The abbreviations used are: G6PD, glucose-6-phosphate dehydrogenase; HPLC, high performance liquid chromatography; M3+, Fe3+ or Ga3+; NMNH, nicotinamide mononucleotide, reduced form.1The abbreviations used are: G6PD, glucose-6-phosphate dehydrogenase; HPLC, high performance liquid chromatography; M3+, Fe3+ or Ga3+; NMNH, nicotinamide mononucleotide, reduced form. increases, resulting in the conversion of NADP+ to NADPH and making these cells resistant to oxidants (23Demple B. Amabile-Cuevas C.F. Cell. 1991; 67: 837-839Abstract Full Text PDF PubMed Scopus (196) Google Scholar, 24Nunoshiba T. Hidalgo E. Amabile-Cuevas C.F. Demple B. J. Bacteriol. 1992; 174: 6054-6060Crossref PubMed Scopus (203) Google Scholar). Although it was initially believed that reduced glutathione was responsible for the antioxidant effects of the soxRS response, it was shown that the redox state of NADPH, not glutathione, modulates oxidative sensitivity (25Scott M.D. Zuo L. Lubin B.H. Chiu D.T.-Y. Blood. 1991; 77: 2059-2064Crossref PubMed Google Scholar). This antioxidant role of G6PD was substantiated further by observations that disruption of the gene encoding G6PD in mouse embryonic stem cells resulted in greatly enhanced sensitivity to oxidative stresses (26Pandolfi P.P. Sonati F. Rivi R. Mason P. Grosveld F. Luzzatto L. EMBO J. 1995; 14: 5209-5215Crossref PubMed Scopus (460) Google Scholar) and that Saccharomyces cerevisiae G6PD null mutants also are sensitive and unable to adapt to hydrogen peroxide (27Nogae I. Johnston M. Gene (Amst.). 1990; 96: 161-169Crossref PubMed Scopus (152) Google Scholar, 28Slekar K.H. Kosman D.J. Culotta V.C. J. Biol. Chem. 1996; 271: 28831-28836Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). The introduction of an intracellular NADPH-generating system restored substantial oxidative stress resistance to the G6PD-deficient yeast (25Scott M.D. Zuo L. Lubin B.H. Chiu D.T.-Y. Blood. 1991; 77: 2059-2064Crossref PubMed Google Scholar). In sum, NADPH is clearly important for protection against oxidative stress (26Pandolfi P.P. Sonati F. Rivi R. Mason P. Grosveld F. Luzzatto L. EMBO J. 1995; 14: 5209-5215Crossref PubMed Scopus (460) Google Scholar).To understand better the role of NADPH in resistance to oxidative stress, the effects of H2O2 on the activities of several enzymes involved in the synthesis and utilization of NAD(P)H and changes in the nicotinamide nucleotide pools have been monitored following exposure of E. coli to H2O2. In addition, the relative abilities of NADH and NADPH to reduce Fe3+ to Fe2+, as well as the nature of Fe3+ interactions with NAD(P)H, were studied.EXPERIMENTAL PROCEDURESBacterial Strains, Buffers, and Reagents—E. coli strains AB1157 (F-thr-1 leuB proA2 his-4 thi-1 argE2 lacY1 galK2 rpsL supE) and UM1 (LacY rpsL thi-1 katE1 katG14) were grown with vigorous shaking in K medium (1% glucose, 1% casamino acids, 1 mm MgSO4, 0.1 mm CaCl2, and M9 salts) unless otherwise noted. Water was double distilled before use. H2O2 (30%) was from Fisher Scientific, NADH (disodium salt), NADPH (tetrasodium salt), and NMNH (disodium salt) were from Sigma. All other chemicals were from Sigma unless otherwise specified. Solutions of Fe3+ and Ga3+ salts were freshly prepared prior to use and stored at pH 2.5. Elemental analyses were performed by Desert Analytics (Tucson, AZ).Spectroscopic Measurements—1H, 13C{1H}, and 31P{1H} NMR spectra were acquired on a Bruker DRX-500 spectrometer at 500, 125, and 202 MHz, respectively. Chemical