Catalytic Mechanism of Thiol Peroxidase from Escherichia coli
2003; Elsevier BV; Volume: 278; Issue: 11 Linguagem: Inglês
10.1074/jbc.m209888200
ISSN1083-351X
AutoresLaura M.S. Baker, Leslie B. Poole,
Tópico(s)Cholinesterase and Neurodegenerative Diseases
ResumoEscherichia coli thiol peroxidase (Tpx, p20, scavengase) is part of an oxidative stress defense system that uses reducing equivalents from thioredoxin (Trx1) and thioredoxin reductase to reduce alkyl hydroperoxides. Tpx contains three Cys residues, Cys95, Cys82, and Cys61, and the latter residue aligns with the N-terminal active site Cys of other peroxidases in the peroxiredoxin family. To identify the catalytically important Cys, we have cloned and purified Tpx and four mutants (C61S, C82S, C95S, and C82S,C95S). In rapid reaction kinetic experiments measuring steady-state turnover, C61S is inactive, C95S retains partial activity, and the C82S mutation only slightly affects reaction rates. Furthermore, a sulfenic acid intermediate at Cys61 generated by cumene hydroperoxide (CHP) treatment was detected in UV-visible spectra of 4-nitrobenzo-2-oxa-1,3-diazole-labeled C82S,C95S, confirming the identity of Cys61 as the peroxidatic center. In stopped-flow kinetic studies, Tpx and Trx1 form a Michaelis complex during turnover with a catalytic efficiency of 3.0 × 106m−1 s−1, and the low K m (9.0 μm) of Tpx for CHP demonstrates substrate specificity toward alkyl hydroperoxides over H2O2 (K m > 1.7 mm). Rapid inactivation of Tpx due to Cys61overoxidation is observed during turnover with CHP and a lipid hydroperoxide, 15-hydroperoxyeicosatetraenoic acid, but not H2O2. Unlike most other 2-Cys peroxiredoxins, which operate by an intersubunit disulfide mechanism, Tpx contains a redox-active intrasubunit disulfide bond yet is homodimeric in solution. Escherichia coli thiol peroxidase (Tpx, p20, scavengase) is part of an oxidative stress defense system that uses reducing equivalents from thioredoxin (Trx1) and thioredoxin reductase to reduce alkyl hydroperoxides. Tpx contains three Cys residues, Cys95, Cys82, and Cys61, and the latter residue aligns with the N-terminal active site Cys of other peroxidases in the peroxiredoxin family. To identify the catalytically important Cys, we have cloned and purified Tpx and four mutants (C61S, C82S, C95S, and C82S,C95S). In rapid reaction kinetic experiments measuring steady-state turnover, C61S is inactive, C95S retains partial activity, and the C82S mutation only slightly affects reaction rates. Furthermore, a sulfenic acid intermediate at Cys61 generated by cumene hydroperoxide (CHP) treatment was detected in UV-visible spectra of 4-nitrobenzo-2-oxa-1,3-diazole-labeled C82S,C95S, confirming the identity of Cys61 as the peroxidatic center. In stopped-flow kinetic studies, Tpx and Trx1 form a Michaelis complex during turnover with a catalytic efficiency of 3.0 × 106m−1 s−1, and the low K m (9.0 μm) of Tpx for CHP demonstrates substrate specificity toward alkyl hydroperoxides over H2O2 (K m > 1.7 mm). Rapid inactivation of Tpx due to Cys61overoxidation is observed during turnover with CHP and a lipid hydroperoxide, 15-hydroperoxyeicosatetraenoic acid, but not H2O2. Unlike most other 2-Cys peroxiredoxins, which operate by an intersubunit disulfide mechanism, Tpx contains a redox-active intrasubunit disulfide bond yet is homodimeric in solution. Oxidative stress defenses combat reactive oxygen species (1Storz G. Tartaglia L.A. Farr S.B. Ames B.N. Trends Genet. 1990; 6: 363-368Google Scholar, 2Carmel-Harel O. Storz G. Annu. Rev. Microbiol. 2000; 54: 439-461Google Scholar) such as superoxide (O 2⨪), hydrogen peroxide (H2O2) and the hydroxyl radical (OH⋅) generated by the host immune response, environmental factors, and the incomplete reduction of oxygen to water during aerobic respiration, all of which are hazardous to proteins, DNA, and lipids (3Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. 