Nitrosylation of Human Glutathione Transferase P1-1 with Dinitrosyl Diglutathionyl Iron Complex in Vitro and in Vivo
2005; Elsevier BV; Volume: 280; Issue: 51 Linguagem: Inglês
10.1074/jbc.m507916200
ISSN1083-351X
AutoresEleonora Cesareo, L.J. Parker, Jens Z. Pedersen, Marzia Nuccetelli, Anna P. Mazzetti, Anna Pastore, Giorgio Federici, Anna Maria Caccuri, Giorgio Ricci, Julian J. Adams, Michael W. Parker, Mario Lo Bello,
Tópico(s)Sulfur Compounds in Biology
ResumoWe have recently shown that dinitrosyl diglutathionyl iron complex, a possible in vivo nitric oxide (NO) donor, binds with extraordinary affinity to one of the active sites of human glutathione transferase (GST) P1-1 and triggers negative cooperativity in the neighboring subunit of the dimer. This strong interaction has also been observed in the human Mu, Alpha, and Theta GST classes, suggesting a common mechanism by which GSTs may act as intracellular NO carriers or scavengers. We present here the crystal structure of GST P1-1 in complex with the dinitrosyl diglutathionyl iron ligand at high resolution. In this complex the active site Tyr-7 coordinates to the iron atom through its phenolate group by displacing one of the GSH ligands. The crucial importance of this catalytic residue in binding the nitric oxide donor is demonstrated by site-directed mutagenesis of this residue with His, Cys, or Phe residues. The relative binding affinity for the complex is strongly reduced in all three mutants by about 3 orders of magnitude with respect to the wild type. Electron paramagnetic resonance spectroscopy studies on intact Escherichia coli cells expressing the recombinant GST P1-1 enzyme indicate that bacterial cells, in response to NO treatment, are able to form the dinitrosyl diglutathionyl iron complex using intracellular iron and GSH. We hypothesize the complex is stabilized in vivo through binding to GST P1-1. We have recently shown that dinitrosyl diglutathionyl iron complex, a possible in vivo nitric oxide (NO) donor, binds with extraordinary affinity to one of the active sites of human glutathione transferase (GST) P1-1 and triggers negative cooperativity in the neighboring subunit of the dimer. This strong interaction has also been observed in the human Mu, Alpha, and Theta GST classes, suggesting a common mechanism by which GSTs may act as intracellular NO carriers or scavengers. We present here the crystal structure of GST P1-1 in complex with the dinitrosyl diglutathionyl iron ligand at high resolution. In this complex the active site Tyr-7 coordinates to the iron atom through its phenolate group by displacing one of the GSH ligands. The crucial importance of this catalytic residue in binding the nitric oxide donor is demonstrated by site-directed mutagenesis of this residue with His, Cys, or Phe residues. The relative binding affinity for the complex is strongly reduced in all three mutants by about 3 orders of magnitude with respect to the wild type. Electron paramagnetic resonance spectroscopy studies on intact Escherichia coli cells expressing the recombinant GST P1-1 enzyme indicate that bacterial cells, in response to NO treatment, are able to form the dinitrosyl diglutathionyl iron complex using intracellular iron and GSH. We hypothesize the complex is stabilized in vivo through binding to GST P1-1. S-Nitrosylation of protein thiol groups by nitric oxide is accepted as being among the most important posttranslational modifications (1Stamler J.S. Simon D.I. Osborne J.A. Mullins M.E. Jaraki O. Michel T. Singel D.J. Loscalzo J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 444-448Crossref PubMed Scopus (1311) Google Scholar). Such modifications can cause modulation of many different functions with recent examples including proteins involved in signaling cascades, apoptosis, ion channels, redox systems, and hemoproteins (2Broillet M.