shifts for 1H, 13C{1H}, and 31P{1H} NMR spectra are reported in ppm (δ) relative to SiMe4 and 40% H3PO4, respectively. Absorbance spectra for NAD(P)H were acquired on a Hewlett-Packard 8453 spectrophotometer with a diode array detector or a Cary 300 dual beam spectrophotometer. Kinetics measurements of NAD(P)H oxidation utilized the Cary spectrophotometer.Chromatography—Nicotinamide nucleotides were resolved on a PerkinElmer 250 HPLC apparatus equipped with a guard column and a Jordi reverse phase C18-DVB column (250-mm length × 4.6-mm inner diameter). The mobile phase was 98% 25 mm tributylammonium bicarbonate, pH 10, 2% acetonitrile (eluent A) and 80% acetonitrile, 20% water (eluent B). A gradient program was initially set to 100% eluent A, increasing over 20 min to 65% eluent A and 35% eluent B, then finally increasing over 40 min to 50% each of eluents A and B. Absorbances of the nucleotides were monitored at 327 nm with a diode array detector.Enzyme Assays—E. coli cells in K medium were challenged with 50 μm H2O2 at a density of 4 × 107 cells/ml. After 15 min at 37 °C, the bacteria were chilled, washed, and harvested by centrifugation. Cell pellets were resuspended in 5 ml of 50 mm KCl, 5 mm dithiothreitol, 100 mm Tris-HCl, pH 8.0, and the suspensions were sonicated four times for 15 s and then cleared by centrifugation. Protein concentration was determined by the method of Bradford (29Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213462) Google Scholar).NAD+ kinase activity was assayed by a procedure similar to that of Zercz et al. (30Zercz C.R. Moul D.E. Gomez E.G. Lopez V.M. Andreoli A.J. J. Bacteriol. 1987; 169: 184-188Crossref PubMed Google Scholar), but [32P]NAD+ was the substrate, and NADP+ was monitored by radioactivity after isolation with thin layer chromatography on silica plates with ethanol and 1 m ammonium acetate (1:1) as solvent. G6PD and isocitrate dehydrogenase activities were assayed according to the protocols described in the Worthington Manual (31Worthington C.C. Worthington Manual: Enzymes and Related Biochemicals. Worthington Biochemical Company, Freehold, NJ1988Google Scholar). The assay of NADH transhydrogenase activity was based on the method of Zercz et al. (30Zercz C.R. Moul D.E. Gomez E.G. Lopez V.M. Andreoli A.J. J. Bacteriol. 1987; 169: 184-188Crossref PubMed Google Scholar). NADH dehydrogenase and NAD(P)H oxidases were assayed as described by Cavari et al. (32Cavari B.Z. Avi-Dor Y. Grossowicz N. J. Bacteriol. 1968; 96: 751-759Crossref PubMed Google Scholar). NAD(P)H-dependent peroxidases were partially purified by ammonium sulfate fractionation and DEAE-cellulose chromatography and assayed following the method of Coves et al. (33Coves J. Eschenbrenner M. Fontecave M. Biochem. Biophys. Res. Commun. 1991; 178: 54-59Crossref PubMed Scopus (4) Google Scholar). The reaction was carried out under aerobic conditions and in the presence of an NADH-generating system. The activity was determined from the appearance of absorbance of NADPH at 340 nm. NAD+ pyrophosphatase activity was determined as described previously (34Kornberg A. Pricer W.E. J. Biol. Chem. 1950; 182: 763-778Abstract Full Text PDF Google Scholar). The assay of DNA ligase activity was based on the method of Olivera et al. (35Olivera B.M. Lehman I.R. Proc. Natl. Acad. Sci. U. S. A. 1967; 57: 1700-1704Crossref PubMed Scopus (71) Google Scholar), and NAD+ glycohydrolase assays were based on the method of Barbieri et al. (36Barbieri J.T. Moloney B.K. Mendo-Mueller L.M. J. Bacteriol. 