3rd Ed. Oxford University Press, Oxford1999: 188-276Google Scholar, 4Storz G. Imlay J.A. Curr. Opin. Microbiol. 1999; 2: 188-194Google Scholar).Escherichia coli protects cellular components from oxidative damage by employing a variety of antioxidant defense enzymes, such as hydroperoxidases (catalases) I and II (gene products of katGand katE, respectively) that decompose H2O2 (5Lynch A.S. Lin E.C.C. Neidhardt F.C. E. coli and Salmonella: Cellular and Molecular Biology VI. 2nd Ed. American Society for Microbiology Press, Washington, D. C.1996: 1526-1538Google Scholar), and superoxide dismutases (manganese superoxide dismutase, sodA; iron superoxide dismutase,sodB; copper-zinc superoxide dismutase, sodC) (6Carlioz A. Touati D. EMBO J. 1986; 5: 623-630Google Scholar) that eliminate O 2⨪. Additional defenses in E. coliagainst alkyl and lipid hydroperoxides are provided by multiple, non-heme peroxidases including 1) the peroxidatic component from the alkyl hydroperoxide reductase system (AhpC) 1The abbreviations used are: AhpC, alkyl hydroperoxide reductase peroxidase component; AhpF, alkyl hydroperoxide reductase flavoprotein oxidoreductase component; Trx, thioredoxin; BCP, bacterioferritin-comigratory protein; Tpx, thiol peroxidase; Prx, peroxiredoxin; Cys-SOH, cysteine sulfenic acid; TrxR, thioredoxin reductase; DTT, dithiothreitol; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); CHP, cumene hydroperoxide; NBD chloride, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole; Cys-SO2H, cysteine sulfinic acid; Cys-SO3H, cysteine sulfonic acid; 15-HPETE, 15-hydroperoxyeicosatetraenoic acid; ESI-MS, electrospray ionization mass spectrometry; HPLC, high pressure liquid chromatography; MES, 2-(N-morpholino)-ethane sulfonic acid; TCEP, Tris[2- carboxyethyl]phosphine; Me2SO, dimethyl sulfoxide (7Costa Seaver L. Imlay J.A. J. Bacteriol. 2001; 183: 7173-7181Google Scholar); 2) a weakly active, thioredoxin (Trx)-dependent bacterioferritin-comigratory protein (BCP) (8Jeong W. Cha M.K. Kim I.H. J. Biol. Chem. 2000; 275: 2924-2930Google Scholar); and 3) the periplasmic thiol peroxidase (Tpx, p20, scavengase) (9Cha M.K. Kim H.K. Kim I.H. J. Biol. Chem. 1995; 270: 28635-28641Google Scholar). In addition, a glutathione peroxidase homologue, the gene product of btuE(10Rioux C.R. Kadner R.J. Mol. Gen. Genet. 1989; 217: 301-308Google Scholar), has been identified in E. coli, and preliminary investigations have indicated Trx-dependent peroxidatic activity against organic hydroperoxides (ROOH) and H2O2. 2L. M. S. Baker and L. B. Poole, unpublished observations. AhpC, BCP, and Tpx are all members of the ubiquitous peroxiredoxin (Prx) family within the Trx superfamily of protein folds; however, the three E. coli Prx members are highly diverged from one another and are representative of three distinct Prx subfamilies (11Poole L.B. Torres M. Fukuto J.M. Forman H.J. Signal Transduction by Reactive Oxygen and Nitrogen Species: Pathways and Chemical Principles. Kluwer Academic Publishers, Dordrecht, The Netherlands2003: 80-101Google Scholar, 12Jin D.-Y. Jeang K.-T. Sen C.K. Sies H. Bauerle P. Antioxidant and Redox Regulation of Genes. Academic Press, Inc., San Diego2000: 381-407Google Scholar, 13Hofmann B. Hecht H.J. Flohé L. Biol. Chem. 2002; 383: 347-364Google Scholar). Generally, the Prx active site contains a disulfide bond composed of a conserved N-terminal Cys (Cys46 fromSalmonella typhimurium AhpC) and the C-terminal Cys (Cys165′ from S. typhimurium AhpC) from the other subunit of the antiparallel dimer, resulting in two symmetrical active sites per dimer (14Ellis H.R. Poole L.B. Biochemistry. 