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• W2013752604 abstract "We have investigated the reaction of peptidylglycine monooxygenase with hydrogen peroxide to determine whether Cu(II)-peroxo is a likely intermediate. When the oxidized enzyme was reacted with the dansyl-YVG substrate and H2O2, the α-hydroxyglycine product was formed. The reaction was catalytic and did not require the presence of additional reductant. When 18O-labeled H2O2 was reacted with peptidylglycine monooxygenase and substrate anaerobically, oxygen in the product was labeled with 18O and must therefore be derived from H2O2. However, when the reaction was carried out with H 162O2 in the presence of 18O2, 60% of the product contained the 18O label. Therefore, the reaction must proceed via an intermediate that can react directly with dioxygen and thus scramble the label. Under strictly anaerobic conditions (in the presence of glucose and glucose oxidase, where no oxygen was released into the medium from nonenzymatic peroxide decomposition), product formation and peroxide consumption were tightly coupled, and the rate of product formation was identical to that measured under aerobic conditions. Peroxide reactivity was eliminated by a mutation at the CuH center, which should not be involved in the peroxide shunt. Our data lend support to recent proposals that Cu(II)-superoxide is the active species. We have investigated the reaction of peptidylglycine monooxygenase with hydrogen peroxide to determine whether Cu(II)-peroxo is a likely intermediate. When the oxidized enzyme was reacted with the dansyl-YVG substrate and H2O2, the α-hydroxyglycine product was formed. The reaction was catalytic and did not require the presence of additional reductant. When 18O-labeled H2O2 was reacted with peptidylglycine monooxygenase and substrate anaerobically, oxygen in the product was labeled with 18O and must therefore be derived from H2O2. However, when the reaction was carried out with H 162O2 in the presence of 18O2, 60% of the product contained the 18O label. Therefore, the reaction must proceed via an intermediate that can react directly with dioxygen and thus scramble the label. Under strictly anaerobic conditions (in the presence of glucose and glucose oxidase, where no oxygen was released into the medium from nonenzymatic peroxide decomposition), product formation and peroxide consumption were tightly coupled, and the rate of product formation was identical to that measured under aerobic conditions. Peroxide reactivity was eliminated by a mutation at the CuH center, which should not be involved in the peroxide shunt. Our data lend support to recent proposals that Cu(II)-superoxide is the active species. Peptidylglycine monooxygenase (PHM) 2The abbreviations used are: PHM, peptidylglycine monooxygenase; ET, electron transfer; PHMcc, PHM catalytic core; GO, glucose oxidase; HPLC, high pressure liquid chromatography; MES, 4-morpholineethanesulfonic acid.2The abbreviations used are: PHM, peptidylglycine monooxygenase; ET, electron transfer; PHMcc, PHM catalytic core; GO, glucose oxidase; HPLC, high pressure liquid chromatography; MES, 4-morpholineethanesulfonic acid. catalyzes the hydroxylation of peptidylglycine substrates at the Cα position, the first step in the amidation of peptides by the bifunctional enzyme peptidylglycine α-amidating monooxygenase (1.Klinman J.P. Chem. Rev. 1996; 1996: 2541-2561Crossref Scopus (800) Google Scholar, 2.Prigge S.T. Mains R.E. Eipper B.A. Amzel L.M. Cell. Mol. Life Sci. 2000; 57: 1236-1259Crossref PubMed Scopus (373) Google Scholar). The enzyme requires two coppers for activity (3.Kulathila R. Consalvo A.P. Fitzpatrick P.F. Freeman J.C. Snyder L.M. Villafranca J.J. Merkler D.J. Arch. Biochem. Biophys. 1994; 311: 191-195Crossref PubMed Scopus (56) Google Scholar) and undergoes redox cycling during catalysis via the intermediacy of both dicopper(II) and dicopper(I) forms (4.Freeman J.C. Villafranca J.J. Merkler D.J. J. Am. Chem. Soc. 1993; 115: 4923-4924Crossref Scopus (49) Google Scholar). Structural (5.Prigge S.T. Kolhekar A.S. Eipper B.A. Mains R.E. Amzel L.M. Science. 