-C. Cell. Mol. Life Sci. 1999; 55: 1036-1042Crossref PubMed Scopus (177) Google Scholar). It has been suggested that NO may play a role in iron homeostasis and/or metabolism based on observations of iron nitrosylation of non-heme iron proteins in bacteria (3D'Autréaux B. Touati D. Bersch B. Latour J. Michaud-Soret I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16619-16624Crossref PubMed Scopus (154) Google Scholar) as well as in mammals (4Kim Y.M. Chung H.T. Simmons R.L. Billiar T.R. J. Biol. Chem. 2000; 275: 10954-10961Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 5Watts R.N. Richardson D.R. Eur. J. Biochem. 2002; 269: 3383-3392Crossref PubMed Scopus (56) Google Scholar, 6Cairo G. Pietrangelo A. Biochem. J. 2000; 352: 241-250Crossref PubMed Scopus (276) Google Scholar). Iron-free proteins, such as albumin (7Boese M. Mordvintcev P.I. Vanin A.F. Busse R. Mulsch A. J. Biol. Chem. 1995; 272: 29244-29249Abstract Full Text Full Text PDF Scopus (214) Google Scholar) and GSH reductase (8Becker K. Savvides S.N. Keese M. Schirmer R.H. Karplus P.A. Nat. Struct. Biol. 1998; 5: 267-271Crossref PubMed Scopus (147) Google Scholar), can also become targets of nitrosylation in the presence of suitable amounts of iron and thiol ligand (mostly GSH under physiological conditions). In these cases the formation of iron-dithiol dinitrosyl complexes are readily detected by EPR spectroscopy. We have recently shown that human glutathione transferase (GST) 5The abbreviations used are: GSTglutathione transferaseCDNB1-chloro-2,4-dinitrobenzeneNONOate2-(N,N-diethylamino)-diazenolate 2-oxideDEANOdiethylamine NONOateDNDGICdinitrosyl diglutathionyl iron complexDNGICdinitrosyl glutathionyl iron complexMES2-(N-morpholino)ethanesulfonic acidWTwild-type enzymeGSNOS-nitrosoglutathione. P1-1 strongly binds dinitrosyl diglutathionyl iron complexes (DNDGIC) in vitro while maintaining its well known detoxifying activity toward dangerous compounds (9Lo Bello M. Nuccetelli M. Caccuri A.M. Stella L. Parker M.W. Rossjohn J. McKinstry W.J. Mozzi A. Federici G. Polizio F. Pedersen J.Z. Ricci G. J. Biol. Chem. 2001; 276: 42138-42145Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). A very high affinity for this complex was also found for other glutathione transferase classes (Mu, Alpha, and Theta), suggesting a common mechanism by which the more recently evolved GSTs may act as intracellular NO carriers or scavengers (10De Maria F. Pedersen J.Z. Caccuri A.M. Antonini G. Turella P. Stella L. Lo Bello M. Federici G. Ricci G. J. Biol. Chem. 2003; 278: 42283-42293Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 11Turella P. Pedersen J.Z. Caccuri A.M. De Maria F. Mastroberardino P. Lo Bello M. Federici G. Ricci G. J. Biol. Chem. 2003; 278: 42294-42299Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). glutathione transferase 1-chloro-2,4-dinitrobenzene 2-(N,N-diethylamino)-diazenolate 2-oxide diethylamine NONOate dinitrosyl diglutathionyl iron complex dinitrosyl glutathionyl iron complex 2-(N-morpholino)ethanesulfonic acid wild-type enzyme S-nitrosoglutathione. The glutathione transferases (EC 2.5.1.18), historically also called glutathione S-transferases, catalyze the nucleophilic attack by reduced glutathione (GSH) on non-polar compounds that contain an electrophilic carbon, nitrogen, or sulfur atom. This classical conjugation reaction toward foreign compounds (e.g. cancer chemotherapeutic agents, insecticides, herbicides, carcinogens) and endogenous compounds (e.g. byproducts of oxidative stress) is considered part of a coordinated