1997; 36: 13349-13356Google Scholar). Most Prxs contain two conserved Cys (2-Cys Prxs); however, in some homologues, only the N-terminal Cys is retained (the 1-Cys Prxs) (15Wood Z.A. Schröder E. Harris J.R. Poole L.B. Trends Biochem. Sci. 2003; 28: 32-40Google Scholar). In many instances, the homodimers assemble into toroid-shaped decamers (15Wood Z.A. Schröder E. Harris J.R. Poole L.B. Trends Biochem. Sci. 2003; 28: 32-40Google Scholar), a redox-dependent process inS. typhimurium AhpC whereby reduced decamers disassociate into dimers upon oxidation (16Wood Z.A. Poole L.B. Hantgan R.R. Karplus P.A. Biochemistry. 2002; 41: 5493-5504Google Scholar). To detoxify peroxides, the reduced N-terminal Cys attacks the peroxide -O–O- bond, with concomitant formation of a Cys sulfenic acid (Cys-SOH) intermediate, which then condenses with the C-terminal Cys to regenerate the stable disulfide bond at the active site (14Ellis H.R. Poole L.B. Biochemistry. 1997; 36: 13349-13356Google Scholar, 17Ellis H.R. Poole L.B. Biochemistry. 1997; 36: 15013-15018Google Scholar). For most bacterial Prxs, disulfide reduction is achieved by a specialized electron donor, AhpF (18Poole L.B. Reynolds C.M. Wood Z.A. Karplus P.A. Ellis H.R. Li Calzi M. Eur. J. Biochem. 2000; 267: 6126-6133Google Scholar), whereas many other Prx systems (both bacterial and eukaryotic) receive electrons from a reducing system composed of Trx and Trx reductase (TrxR) (13Hofmann B. Hecht H.J. Flohé L. Biol. Chem. 2002; 383: 347-364Google Scholar, 15Wood Z.A. Schröder E. Harris J.R. Poole L.B. Trends Biochem. Sci. 2003; 28: 32-40Google Scholar). Homologues of tpx are distributed throughout most or all eubacterial species, both Gram-negative and Gram-positive, and are found in pathogenic strains such as Haemophilus influenzae,Streptococcus pneumoniae, and Helicobacter pylori(19Wan X.-Y. Zhou Y. Yan Z.-Y. Wang H.-L. Hou Y.-D. Jin D.-Y. FEBS Lett. 1997; 407: 32-36Google Scholar), but biochemical and genetic analyses have been limited primarily to E. coli Tpx. In response to oxidative stress, E. coli up-regulates Tpx expression through an oxygen-responsive promoter element that is repressed by the transcriptional regulators ArcA and Fnr under anaerobic conditions (9Cha M.K. Kim H.K. Kim I.H. J. Biol. Chem. 1995; 270: 28635-28641Google Scholar, 20Kim H.K. Kim S.J. Lee J.W. Cha M.K. Kim I.H. Biochem. Biophys. Res. Commun. 1996; 221: 641-646Google Scholar, 21Kim S.J. Han Y.H. Kim I.H. Kim H.K. IUBMB Life. 1999; 48: 215-218Google Scholar). In addition,tpx deletion mutants, while still viable, were more susceptible to oxidative stress and displayed diminished colony sizes and numbers after peroxide exposure (22Cha M.K. Kim H.K. Kim I.H. J. Bacteriol. 1996; 178: 5610-5614Google Scholar). In vitro studies have confirmed that Tpx forms a Trx-linked peroxidase system capable of reducing H2O2 and ROOH and protecting against glutamine synthetase inactivation by a mixed function oxidation system (9Cha M.K. Kim H.K. Kim I.H. J. Biol. Chem. 1995; 270: 28635-28641Google Scholar). Tpx contains three Cys residues in its primary sequence, Cys61, Cys82, and Cys95. Of these, Cys61 aligns with the peroxidatic, N-terminal Cys of other Prxs; whereas Cys95 does not align with the conserved C-terminal Cys of other 2-Cys Prxs, it is conserved among all Tpx homologues (19Wan X.-Y. Zhou Y. Yan Z.-Y. Wang H.-L. Hou Y.-D. Jin D.-Y. FEBS Lett. 1997; 407: 32-36Google Scholar). Previous mutagenesis studies have presented conflicting information about which Cys residues are involved in peroxide attack and have indicated that both Cys61 and Cys95 are essential for activity, whereas loss of Cys82 only slightly attenuates activity (22Cha M.K. Kim H.K. Kim I.H. J. Bacteriol. 