1997; 278: 1300-1305Crossref PubMed Scopus (304) Google Scholar, 6.Prigge S.T. Kolhekar A.S. Eipper B.A. Mains R.E. Amzel L.M. Nat. Struct. Biol. 1999; 6: 976-983Crossref PubMed Scopus (159) Google Scholar, 7.Prigge S.T. Eipper B.A. Mains R.E. Amzel M. Science. 2004; 304: 864-867Crossref PubMed Scopus (358) Google Scholar), spectroscopic (8.Eipper B.A. Quon A.S.W. Mains R.E. Boswell J.S. Blackburn N.J. Biochemistry. 1995; 34: 2857-2865Crossref PubMed Scopus (102) Google Scholar, 9.Boswell J.S. Reedy B.J. Kulathila R. Merkler D.J. Blackburn N.J. Biochemistry. 1996; 35: 12241-12250Crossref PubMed Scopus (92) Google Scholar, 10.Jaron S. Blackburn N.J. Biochemistry. 1999; 38: 15086-15096Crossref PubMed Scopus (70) Google Scholar, 11.Blackburn N.J. Rhames F.C. Ralle M. Jaron S. J. Biol. Inorg. Chem. 2000; 5: 341-353Crossref PubMed Scopus (98) Google Scholar, 12.Chen P. Bell J. Eipper B.A. Solomon E.I. Biochemistry. 2004; 43: 5735-5747Crossref PubMed Scopus (77) Google Scholar), and theoretical (13.Chen P. Solomon E.I. J. Am. Chem. Soc. 2004; 126: 4991-5000Crossref PubMed Scopus (250) Google Scholar, 14.Chen P. Solomon E.I. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13105-13110Crossref PubMed Scopus (144) Google Scholar) studies have provided a detailed description of the ligand environment of the copper centers, which are bound in separate domains, about 11 Å apart (Fig. 1). One copper (CuH, also termed CuA) is bound to three histidines (His107, His108, and His172) in domain 1. The other copper (CuM, also termed CuB) is bound to two histidines (His242 and His244) and a methionine (Met314) in domain 2. X-ray absorption spectroscopy studies have identified large changes in coordination between Cu(II) and Cu(I) states (11.Blackburn N.J. Rhames F.C. Ralle M. Jaron S. J. Biol. Inorg. Chem. 2000; 5: 341-353Crossref PubMed Scopus (98) Google Scholar). In the oxidized enzyme, CuH is further ligated by at least one solvent molecule, whereas CuM coordinates two histidines and two water molecules in the equatorial plane with the methionine in an axial position undetectable by EXAFS (11.Blackburn N.J. Rhames F.C. Ralle M. Jaron S. J. Biol. Inorg. Chem. 2000; 5: 341-353Crossref PubMed Scopus (98) Google Scholar, 12.Chen P. Bell J. Eipper B.A. Solomon E.I. Biochemistry. 2004; 43: 5735-5747Crossref PubMed Scopus (77) Google Scholar). Reduction causes the water ligands to dissociate and the methionine to move close (2.25 Å) to the CuM center (9.Boswell J.S. Reedy B.J. Kulathila R. Merkler D.J. Blackburn N.J. Biochemistry. 1996; 35: 12241-12250Crossref PubMed Scopus (92) Google Scholar, 11.Blackburn N.J. Rhames F.C. Ralle M. Jaron S. J. Biol. Inorg. Chem. 2000; 5: 341-353Crossref PubMed Scopus (98) Google Scholar). In contrast, crystallographic studies have failed to detect large changes in metal coordination during redox (6.Prigge S.T. Kolhekar A.S. Eipper B.A. Mains R.E. Amzel L.M. Nat. Struct. Biol. 1999; 6: 976-983Crossref PubMed Scopus (159) Google Scholar). The detailed mechanism of substrate hydroxylation has been the subject of much debate. It is generally accepted that the enzyme cycles through a reductive phase in which the two copper centers are reduced to Cu(I) and an oxidative phase in which O2 is activated by binding at one of the copper centers and subsequently hydroxylates the substrate. How this chemistry occurs is still unclear. Crystal structures of substrate-bound forms of PHM have located a di-iodo-YG substrate bound in the vicinity of the CuM center (2.Prigge S.T. Mains R.E. Eipper B.A. Amzel L.M. Cell. Mol. Life Sci. 2000; 57: 1236-1259Crossref PubMed Scopus (373) Google Scholar, 5.Prigge S.T. Kolhekar A.S. Eipper B.A. Mains R.E. Amzel L.M. Science. 