defense strategy together with other GSH-dependent enzymes, the cytochrome P450s (Phase I enzymes) and some membrane transporters (Phase III) such as MRP1 and MRP2, to remove glutathione conjugates from the cell. In mammals there are three major families of proteins widely distributed in nature that exhibit glutathione transferase activity. Two of these, the cytosolic and mitochondrial GSTs, comprise soluble enzymes, whereas the third family is microsomal and is referred to as MAPEG (membrane-associated proteins in eicosanoid and glutathione) metabolism (12Hayes D.H. Flanagan J.U. Jowsey I.R. Annu. Rev. Pharmacol. Toxicol. 2005; 45: 51-88Crossref PubMed Scopus (2981) Google Scholar). The human cytosolic GSTs are dimeric proteins that can be grouped into at least seven gene-independent classes (Alpha, Mu, Pi, Sigma, Theta, Omega, and Zeta) on the basis of their amino acid sequence and immunological properties (13Mannervik B. Ålin P. Guthenberg C. Jensson H. Tahir M.K. Warholm M. Jörnvall H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7202-7206Crossref PubMed Scopus (1077) Google Scholar, 14Meyer D.J. Coles B. Pemble S.E. Gilmore K.S. Fraser G.M. Ketterer B. Biochem. J. 1991; 274: 409-414Crossref PubMed Scopus (749) Google Scholar, 15Buetler T.M. Eaton D.L. J. Environ. Sci. Health C Environ. Carcinog. Ecotoxicol. Rev. 1992; 10: 181-200Crossref Scopus (175) Google Scholar, 16Meyer D.J. Thomas M.R. Biochem. J. 1995; 311: 739-742Crossref PubMed Scopus (143) Google Scholar). Their three-dimensional structures do not differ significantly despite low sequence homology (17Dirr H.W. Reinemer P. Huber R. Eur. J. Biochem. 1994; 220: 645-661Crossref PubMed Scopus (389) Google Scholar, 18Wilce M.C.J Parker M.W. Biochim. Biophys. Acta. 1994; 205: 1-18Crossref Scopus (548) Google Scholar, 19Rossjohn J. McKinstry W.J. Oakley A.J. Verger D. Flanagan J. Chelvanayagam G. Tan K.-L. Board P.G. Parker M.W. Structure. 1998; 6: 309-322Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 20Wilce M.C.J Board P.G. Feil S.C. Parker M.W. EMBO J. 1995; 14: 2133-2143Crossref PubMed Scopus (218) Google Scholar). Each subunit contains a very similar binding site for GSH (G-site) and a second one for the hydrophobic cosubstrate (H-site). Structural differences at the H-site confer a certain degree of substrate selectivity. Despite a common structure they appear to play multiple functions. For example, Zeta class GST Z1-1 is involved in the catabolism of phenylalanine (21Polekhina G. Board P.G. Blackburn A.C. Parker M.W. Biochemistry. 2001; 40: 1567-1576Crossref PubMed Scopus (104) Google Scholar), Pi class GST P1-1 and Mu class GST M1-1 are involved in signaling pathways through physical interaction with some kinases (22Adler V. Yin Z. Fuchs S.Y. Benezra M. Rosario L. Tew K.D. Pincus M.R. Ardana M. Henderson C.J. Wolf C.R. Davis R.J. Ronai Z. EMBO J. 1999; 18: 1321-1334Crossref PubMed Scopus (966) Google Scholar, 23Cho S-G. Lee Y.H. Park H-S. Ryoo K. Kang K.W. Park J. Eom S.J. Kim M.J. Chang T.S. Choi S.Y. Shim J. Kim Y. Dong M.-S. Lee M.-J. Kim S.G. Ichijo H. Choi E.-J. J. Biol. Chem. 2001; 276: 12749-12755Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar), and Omega class GST O1-1 modulates calcium channels, thus protecting mammalian cells from apoptosis induced by Ca2+ mobilization (24Dulhunty A. Gage P. Curtis S. Chelvanayagam G. Board P. J. Biol. Chem. 2001; 276: 3319-3323Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). In this paper we report the crystallographic structure of GST P1-1 with the DNDGIC bound in the active site, which together with site-directed mutagenesis studies demonstrate a crucial role for the catalytic residue Tyr-7 acting as a ligand for the iron