1996; 178: 5610-5614Google Scholar, 23Zhou Y. Wan X.Y. Wang H.L. Yan Z.Y. Hou Y.D. Jin D.Y. Biochem. Biophys. Res. Commun. 1997; 233: 848-852Google Scholar). In this report, we identify Cys61 as the peroxidatic Cys forming a Cys-SOH intermediate and firmly establish its intrasubunit linkage to Cys95 in the redox-active disulfide. Although Tpx monomers are not covalently linked, analytical ultracentrifugation studies reported herein demonstrate that the enzyme is a homodimer in solution. SDS, ultrapure glycine, ultrapure urea, EDTA disodium salt, dithiothreitol (DTT), ammonium sulfate, β-mercaptoethanol, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), Tris base, and other buffer reagents were purchased from Research Organics (Cleveland, OH). Bacteriological media components were from Difco. Ethanol was obtained from Warner Graham Company (Cockeysville, MD). Isopropyl β-d-thiogalactopyranoside and X-gal (5-bromo-4-chloro-3-inolyl-β-d-galactopyranoside) were from Inalc (Milan, Italy). Vent DNA polymerase was purchased from New England Biolabs (Beverly, MA). Restriction enzymes, T4 DNA ligase, calf intestinal phosphatase, Taq DNA polymerase, MgCl2 solutions, and restriction buffers were obtained fromPromega (Madison, WI). Agarose medium EEO (electrophoresis grade), organic solvents (high pressure liquid chromatography (HPLC) grade), water (optima grade), sodium borate, acetic acid, sodium chloride, and H2O2 were from Fisher. Acrylamide/bis (40%) solution was purchased from Bio-Rad. Ampicillin powder, chloramphenicol, streptomycin sulfate, formic acid, calcium chloride, cumene hydroperoxide (CHP), dimethyl sulfoxide (Me2SO), protocatechuic acid, protocatechuate-3,4dioxygenase, lipoxygenase,Saccharomyces cerevisiae glutathione reductase, E. coli glutaredoxin 1, and insulin were from Sigma. Ethyl hydroperoxide was from Polysciences, Inc. (Warrington, PA). Glutathione, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD chloride), 4-vinylpyridine, and tert-butyl hydroperoxide were from Aldrich. NADPH and NADH were from Roche Molecular Biochemicals.l-1-(Tosylamino)-2-phenylethyl chloromethyl ketone-treated trypsin was obtained from Worthington. Pierce supplied the immobilized Tris[2-carboxyethyl]phosphine (TCEP) disulfide-reducing gel, Gel Code Blue stain, and trifluoroacetic acid ampules. Molecular Probes, Inc. (Eugene, OR) supplied the methyl methanethiolsulfonate and fluorescein-5-maleimide. Arachidonic acid was from NuCheck Prep (Elysian, MN). Stratagene (La Jolla, CA) supplied the PfuTurbo polymerase, DpnI, and E. coli XL-1 Blue competent cells. All ultrafiltration was carried out using Apollo 7-ml high performance centrifugal concentrators (Orbital Biosciences, Topsfield, MA) unless otherwise noted. Genomic DNA prepared from a 400-ml overnight culture of an E. coli K-12 strain (XL-1 Blue) was used as the PCR template (24Ausubel F.M. Brent R.B. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Short Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1999: 212-213Google Scholar). The gene of interest was amplified with the following PCR primers: forward primer, 5′-GCGAATTCAGGAGGAAGAATAGATGTCACAAACCGTACATTTCCAGGGC-3′ and reverse primer 5′-GCCTGCAGTTATGCTTTCAGTACAGCC-3′ (engineered restriction sites underlined) synthesized in the DNA Synthesis Core Laboratory of the Comprehensive Cancer Center of Wake Forest University. PCR mixtures (50 μl) contained 200 μm dNTPs, 2 units of Vent DNA polymerase, 20 pmol each of forward and reverse oligonucleotides, 1 mmMgCl2, and 0.5 μg of genomic DNA. The reaction was carried out in a Mini Cycler (MJ Research, Waltham, MA) as follows: 95 °C at 30 s, 55 °C for 45 s, and 72 °C for 1.5 min (35 cycles) and then 72 °C for 15 min. The 507-bp tpx PCR product purified with the QIAquick