1989; 171: 4362-4369Crossref PubMed Google Scholar).Measurement of E. coli Nicotinamide Nucleotide Pools—Intracellular NAD(P)H and NAD(P)+ concentrations were determined by a modification of the method of Klaidman et al. (37Klaidman L.K. Leung A.C. Adams Jr., J.D. Anal. Biochem. 1995; 228: 312-317Crossref PubMed Scopus (132) Google Scholar). E. coli AB1157 was grown in 50 ml of K medium plus 1 μg/ml thiamine at 37 °C to an A 600 of ∼0.5, and then H2O2 was added to the culture medium, and incubation was continued for 15 min. Anaerobic cultures were grown with bubbling 95% N2 and 5% CO2 prior to and after the addition of H2O2. After H2O2 challenge, cell pellets were collected by centrifugation and resuspended in 1 m KCN, 0.3 n KOH, 5 mm EDTA and then mixed with an equal volume of 50 mm tributylammonium bicarbonate, pH 10, to give a final volume of ∼2.5 ml. The lysate was cleared by centrifugation, filtered through a 0.2-μm filter, and analyzed by HPLC. Amounts of nicotinamide nucleotides were determined by measuring areas of the HPLC peak traces at 327 nm and comparing these with standard 3-nmol samples of each of the nicotinamide nucleotides run under identical conditions. Sample traces are given in the supplementary data (Fig. S1). Cellular concentrations were calculated assuming that an A 600 = 1.0 corresponds to 109 cells/ml and that the volume of a cell is 10-12 ml (38Norland S. Hedal M. Tumyr O. Microb. Ecol. 1987; 13: 95-101Crossref PubMed Scopus (91) Google Scholar).Metal Titrations of NADH and Its Analogs Monitored by NMR Spectroscopy—NMR spectra of freshly prepared 0.5-ml solutions of NAD(P)H, NMNH, or ADP-ribose between 10 and 100 mm in D2O, pD ∼ 7.5, were acquired after incremental addition of FeCl3·6 H2O or Ga(NO3)3 in D2O as indicated. Addition of DCl at pH 2.5 to a D2O solution of NADPH in the same amounts as those added for Fe3+ or Ga3+ resulted in no significant shifting of the NMR resonances. Spectra were acquired within 30 min of the addition of Fe3+. Percent broadening of NMR resonances upon addition of Fe3+ was calculated using Equation 1. &x0025;broadening=100×(1-(peakheightwithFe3+)/(initialpeakheight))(Eq. 1) A broadening of 100% indicates that no resonance was visible. K d values and fractional occupancy of Ga3+ were determined as described previously (39Rai P. Cole T.D. Wemmer D.E. Linn S. J. Mol. Biol. 2001; 312: 1089-1101Crossref PubMed Scopus (60) Google Scholar). Percentages of NAD(P)H in linear and folded forms were calculated as described by Oppenheimer et al. (40Oppenheimer N.J. Arnold Jr., L.J. Kaplan N.O. Biochemistry. 1978; 17: 2613-2619Crossref PubMed Scopus (49) Google Scholar).Elemental Analyses of Ga 3+ /NAD(P)H Precipitates—20 mg of NAD(P)H was dissolved in 0.4 ml of H2O, and 1 eq of Ga(NO3)3 in 7.5 μl was added. A white precipitate formed immediately for the NADH sample, but an additional 1 eq of Ga(NO3)3 was added to the NADPH sample to obtain sufficient precipitate for analysis. Both samples were centrifuged at 14,000 rpm for 5 min, and the supernatant was discarded. The precipitates were washed three times by centrifugation with 1 ml of water and dried under vacuum.For the NADH sample, the best fit formula was Na[(NADH)Ga2(OH)5]·3H2OCalculated:x0026;C26.1&x0025;H3.97&x0025;N10.2&x0025;Ga14.5&x0025;Found:x0026;C26.7&x0025;H3.87&x0025;N10.1&x0025;Ga13.7&x0025;For the NADH sample, the best fit formula was