complex in the active site (Scheme 1). These data provide direct support that GSTs can be nitrosylated in vitro. Further studies on intact Escherichia coli cells upon exposure to either GSNO or diethylamine NONOate suggest that human GST P1-1 can also be nitrosylated inside the cell. Chemicals—Diethylamine NONOate (DEANO) was from Calbiochem. GSH, 1-chloro-2,4-dinitrobenzene (CDNB), and other reagents used were from Sigma. DEANO solutions were prepared in phosphate-buffered saline buffer, pH 7.4, at room temperature; under these conditions the half-life of NO release is 16 min. GSNO Synthesis—GSNO was prepared by a modification of the original published procedure (9Lo Bello M. Nuccetelli M. Caccuri A.M. Stella L. Parker M.W. Rossjohn J. McKinstry W.J. Mozzi A. Federici G. Polizio F. Pedersen J.Z. Ricci G. J. Biol. Chem. 2001; 276: 42138-42145Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 27Hart T.W. Tetrahedron Lett. 1985; 26: 2013-2016Crossref Scopus (386) Google Scholar, 28Cavero M. Hobbs A. Madge D. Motherwell W.B. Selwood D. Potier P. Bioorg. Med. Chem. Lett. 2000; 10: 641-644Crossref PubMed Scopus (7) Google Scholar). 3 ml of equimolar GSH and NaNO2 (33 mm) were vortexed and placed on ice for an hour. To this solution 45 μl of 11 m HCl was added and vortexed, and the solution was again placed on ice for a further 2 h. 40 μl of 10 m NaOH was then added to neutralize the solution. 4 ml of acetone were added, and the GSNO sank to the bottom of the tube as a pink oil. The upper layer of acetone/H2O was aspirated off. GSNO was re-suspended in 500 μl of H2O, and again 4 ml of acetone was added. The solution was mixed and then left to sit for an hour until the GSNO oil sank to the bottom. The upper solution was again aspirated away. The resulting pink oil was frozen to -196 °C and placed on a freeze dryer for 4 h. The resulting pink solid (yield of ∼0.89 mmol) had a UV spectrum consistent with published reports (27Hart T.W. Tetrahedron Lett. 1985; 26: 2013-2016Crossref Scopus (386) Google Scholar, 29Hogg N. Singh R.J. Kalyanaraman B. FEBS Lett. 1996; 382: 223-228Crossref PubMed Scopus (249) Google Scholar). The compound was wrapped in aluminum foil to exclude light and stored at 4 °C. The GSNO concentration was determined by UV-visible spectroscopy as described (9Lo Bello M. Nuccetelli M. Caccuri A.M. Stella L. Parker M.W. Rossjohn J. McKinstry W.J. Mozzi A. Federici G. Polizio F. Pedersen J.Z. Ricci G. J. Biol. Chem. 2001; 276: 42138-42145Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar); the purity was comparable with that of commercially available GSNO. Dinitrosyl Diglutathionyl Iron Complex Synthesis—DNDGIC was prepared by a modification of the original published procedure (9Lo Bello M. Nuccetelli M. Caccuri A.M. Stella L. Parker M.W. Rossjohn J. McKinstry W.J. Mozzi A. Federici G. Polizio F. Pedersen J.Z. Ricci G. J. Biol. Chem. 2001; 276: 42138-42145Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). To 50 μl of 100 mm MES buffer, pH 6.0, and 200 μl of 100 mm GSH, 20 μl of 100 mm GSNO was added. Finally, 2 μl of 1 m FeSO4 solution containing 0.5 mm vitamin C was added. The final concentrations were 40 mm GSH, 4 mm GSNO, 4 mm FeSO4, and 1 μm vitamin C in a 500-μl volume. After several hours on ice wrapped in aluminum foil to exclude light, the solution turned a strong yellow color that was stable, contrary to previous reports, for up to a week (9Lo Bello M. Nuccetelli M. Caccuri A.M. Stella L. Parker M.W. Rossjohn J. McKinstry W.J. Mozzi A. Federici G. Polizio F. Pedersen J.Z. Ricci G. J. Biol. Chem. 2001; 276: 42138-42145Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 10De Maria F. Pedersen J.Z. Caccuri A.M. Antonini G. Turella P. Stella