PCR Cleanup kit (Qiagen, Studio City, CA) was then ligated into the pCR2.1 TA cloning vector (Invitrogen) after Taq DNA polymerase/dNTP treatment. Plasmid DNA purified from the E. coli host using the Wizard Miniprep Kit (Promega) was screened for the presence of insert by digestion with the appropriate restriction enzymes (EcoRI and PstI). The DNA fragment containing tpx was excised from agarose gels and purified using the Gene Clean II Kit (Bio 101, Inc., Vista, CA). The fragment was ligated using T4 DNA ligase into the pPROK1 expression vector (Clontech, Palo Alto, CA; expression under control of the tac promoter) that had been similarly digested, isolated from an agarose gel, and pretreated with calf intestinal phosphatase to generate pPROK1/tpx. The four mutant E. coli Tpx enzymes, C61S, C82S, C95S, and C82S,C95S were created by following the protocol outlined in the QuikChange site-directed mutagenesis kit (Stratagene) using primers complementary to the coding and noncoding template sequence (pPROK1/tpx) containing a double-base mismatch. To generate the C61S mutation, the forward primer 5′-GATACCGGTGTTTCGGCCGCATCAGTACG-3′ and a complementary reverse primer were used (underlined letters indicate the base pair mismatch). To generate the C95S mutation, the forward primer 5′-CGCCCAGTCTCGTTTCTCGGGCGCAGAAGG-3′ and a complementary reverse primer were employed, and C82S was constructed using the forward primer 5′-CACCGTTGTGCTGTCTATCTCTGCCGATCTGCC-3′ and its complementary reverse primer. C82S,C95S was created using C95S plasmid as template with the forward and reverse primers for the C82S mutation. Reaction mixtures (50 μl) contained 10–50 ng of template DNA (pPROK1/tpx or pPROK1/tpxC95S), 125 ng of each primer, 200 μm dNTPs, and 1 μl of PfuTurbo polymerase. Twelve cycles of 95 °C for 30 s and 55 °C for 13 min were carried out in a Mini Cycler followed by 55 °C for 10 min to finish extending products. To digest methylated template, each reaction mixture was treated with 1 μl of DpnI at 37 °C for 1 h. Ligated DNA (pPROK1/tpx) and mutagenesis products were transformed into XL-1 Blue cells. Single colonies were selected on Luria-Bertani (LB) plates containing ampicillin (50 μg/ml), and those containing the recombinant DNA were evaluated for protein expression by SDS-PAGE after induction with 0.4 mm isopropyl β-d-thiogalactopyranoside. Isolated plasmid DNA for each construct was sequenced throughout the coding region by automated DNA sequencing at the Comprehensive Cancer Center of Wake Forest University. Bacterial stocks containing each plasmid with the subcloned gene were prepared from a single colony and stored at −80 °C in LB broth containing 15% (v/v) glycerol. Culture procedures were generally the same as reported earlier (25Poole L.B. Ellis H.R. Biochemistry. 1996; 35: 56-64Google Scholar). A modification of a previous Tpx purification protocol was used for this study (9Cha M.K. Kim H.K. Kim I.H. J. Biol. Chem. 1995; 270: 28635-28641Google Scholar). All procedures were carried out in a standard buffer (pH 7.0) consisting of 25 mm potassium phosphate with 1.0 mm EDTA. Briefly, 100 ml of XL-1 Blue E. coliharboring the pPROK1/tpx plasmid were added to 10 liters of LB medium containing 0.5 g of ampicillin supplemented with 0.2% glucose in a BioFlo 2000 fermentor (New Brunswick Scientific, Edison, NJ). Isopropyl β-d-thiogalactopyranoside (0.4 mm) was added at A 600 = 0.9, and bacteria were harvested by centrifugation 16 h after induction. Pelleted bacteria were disrupted with a Bead Beater (BioSpec Products, Bartlesville, OK), and cell extracts treated with streptomycin sulfate to precipitate nucleic acids were subjected to 30 and 75% (NH4)2SO4 treatments to precipitate proteins (25Poole L.B. Ellis H.R. Biochemistry. 