Na0.7[(NADH)Ga2.3(OH)3.6]·4H2OCalculated:x0026;C24.0&x0025;H3.61&x0025;N9.33&x0025;Ga15.3&x0025;Found:x0026;C23.4&x0025;H3.40&x0025;N8.90&x0025;Ga15.7&x0025;Metal Titrations of NAD(P)H Monitored by Absorbance Spectroscopy—Absorbance spectra of freshly prepared solutions of NAD(P)H were acquired after incremental addition of Fe(NO3)3 to 50 or 500 μm solutions of NAD(P)H, or Ga(NO3)3 to 66 μm solutions of NADPH. All spectra were acquired within 30 min of the addition of M3+.Kinetic Measurements of NAD(P)H Oxidation—Freshly prepared solutions of 16 μm NAD(P)H were brought to 100 mm ethanol, 1.25 μm H2O2, and/or 80 μm FeCl3 as indicated. A 340 was measured at 25 °C every 0.2 s for 7 min and corrected with appropriate blanks. Best fit lines were calculated from the initial rates. For anaerobic measurements, the water used to prepare the samples was degassed with argon for 3 h prior to use, and the samples were prepared and sealed under an argon atmosphere. The initial rates were very dependent on concentration, so care was taken to use the same solutions and order of addition of reactants for both the NADH and NADPH trials.Molecular Modeling—Molecular models for Fe3+ binding to NADPH were constructed using Spartan 02 (Wavefunction, Inc., Irvine, CA). The NADPH conformation was obtained from the most probable folded conformation as predicted by NMR spectroscopy (41Oppenheimer N.J. Dolphin D. Poulson R. Avramovic O. Pyridine Nucleotide Coenzymes. Vol. A. John Wiley and Sons, New, York1987: 185-230Google Scholar): 4′-5′-gg and 5′-O-gg rotamers for both the adenyl and reduced nicotinamide ribose units, 2′-endo conformation for the nicotinamide ribose, and 3′-endo conformation for the adenine ribose. To ensure that the molecule remained in the folded conformation, the distance between the adenine and reduced nicotinamide rings was restrained to 3.6 Å. This NADPH conformation was energy minimized using MMFF94 force field calculations (42Kong J. White C.A. Krylov A.I. Sherrill D. Adamson R.D. Furlani T.R. Lee M.S. Lee A.M. Gwaltney S.R. Adams T.R. Ochsenfeld C. Gilbert A.T.B. Kedziora G.S. Rassolov V.A. Maurice D.R. Nair N. Shao Y. Besley N.A. Maslen P.E. Dombroski J.P. Daschel H. Zhang W. Korambath P.P. Baker J. Byrd E.F.C. Van Voorhis T. Oumi M. Hirata S. Hsu C.-P. Ishikawa N. Florian J. Warshel A. Johnson B.G. Gill P.M.W. Head-Gordon M. Pope J.A. J. Comput. Chem. 2000; 21: 1532-1548Crossref Scopus (632) Google Scholar) before adding the Fe3+ ion at a restrained distance of 2.1 Å (43Obrey S.J. Bott S.G. Barron A.R. J. Chem. Cryst. 2000; 30: 61-63Crossref Scopus (8) Google Scholar) to the adenine N7. Because of steric hindrance resulting from the close contacts between the adenine N7-bound Fe3+ and the adenine amino group, a bond between the Fe3+ and the amino group was included and restrained to a distance of 2.3 Å.RESULTSNicotinamide Nucleotide Enzyme Levels after H 2 O 2 Challenge of E. coli—Because of a possible involvement of NAD(P)H in mediating H2O2 toxicity, the activities of several enzymes involved in nicotinamide nucleotide metabolism (Table I) were measured before and after challenge with H2O2 (Table II). In consideration of the existence of the “early” H2O2 induction of proteins, the activities of which are induced during the first 10-18 min but return to normal within 30 min after the addition of hydrogen peroxide (44Storz G. Tartaglia L.A. Farr S.B. Ames B.N. Trends Genet. 