L. Lo Bello M. Federici G. Ricci G. J. Biol. Chem. 2003; 278: 42283-42293Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 11Turella P. Pedersen J.Z. Caccuri A.M. De Maria F. Mastroberardino P. Lo Bello M. Federici G. Ricci G. J. Biol. Chem. 2003; 278: 42294-42299Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). UV spectra were consistent with previous reports (9Lo Bello M. Nuccetelli M. Caccuri A.M. Stella L. Parker M.W. Rossjohn J. McKinstry W.J. Mozzi A. Federici G. Polizio F. Pedersen J.Z. Ricci G. J. Biol. Chem. 2001; 276: 42138-42145Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 10De Maria F. Pedersen J.Z. Caccuri A.M. Antonini G. Turella P. Stella L. Lo Bello M. Federici G. Ricci G. J. Biol. Chem. 2003; 278: 42283-42293Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 11Turella P. Pedersen J.Z. Caccuri A.M. De Maria F. Mastroberardino P. Lo Bello M. Federici G. Ricci G. J. Biol. Chem. 2003; 278: 42294-42299Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). DNDGIC cannot be isolated as a solid due to dimerization (30McDonald C.C. Phillips W. Mower H.F. J. Am. Chem. Soc. 1965; 87: 3319-3326Crossref Scopus (182) Google Scholar) but is stabilized in solution by the large excess of GSH present. Crystallization—Wild-type human Pi class GST P1-1 was expressed and purified as previously described (25Battistoni A. Mazzetti A.P. Petruzzelli R. Muramatzu M. Ricci G. Federici G. Lo Bello M. Protein Expression Purif. 1995; 6: 579-587Crossref PubMed Scopus (41) Google Scholar). The protein was crystallized using the hanging drop vapor diffusion method as described elsewhere (26Oakley A.J. Lo Bello M. Battistoni A. Ricci G. Rossjohn J. Villar H.O. Parker M.W. J. Mol. Biol. 1997; 274: 84-100Crossref PubMed Scopus (154) Google Scholar). Briefly, a 2-μl drop of protein (concentration of 6.7 mg/ml in 1 mm EDTA, 1 mm mercaptoethanol, and 10 mm phosphate buffer, pH 7.0) was mixed with the same volume of reservoir buffer composed of 100 mm MES buffer, pH 5.5 or 6.0, 22% (w/v) polyethylene glycol 8000, 20 mm CaCl2, 10 mm dithiothreitol, and 10 mm GSH. Crystals grew at 22 °C and reached a suitable size in approximately a week. Dinitrosyl Diglutathionyl Iron Complex Soak—Wild-type crystals were transferred into a new drop containing 200 μl of the DNDGIC solution described above, 100 mm MES buffer, pH 6.0, 22% (w/v) polyethylene glycol 8000, and 20 mm CaCl2. These crystals were soaked for several days. Data Collection and Processing—The x-ray diffraction data were collected at the Advanced Photon Source (Chicago, Illinois), beam line 14-ID-B using a MAR165 CCD MARResearch detector. The wavelength was set to 0.99 Å. For cryoprotection the crystals were soaked for 2 min in the well solution containing 5% (v/v) methyl-2,4-pentanediol (MPD), then dipped briefly in well solution containing 10% (v/v) MPD. The crystals were then snap-frozen at 100 K in the cryostream. Diffraction data were processed and scaled with HKL (31Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar). The crystals were shown to belong to a monoclinic lattice, with the space group C2, as seen previously for the wild-type GST P1-1 (26Oakley A.J. Lo Bello M. Battistoni A. Ricci G. Rossjohn J. Villar H.O. Parker M.W. J. Mol. Biol. 1997; 274: 84-100Crossref PubMed Scopus (154) Google Scholar). Structure Determination and Refinement—Refinement began with the Pi class GST in the C2 space group (5GSS (26Oakley A.J. Lo Bello M. Battistoni A. Ricci G. Rossjohn J. Villar H.O. Parker M.W. J. Mol. Biol. 1997; 274: 84-100Crossref PubMed Scopus (154) Google Scholar)) that had GSH and water molecules removed. Rigid body refinement in CNS (32Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.