1996; 35: 56-64Google Scholar). The protein mixture resuspended in standard buffer containing 10% (NH4)2SO4 was applied to a 24 × 2.5-cm Phenyl-Sepharose 6 Fast Flow Column (Amersham Biosciences), washed with 10% (NH4)2SO4 buffer, and eluted with deionized H2O. Protein fractions were evaluated for contamination of overexpressed Tpx by SDS-PAGE, and the purest fractions were pooled. After dialysis against 5 mmpotassium phosphate buffer (pH 7.0), the protein was loaded onto a Q-Sepharose column (Amersham Biosciences) pre-equilibrated in 5 mm potassium phosphate and eluted with a linear gradient from 5 to 30 mm potassium phosphate (1 liter total volume). Again, fractions were analyzed for Tpx by SDS-PAGE, and the pure fractions were pooled, concentrated, and aliquotted for storage at −20 °C. Purifications of mutant Tpx proteins were carried out in the same manner as for wild type Tpx with a few exceptions. The pooled C61S mutant Tpx protein from the Phenyl-Sepharose column was loaded onto a 24 × 2.5-cm DEAE-cellulose column (DE52; Whatman, Kent, UK) equilibrated with 30 mm potassium phosphate, and eluted with a linear gradient from 30 to 80 mm potassium phosphate (1 liter total volume). To completely purify C61S, fractions containing the mutant protein were reapplied to the Phenyl-Sepharose and DEAE-cellulose columns. Initial attempts at purifying the C95S mutant under the same conditions as wild type resulted in aggregation of the mutant protein, as observed by multiple bands during SDS-PAGE of the subsequent fractions, even after many rounds of purification over the two columns. DTNB titration of the isolated protein even after DTT treatment gave less than one thiol (data not shown), suggesting that Cys61 had become irreversibly overoxidized to a sulfinic (Cys-SO2H) or sulfonic (Cys-SO3H) acid. The addition of 2 mm DTT to all buffers prior to bacterial disruption and over the course of the purification of C95S gave pure protein after one round of purification on the two columns. C82S was purified in buffers containing 2 mm DTT according to the same protocol as C95S; however, pure C82S,C95S was obtained using a slightly altered protocol. 20% instead of 10% (NH4)2SO4 was used to treat C82S,C95S prior to its application to a Phenyl-Sepharose column equilibrated with 20% (NH4)2SO4, and C82S,C95S was eluted using a gradient of 20% (NH4)2SO4 in 25 mmpotassium phosphate with 2 mm DTT to 0% (NH4)2SO4 in deionized water with 2 mm DTT. Elution from the Q Sepharose column was achieved with a gradient of 20–120 mm potassium phosphate in 2 mm DTT. Prior to assay, each mutant was subjected to ultrafiltration and washed with standard buffer to remove DTT and then immediately incubated with equal volumes of TCEP gel. Purifications of E. coli TrxR (26Poole L.B. Godzik A. Nayeem A. Schmitt J.D. Biochemistry. 2000; 39: 6602-6615Google Scholar) and E. coli Trx1 (27Reynolds C.M. Poole L.B. Biochemistry. 2000; 39: 8859-8869Google Scholar) were carried out as described previously. S. typhimurium AhpF and S. typhimurium AhpC were purified as reported previously (25Poole L.B. Ellis H.R. Biochemistry. 1996; 35: 56-64Google Scholar). Wild type, C61S, C82S, C95S, and C81S,C95S Tpx (100 μg each), prereduced with DTT that was removed by ultrafiltration, were reacted with fluorescein- 5-maleimide (10 equivalents) under denaturing (4 m urea) and reducing (100 eq of TCEP) conditions for 16 h at 4 °C in standard buffer. Protein samples (5 μg) were separated on 18%, 20-cm-long SDS-polyacrylamide gels. Densitometry to assess the protein contents of fluorescein-labeled bands was conducted using the Quantity One quantitation software from Bio-Rad, and image files were generated with a ChemiImager 5500 digital imaging system from Alpha Innotech