1990; 6: 363-368Abstract Full Text PDF PubMed Scopus (265) Google Scholar), the activities were measured 15 min after challenge of catalase-deficient cells with 50 μm H2O2. Catalase is the major enzyme for removing hydrogen peroxide, but catalase-deficient mutants are not more sensitive to killing by H2O2 than are wild type cells (22Imlay J.A. Linn S. J. Bacteriol. 1987; 169: 2967-2976Crossref PubMed Scopus (292) Google Scholar). Thus, catalase mutants were used in this study to elucidate other defense systems against hydrogen peroxide. E. coli G6PD, which specifically reduces NADP+, is one of the major sources of cellular NADPH. Upon H2O2 challenge, the activity of G6PD was induced by about 2.9-fold (Table II). Meanwhile, the activity of isocitrate dehydrogenase, which also reduces NADP+, was not induced.Table IEnzymatic activities assayedEnzymes that synthesize NADPHGlucose-6-phosphate dehydrogenased-glucose 6-phosphate + NADP+ → NADPH + d-glucono-δ-lactone 6-phosphateIsocitrate dehydrogenaseIsocitrate + NADP+ → α-ketoglutarate + NADPHNADH/NADP+ transhydrogenaseRespiration-dependentNADH + NADP+ + energy produced by respiration → NAD+ + NADPHATP-dependentNADH + NADP+ + ATP → NAD+ + NADPH + ADP + PiEnzymes that oxidize NAD(P)HIn the respiratory chainNADH dehydrogenaseNADH + ubiquinone → NAD+ + ubiquinolIn the peroxidase reactionNAD(P)H-dependent peroxidasesNAD(P)H + 2 H2O2 → NAD(P)+ + 2 H2O + O2NAD(P)H oxidaseNAD(P)H + O2 + H+ → NAD(P)+ + H2O2Enzymes that deplete NAD+NAD+ glycohydrolaseNAD+ → nicotinamide + ADP-riboseNAD+ pyrophosphataseNAD+ → NMN + AMPDNA ligaseNAD+ → NMN + AMPNAD+ kinaseNAD+ + ATP → NADP+ + ADP Open table in a new tab Table IIChanges in enzymatic activities after H2O2 challengeEnzymePercentage activity 15 Min after challenge%Glucose-6-phosphate dehydrogenaseaAssays were performed using catalase-deficient cells.286 ± 28Isocitrate dehydrogenase100 ± 4NADH/NADP+ transhydrogenaseaAssays were performed using catalase-deficient cells.Respiration-dependent280 ± 16ATP-dependent99 ± 7NADH dehydrogenase (ubiquitin-dependent)aAssays were performed using catalase-deficient cells.94 ± 8PeroxidaseaAssays were performed using catalase-deficient cells.NADH-dependent (195 units/mg)138 ± 10NADPH-dependent (46 units/mg)248 ± 29NAD(P)H oxidaseaAssays were performed using catalase-deficient cells.NADH (240 units/mg)140 ± 13NADPH (39 units/mg)145 ± 7NAD+ glycohydrolaseaAssays were performed using catalase-deficient cells.93 ± 3NAD+ pyrophophatase98 ± 7DNA ligaseaAssays were performed using catalase-deficient cells.102 ± 18NAD+ kinaseaAssays were performed using catalase-deficient cells.99 ± 8a Assays were performed using catalase-deficient cells. Open table in a new tab NADH/NADP+ transhydrogenases reversibly transfer a hydrogen atom between NADH and NADP+ (Table I). Upon membrane energization either by respiration or by ATP hydrolysis, the rate of reduction of NADP+ by NADH is increased severalfold, and the equilibrium constant for the reaction increased from 0.79 for the non-energy-dependent reaction to 480 (45Lee C.-P. Ernster L. Biochim. Biophys. Acta. 1964; 81: 187-190Google Scholar). Presumably the physiological function of these transhydrogenases is to provide NADPH for biosynthesis and detoxification (46Bragg P.D. Davies P.L. Hou C. Biochem. Biophys. Res. Commun. 