-S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren J.L. Acta Crystallogr. Sect. D. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar) was used to compensate for any possible changes in crystal packing. The starting model gave an R-factor of 33.0% (Rfree = 35.3%). The model was then refined by a round of simulated annealing using CNS. Because the asymmetric unit of the crystal contained two GST monomers, use was made of the non-crystallographic symmetry restraints on all non-hydrogen atoms in the initial rounds of the refinement. The model was rebuilt with TURBO (33Roussel A. Cambillau C. Silicon Graphics Partners Directory. Silicon Graphics, Mountain View, CA1989: 72-78Google Scholar), and GSH, MES, and water molecules were added. The model was further refined with cycles of positional and isotropically restrained B-factor refinement. After several rounds of refinement, the density for the iron complex was evident in a Fo - Fc map and was subsequently built into the model. An absorption scan at the iron edge, at 7.13 keV or 1.74 Å, demonstrated the presence of the metal in the crystal. However, data collected from this crystal were limited to a resolution of 2.7 Å because of radiation damage at this wavelength. Thus, the data were recollected at 0.99 Å off a fresh crystal. After multiple rounds of refinement and rebuilding the final R-factor was 18.2% (Rfree = 24.4%) for all data to 2.1 Å of resolution. The stereochemistry was analyzed with the program PROCHECK (34Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) and gave values either similar or better than expected for structures refined at similar resolutions. Data collection results and a summary of the refinement statistics are given in TABLE ONE.TABLE ONESummary of data collection and structure refinement for the DNGIC-GST P1-1 complex The values in parentheses are for the highest resolution bin.Data collection Temperature (K)100 Space groupC2 Cell dimensionsa (Å)76.2b (Å)89.8c (Å)68.7β (°)97.6 Maximum resolution (Å)2.1 (2.18-2.10) No. of crystals1 No. of observations2,076,942 No. of unique reflections24,224 (1,811) Data completeness (%)90.1 (68.0) I/σ27.1 (8.33) Multiplicity85.7 (13.5) RmergeaRmerge = ΣhklΣi|Ii–〈I〉|/|〈I〉|, where Ii is the intensity for the ith measurement of an equivalent reflection with indices h, k, and l (%)6.1 (17.1)RefinementNon-hydrogen atoms Protein3,260 DNGIC50 MES24 Solvent (H2O)242Resolution (Å)2.1 (2.18-2.10)RconvbRconv = Σ||Fobs|–Fcalc||/Σ|Fobs|, where Fobs and Fcalc are the observed and calculated structure factor amplitudes, respectively (%)18.2 (20.7)RfreecRfree was calculated with 5% of the diffraction data that were selected randomly and not used throughout refinement (%)24.4 (28.4)Reflections used in Rconv calculations Number22,998 (1729) Completeness (%)85.8 (67.9)Root mean square deviation from ideal geometry Bonds (Å)0.005 Angles (°)1.2Mean B (protein) (Å2)29.2 Main chain27.7 Side chain30.8 Iron43.3 NO33.1 GSH31.1Mean B (solvent) (Å2)33.0Residues in most favored regions of Ramachandran plot (%)92.5Residues in allowed regions of Ramachandran plot (%)5.8Residues in generously allowed regions of Ramachandran plot (%)1.7Residues in disallowed regions of Ramachandran plot (%)0a Rmerge = ΣhklΣi|Ii–〈I〉|/|〈I〉|, where Ii is the intensity for the ith measurement of an equivalent reflection with indices h, k, and lb Rconv = Σ||Fobs|–Fcalc||/Σ|Fobs|, where Fobs and Fcalc are the observed and calculated structure factor amplitudes, respectivelyc Rfree was calculated with 5% of the diffraction data that were selected randomly and not used throughout refinement Open table in a new tab Expression Plasmids and Site-directed Mutagenesis—The plasmid pGST-1, producing large amounts of recombinant wild-type GST P1-1 in the cytoplasm of E. coli, has been described previously (25Battistoni A. Mazzetti A.P. Petruzzelli R. Muramatzu M. Ricci G. Federici G. Lo Bello M. Protein Expression Purif. 