Corp. (San Leandro, CA) and a near UV light source and UV filter. For quantitation, six amounts of wild type Tpx (0.03–5 μg) were electrophoresed along with each of the mutant samples. Electrophoresis samples also included one with 5 μg of C61S, to which 0.25 μg of wild type Tpx was intentionally added, and one into which 1.7 μg each of wild type Tpx and C61S and C82S,C95S mutants were loaded to assess the quality of the separation. The labeled proteins were also submitted for electrospray mass spectrometry, as described below, following ultrafiltration into water to remove buffer using Apollo concentrators (Orbital Biosciences). To determine the oligomeric state of wild type and mutant proteins, samples of different concentrations were analyzed by sedimentation equilibrium at various speeds on an Optima XL-A analytical ultracentrifuge (Beckman Instruments, Palo Alto, CA) outfitted with absorbance optics. Tpx, C61S, and C95S (purified in DTT) were exchanged into a buffer of 25 mm potassium phosphate, 1 mm EDTA, and 0.15m NaCl, pH 7.05, via ultrafiltration. Three different concentrations of each protein (39.5, 184.2, and 342.1 μm) in 110 μl were loaded into three of the six sectors of each cell, and buffer (125 μl) was loaded into the remaining sectors as a reference. Data were obtained at 11,000, 16,000, and 20,000 rpm at 20 °C following equilibration for 8, 10, and 12 h at each speed. Data with absorbances higher than 1.4 were excluded from data analysis. The partial specific volumes for Tpx and the mutants were calculated from the amino acid composition to be 0.7433 cm3 g−1 and 0.7434 cm3g−1, respectively (28Laue T.M. Shah B.D. Ridgeway T.M. Pelletier S.L. Harding S.E. Rowe A.J. Horton C.J. Analytical Ultracentrifugation in Biochemistry and Polymer Science. The Royal Society of Chemistry, Cambridge, UK1992: 90-125Google Scholar). Multiple data sets were globally fit to the equation for a single ideal species using WinNonLin2 (29Johnson M.L. Correia J.J. Yphantis D.A. Halvorson H.R. Biophys. J. 1981; 36: 575-588Google Scholar). The buffer density of 1.00773 g/cm3 was determined using a DA-310 M precision density meter (Mettler Toledo, Hightstown, NJ) at 20 °C. Protein samples were extensively dialyzed in deionized water (6 liters) in a Slide-A-Lyzer cassette (Pierce) overnight prior to analysis by electrospray ionization mass spectrometry (ESI-MS; Micromass, Manchester, UK) precalibrated with horse heart myoglobin. The protein sample (1 μm) in 1% formic acid was injected at a flow rate of 300 μl/h, and positively charged ions in the m/zrange of 800–1800 were analyzed using MassLynx software (version 3.5; Micromass). Most spectral assays were conducted on a single wavelength, thermostatted (25 °C) Gilford 220 updated recording spectrophotometer (Oberlin, OH) with a Beckman DU monochrometer (Fullerton, CA) unless otherwise noted. Microbiuret assays for proteins to determine extinction coefficients, disulfide assays with 2-nitro-5-thiosulfobenzoate, and thiol quantification with DTNB were conducted as described previously (25Poole L.B. Ellis H.R. Biochemistry. 1996; 35: 56-64Google Scholar, 30Poole L.B. Biochemistry. 1996; 35: 65-75Google Scholar). To further quantify Tpx proteins by absorbance, the following experimentally determined extinction coefficients at 280 nm were used: Tpx and C82S,C95S, 3800 ± 300 m−1cm−1; C95S, 3500 ± 200 m−1cm−1; C82S, 4200 ± 600 m−1cm−1; C61S, 5300 ± 700 m−1cm−1. Other extinction coefficients used were as follows:E. coli TrxR, 11,300 m−1cm−1 (454 nm) (31Williams Jr., C.H. Zanetti G. Arscott L.D. McAllister J.K. J. Biol. Chem. 1967; 242: 5226-5231Google Scholar); E. coli Trx1, 13,700m−1 cm−1 (280 nm) (32Holmgren A. Reichard P. Eur. J. Biochem. 