1997; 278: 1300-1305Crossref PubMed Scopus (304) Google Scholar, 6.Prigge S.T. Kolhekar A.S. Eipper B.A. Mains R.E. Amzel L.M. Nat. Struct. Biol. 1999; 6: 976-983Crossref PubMed Scopus (159) Google Scholar) (Fig. 1A), whereas a precatalytic complex of PHM with the slow substrate tyrosyl-d-threonine and dioxygen shows O2 bound at CuM but rotated away from the Cu-Cα(substrate) vector (7.Prigge S.T. Eipper B.A. Mains R.E. Amzel M. Science. 2004; 304: 864-867Crossref PubMed Scopus (358) Google Scholar). These structures strongly support the premise that oxygen activation occurs at the CuM center but provide no information on the chemical identity of the reactive species. If the reactive oxygen species is a CuM-peroxo or hydroperoxo complex, an electron must be transferred from CuH to CuM to complete the 2-electron reduction of O2 to peroxide, but this itself presents a mechanistic challenge, since the two coppers are separated across an 11-Å solvent-filled cavity, and the shortest through-bond pathway is >80 Å. To overcome this problem, Prigge et al. (6.Prigge S.T. Kolhekar A.S. Eipper B.A. Mains R.E. Amzel L.M. Nat. Struct. Biol. 1999; 6: 976-983Crossref PubMed Scopus (159) Google Scholar) identified a potential electron transfer (ET) pathway involving the CuH ligand His108, Gln170, a hydrogen-bonded water molecule, and the peptide substrate, which reduced the ET pathway to ∼20 Å. Invoking a different strategy, Jaron and Blackburn (10.Jaron S. Blackburn N.J. Biochemistry. 1999; 38: 15086-15096Crossref PubMed Scopus (70) Google Scholar) suggested that O2 might react initially at CuH and that the superoxide so formed could channel to CuM providing a carrier for the electron and possibly a proton. Neither of these mechanisms is consistent with all of the available data. Glutamine 170, a critical residue in the substrate-mediated ET pathway, can be mutated to alanine with no loss of catalytic activity (15.Bell J. El Meskini R. D'Amato D. Mains R.E. Eipper B.A. Biochemistry. 2003; 42: 7133-7142Crossref PubMed Scopus (38) Google Scholar), whereas in the related enzyme dopamine β-monooxygenase, oxygen reduction and substrate hydroxylation remain tightly coupled even in the case of extremely slow substrates, apparently ruling out superoxide channeling, where some leakage of superoxide into bulk solution would be expected (16.Evans J.P. Ahn K. Klinman J.P. J. Biol. Chem. 2003; 278: 49691-49698Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). These results led Klinman and co-workers (16.Evans J.P. Ahn K. Klinman J.P. J. Biol. Chem. 2003; 278: 49691-49698Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 17.Francisco W.A. Knapp M.J. Blackburn N.J. Klinman J.P. J. Am. Chem. Soc. 2002; 124: 8194-8195Crossref PubMed Scopus (120) Google Scholar, 18.Francisco W.A. Wille G. Smith A.J. Merkler D.J. Klinman J.P. J. Am. Chem. Soc. 2004; 126: 13168-13169Crossref PubMed Scopus (57) Google Scholar) to argue against peroxide as a viable intermediate in both PHM and dopamine β-monooxygenase and to propose that the reactive oxygen species is a Cu(II)-superoxo species, which abstracts a hydrogen atom from substrate prior to the electron transfer step. IfaCuM(II)-peroxo or hydroperoxo species is an intermediate, then it should be possible to generate product by reacting the oxidized enzyme with hydrogen peroxide and substrate as depicted schematically in the oxidative phase of the mechanism shown in Fig. 1B. This “peroxide shunt” has been shown to occur in other oxygenase systems such as cytochrome P450 (19.Hrycay E.G. Gustafsson J.A. Ingelman-Sundberg M. Ernster L. Eur. J. Biochem. 