1995; 6: 579-587Crossref PubMed Scopus (41) Google Scholar). Site-directed mutagenesis of Tyr-7 into Phe, His, and Cys residues was accomplished using the same strategy adopted for the plasmid pGST-1 except that the synthetic linkers with NcoI-TaqI-compatible ends were obtained by annealing the following complementary oligonucleotides: 5′-CATGCCACCGTACACCGTTGTTTTCTTCCCGGTT and 5′-CGAACCGGGAAGAAAACAACGGTGTACGGTGG (Phe-7); 5′-CATGCCACCGTACACCGTTGTTCATTTCCCGGTT and 5′-CGAACCGGGAAATGAACAACGGTGTACGGTGG (His-7); 5′-CATGCCACCGTACACCGTTGTTTGCTTCCCGGTT and 5′-CGAACCGGGAAGCAAACAACGGTGTACGGTGG (Cys-7). Protein Expression and Purification—Native and mutant GST P1-1 enzymes were produced as described previously (25Battistoni A. Mazzetti A.P. Petruzzelli R. Muramatzu M. Ricci G. Federici G. Lo Bello M. Protein Expression Purif. 1995; 6: 579-587Crossref PubMed Scopus (41) Google Scholar, 35Lo Bello M. Battistoni A. Mazzetti A.P. Board P.G. Muramatsu M. Federici G. Ricci G. J. Biol. Chem. 1995; 270: 1249-1253Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Briefly, E. coli strain TOP 10 cells, harboring plasmid pGST-1 or plasmids expressing Phe-7, His-7, or Cys-7 mutant enzymes (pGST-F7, pGST-H7, pGST-C7), were grown in Luria broth containing 100 μg/ml ampicillin and 50 μg/ml streptomycin. The expression of GST was induced by the addition of 0.2 mm isopropyl-1-thio-β-galactopyranoside when the absorbance at 600 nm was 0.5. Eighteen hours after induction cells were harvested by centrifugation and lysed as previously described (25Battistoni A. Mazzetti A.P. Petruzzelli R. Muramatzu M. Ricci G. Federici G. Lo Bello M. Protein Expression Purif. 1995; 6: 579-587Crossref PubMed Scopus (41) Google Scholar). Native and GST mutant enzymes were purified by affinity chromatography on immobilized glutathione (36Simons P.C. Vander Jagt D.L. Anal. Biochem. 1977; 82: 334-341Crossref PubMed Scopus (440) Google Scholar). After affinity purification, the native and the mutant enzymes (Y7F, Y7H, and Y7C) were homogeneous as judged by SDS-PAGE (37Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). Protein concentration was determined by the method of Lowry et al. (38Lowry O.H. Rosebrough N.J. Farr A.L. Randall R. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Kinetic Studies—The enzymatic activities were determined spectrophotometrically at 25 °C with CDNB as cosubstrate following the product formation at 340 nm, ϵ = 9600 m-1 cm-1 (39Habig W.H. Jakoby W.B. Methods Enzymol. 1981; 77: 398-405Crossref PubMed Scopus (2098) Google Scholar). Spectrophotometric measurements were performed in a double beam Uvicon 940 spectrophotometer (Kontron Instruments) equipped with a thermostatted cuvette compartment. Initial rates were measured at 0.1-s intervals for a total period of 12 s after a lag time of 5 s. Enzymatic rates were corrected for the spontaneous reaction. Apparent kinetic parameters, kcat, KmCDNB were determined in 0.1 m potassium phosphate buffer, pH 6.5, and 0.1 mm EDTA, containing fixed concentrations of GSH (10 mm) and variable concentrations of CDNB (0.1-2 mm). The collected data were fitted to the Michaelis-Menten equation by non-linear regression analysis using the GraphPad Prism (GraphPad Software, San Diego, CA). The apparent KmGSH was also determined at a fixed CDNB concentration (1 mm) and variable GSH concentrations (from 0.02-10 mm). Kinetic parameters reported in this paper represent the mean of at least three different experimental data sets. Cell Growth