1967; 2: 187-196Google Scholar); S. typhimurium AhpC, 24,300 m−1cm−1 (280 nm) (25Poole L.B. Ellis H.R. Biochemistry. 1996; 35: 56-64Google Scholar); S. typhimurium AhpF, 13,100m−1 cm−1 (450 nm) (25Poole L.B. Ellis H.R. Biochemistry. 1996; 35: 56-64Google Scholar); E. coli glutaredoxin 1, 12,400 m−1cm−1 (280 nm) (33Holmgren A. Methods Enzymol. 1985; 113: 525-540Google Scholar); NADPH, 6200m−1 cm−1 (340 nm); NADH, 6220m−1 cm−1 (340 nm); 2-nitro-5-thiobenzoate (TNB2−), 14,150m−1 cm−1 (412 nm) (34Riddles P.W. Blakeley R.L. Zerner B. Anal. Biochem. 1979; 94: 75-81Google Scholar). Michaelis constants (K m) and turnover numbers (k cat) with CHP as the substrate for Tpx were obtained as described previously for H. pylori TrxR (35Baker L.M. Raudonikiene A. Hoffman P.S. Poole L.B. J. Bacteriol. 2001; 183: 1961-1973Google Scholar). Briefly, reactions were conducted on an Applied Photophysics DX-17 MV stopped-flow spectrophotometer (Surrey, UK) at 25 °C, and activity was followed by monitoring the decrease in fluorescence or absorbance of NADPH over time. Use of the stopped-flow spectrophotometer for mixing and data acquisition helped alleviate nonlinearity and reproducibility problems inherent in the manual mixing methods (14Ellis H.R. Poole L.B. Biochemistry. 1997; 36: 13349-13356Google Scholar). Reaction mixtures in one syringe contained NADPH (150 μm), Trx1 (2–80 μm), TrxR (0.25–10 μm, in a molar ratio of 1:8 TrxR to Trx1, so that the electron transfer to Trx1 is not rate-limiting), and Tpx (0.2 μm) in peroxidase buffer consisting of 50 mmpotassium phosphate (pH 7.0), 0.1 m(NH4)2SO4, and 0.5 mmEDTA. CHP (1–50 μm) was prediluted into Me2SO (in a ratio of 1:50 with the solvent) and mixed with peroxidase buffer in the other syringe. In all cases, concentrations given are final concentrations achieved after mixing of the contents of the two different syringes. Under the conditions given, Tpx was supplied in rate-limiting concentrations, and due to the saturable interaction between Tpx and Trx1, the reaction was linearly dependent on Trx1, provided that concentrations supplied were below theK m. Assays were designed so that Trx1 levels were at least 10-fold higher than those of Tpx to maintain the peroxidase as the limiting enzyme. Flavin-dependent oxidase activity did not contribute significantly to NADPH oxidation (0.05 s−1) and was not subtracted from initial rates. After conversion to concentration units/min, the primary rate data were fit to a rectangular hyperbolic curve function in Sigma Plot (Jandel Scientific, San Rafael, CA). The data obtained for each substrate concentration were plotted according to the Hanes-Woolf representation of the Michaelis-Menten equation to give intersecting lines at they axis, indicating a substituted (ping-pong) mechanism and were further evaluated using the Hanes-Woolf equation for a two-substrate, substituted reaction as previously described (35Baker L.M. Raudonikiene A. Hoffman P.S. Poole L.B. J. Bacteriol. 2001; 183: 1961-1973Google Scholar). Because of the much larger K m of Tpx for H2O2, larger amounts of substrates were present in the reaction mixtures, and the sensitivity required previously in the fluorescence assay was no longer necessary. Therefore, all H2O2 data were collected in absorbance mode on the stopped flow spectrophotometer with the same protein assay mixtures as above, except that the second syringe contained varying H2O2 concentrations (0.25–10 mm). Synthesis of 15-hydroperoxyeicosatetraenoic acid (15-HPETE) was carried out as described by O'Flaherty et al. (36O'Flaherty J.T. Wykle R.L. Thomas M.J. McCall C.E. Res. Commun. Chem. Pathol. Pharmacol. 1984; 43: 3-23Google Scholar). To observe the formation of Cys-SOH as a reaction intermediate, experiments with
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