1972; 47: 1248-1255Crossref PubMed Scopus (56) Google Scholar). To investigate whether either of these energy-linked transhydrogenase activities is increased upon hydrogen peroxide exposure, they were assayed in catalase-deficient E. coli cells after exposure to 50 μm H2O2 for 15 min (Table II). The assays included an NADH-regenerating system. The respiration-driven transhydrogenase activity was increased almost 3-fold, but the ATP-dependent transhydrogenase activity showed no increase. Likewise, no induction of NADH dehydrogenase activity was observed.In addition to alkylhydroperoxidase (19McKie J.H. Douglas K.T. FEBS Lett. 1991; 279: 5-8Crossref PubMed Scopus (64) Google Scholar) and o-dianisidine peroxidase (47Claiborne A. Fridovich I. J. Biol. Chem. 1979; 254: 4245-4254Abstract Full Text PDF PubMed Google Scholar), E. coli contains an NADH-dependent peroxidase (33Coves J. Eschenbrenner M. Fontecave M. Biochem. Biophys. Res. Commun. 1991; 178: 54-59Crossref PubMed Scopus (4) Google Scholar). Although the former enzymes do not utilize H2O2 as substrate, the latter one does. The NADH-dependent peroxidase activity was elevated by only 38% after exposure to peroxide. However, NADPH-dependent peroxidase activity increased by 248% upon H2O2 challenge. NADH and NADPH oxidase activities were induced to only 140% of normal activity.The turnover of NAD+ is very rapid in the cell, and it is 4-fold faster under aerobic conditions than anaerobic conditions (48Park U.E. Olivera B.M. Hughes K.T. Roth J.R. Hillyard D.R. J. Bacteriol. 1989; 171: 2173-2180Crossref PubMed Google Scholar). Moreover, in mammalian cells, DNA breaks caused by oxidative stresses can deplete NAD+ via the NAD+ glycohydrolase activity of poly(ADP-ribose) polymerase, whereas in E. coli, DNA ligase hydrolyzes NAD+. Thus, it was proposed that the turnover of NAD+ might have important functions under conditions of oxidative stress (48Park U.E. Olivera B.M. Hughes K.T. Roth J.R. Hillyard D.R. J. Bacteriol. 1989; 171: 2173-2180Crossref PubMed Google Scholar). Three enzymatic activities initiate NAD+ turnover in E. coli: NAD+ glycohydrolase, NAD+ pyrophosphatase, and DNA ligase. However, none of the activities of these enzymes changed after hydrogen peroxide exposure (Table II). Finally, NAD+ kinase activity was unchanged. In conclusion, the changes in the enzyme activities indicate that H2O2 is depleted by NADPH-dependent peroxidases in the absence of catalase and predict that cellular NADPH levels might be increased relative to those of NADH.Measurement of Nicotinamide Nucleotide Pools in E. coli—The determination of nicotinamide nucleotide pools is complex because of the sensitivity of NAD(P)H to degradation or oxidation during isolation. Lundquist and Olivera (49Lundquist R. Olivera B.M. J. Biol. Chem. 1971; 246: 1107-1116Abstract Full Text PDF PubMed Google Scholar) estimated the concentrations of NAD(P)+ in E. coli but did not include the reduced nicotinamide nucleotides. Bochner and Ames (50Bochner B.R. Ames B.N. J. Biol. Chem. 1982; 257: 9759-9769Abstract Full Text PDF PubMed Google Scholar) used an acid extraction procedure for purification of the nicotinamide nucleotides which probably resulted in oxidation of NAD(P)H prior to quantitation. Therefore, we modified the method used for brain cells by Klaidman et al. (37Klaidman L.K. Leung A.C. Adams Jr., J.D. Anal. Biochem. 1995; 228: 312-317Crossref PubMed Scopus (132) Google Scholar) to measure the effect of oxidative stress upon the free pools" @default.
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• W2006365527 title "Effects of Hydrogen Peroxide upon Nicotinamide Nucleotide Metabolism in Escherichia coli" @default.
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