and NO Treatment of Intact Cells—Single colonies of freshly plated E. coli strain TOP 10 harboring plasmid pGST-1 or pGST-F7 were used to inoculate 25 ml of overnight cultures. These cultures were diluted 1:100 into Luria-Bertani medium containing 100 μg/ml ampicillin and 50 μg/ml streptomycin sulfate, grown at 37 °C to an A600 value of 0.5, and induced by the addition of 0.5 mm isopropyl β-d-thiogalactoside. Cells (1 liter) were grown at 37 °C for 4 h, divided in 4 aliquots (0.25 liter each), and treated as follows. (a) two aliquots were incubated with either 2 mm GSNO or 50 μm FeSO4, one aliquot was incubated with 2 mm GSNO and 50 μm FeSO4, and the last aliquot was used as the control. All the aliquots were incubated under the same conditions, at 37 °C for 15 min. (b) Another experimental set was established to monitor the time course of DNDGIC complex formation in which two aliquots were incubated with either 2 mm GSNO alone or 2 mm GSNO plus 50 μm FeSO4 at different times (5, 15, 30, 60 min). At the end of the incubation cells of different aliquots were harvested by centrifugation for 15 min at 7000 rpm, washed with 10 mm phosphate buffer, pH 7.0, containing 0.1 mm EDTA, and after centrifugation resuspended in a suitable volume of 10 mm phosphate buffer, pH 7.0, for electron paramagnetic resonance (EPR) analysis. The same cells were also lysed by sonication, and cell membranes were removed by centrifugation at 14,000 rpm for 10 min, and the resulting supernatant was tested for GST activity assay, protein concentration, and further EPR analysis. A similar set of experiments was also carried out using 0.5 mm DEANO (final concentration) as the NO donor instead of GSNO. We used the concentration of 2 mm GSNO throughout this work, which is the same reported previously for the DNDGIC synthesis in vitro (9Lo Bello M. Nuccetelli M. Caccuri A.M. Stella L. Parker M.W. Rossjohn J. McKinstry W.J. Mozzi A. Federici G. Polizio F. Pedersen J.Z. Ricci G. J. Biol. Chem. 2001; 276: 42138-42145Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). However, we have also exposed E. coli cells to different concentrations of GSNO (in a range between 0.5 and 10 mm) and obtained quite similar results to those shown in Figs. 3 and 4. As an example, after 15 min of exposure to 0.5 mm GSNO or DEANO, the GST inactivation was about 20% (despite 35% with 2 mm GSNO), whereas the EPR signal was only slightly decreased in comparison with that shown in Fig. 3Ba (2 mm GSNO exposure). E. coli cell exposure to 10 mm GSNO (a sledge hammer) only reduces GST activity 40%. To check if 2 mm GSNO concentration could inhibit cell growth, we followed bacterial growth of cells exposed to 2 mm GSNO for 24 h at 600 nm, and we found no significant difference with E. coli untreated cells under the same conditions (data not shown).FIGURE 4GST specific activity in E. coli cells upon exposure to NO and/or iron before (black) and after cysteine (white) and cyanide (gray) treatment. The experimental values of specific activity, expressed inμmol of product/min, were normalized for those of the untreated cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT) EPR Detection of Dinitrosyl Diglutathionyl Iron Complex—EPR spectra were recorded using 80-μl samples in flat glass capillaries (inner cross-section 5 × 0.3 mm) to optimize instrument sensitivity as previously described (40Pedersen J.Z. Cox R.P. J. Magn. Reson. 1988; 77: 369-371Google Scholar). All measurements were made at room temperature with an ESP300 X-band instrument (Bruker, Karlsruhe, Germany) equipped with a high sensitivity TM110-mode cavity. Spectra were measured over a 200 G range using 20
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