1976; 61: 43-52Crossref PubMed Scopus (89) Google Scholar, 20.Davydov R. Makris T.M. Kofman V. Werst D.E. Sligar S.G. Hoffman B.M. J. Am. Chem. Soc. 2001; 123: 1403-1415Crossref PubMed Scopus (404) Google Scholar), methane monooxygenase (21.Froland W.A. Andersson K.K. Lee S.K. Liu Y. Lipscomb J.D. J. Biol. Chem. 1992; 267: 17588-17597Abstract Full Text PDF PubMed Google Scholar), and naphthalene 1,2-dioxygenase (22.Wolfe M.D. Lipscomb J.D. J. Biol. Chem. 2003; 278: 829-835Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Peroxide shunt reactions have the characteristic that when labeled peroxide is used as the source of the hydroxylating oxygen atom, the label is quantitatively transferred to the products (22.Wolfe M.D. Lipscomb J.D. J. Biol. Chem. 2003; 278: 829-835Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). In this paper, we have investigated the reactivity of hydrogen peroxide with the oxidized form of the PHM catalytic core (residues 42–356, PHMcc) and find that hydroxylated product is indeed formed in the reaction. However, when 18O-labeled peroxide was used as the source of oxygen, we observed scrambling of the label with atmospheric O2. We also observed that peroxide reactivity is eliminated by a mutation at the CuH center, which should not be involved in the peroxide shunt chemistry. The results have led to the conclusion that Cu(II)-peroxo or -hydroperoxo species are probably not involved in the reaction pathway, and an alternative mechanism involving Cu(II)-superoxo species appears more likely. Chemicals and Reagents—Unless otherwise stated, chemicals and buffers were obtained from Sigma or Fisher and were used without further purification. 18O-Labeled hydrogen peroxide (2% (v/v) solution) and oxygen gas were obtained from Icon Isotopes, New Jersey, at 90 and 99 atom %, respectively. Expression and Purification of PHMcc and Its H242A and H172A Mutants—PHMcc and the H242A and H172A mutants were expressed and purified as described previously (10.Jaron S. Blackburn N.J. Biochemistry. 1999; 38: 15086-15096Crossref PubMed Scopus (70) Google Scholar, 11.Blackburn N.J. Rhames F.C. Ralle M. Jaron S. J. Biol. Inorg. Chem. 2000; 5: 341-353Crossref PubMed Scopus (98) Google Scholar, 23.Jaron S. Mains R.E. Eipper B.A. Blackburn N.J. Biochemistry. 2002; 41: 13274-13282Crossref PubMed Scopus (27) Google Scholar) using a Cellmax 100 (Spectrum Laboratories) hollow fiber bioreactor with a 1.1-m2 cellulose acetate cartridge. In some experiments, wild type PHMcc was expressed using a Biovest Minimax automated cell culture system (Biovest International). Typically, 5 days of bioreactor harvest were pooled. Ammonium sulfate was added to 50% saturation, and the solution was stirred for 1 h. The resulting precipitate was centrifuged at 12,000 × g and redissolved, with gentle shaking, in 10 ml of 50 mm sodium phosphate buffer (pH 7.5), containing 0.001% Triton X-100. The sample was then centrifuged to remove insoluble material, filtered through a 0.45-μm sterile filter, and applied to a 26/60 Hiload Superdex 75 prep grade filtration column (Amersham Biosciences) at a flow rate of 2.5 ml/min. The enzyme began elution at 0.6 column volumes and continued to elute over 45 ml. At this stage of purification, SDS-PAGE revealed a purity level of 90–95%. The enzyme was further purified by anion exchange chromatography on a Biocad 700E perfusion chromatography system (Applied Biosystems), using a 10 × 100-ml Peek column packed with Poros 20-μm HQ anion exchange resin. Partially purified PHM from size exclusion chromatography was loaded in 20 mm Tris acetate buffer, pH 8.2, and then washed with 2 column volumes of the buffer. The column was eluted by a 0–300 mm NaCl gradient in loading buffer, over 10 column volumes. Purified PHM eluted close to 100 mm NaCl. Yields of pure PHMcc for 5 days of harvest ranged from 30 to 50 mg. Copper Reconstitution—The purified protein in 20 mm sodium phosphate, pH 8.0, was placed in a 50-ml conical centrifuge tube to which 100 mm copper sulfate was added at 1 μl/min on ice with gentle stirring until the molar ratio of copper added per protein was 2.5:1. The protein was then concentrated in an Amicon ultrafiltration device (10,000 Da cut-off) from 30 to 1 ml. This was followed by three wash sequences to remove excess and/or adventitiously bound copper. During each wash, 10 ml of 20 mm sodium phosphate buffer, pH 8, containing 25 μm Cu(II) (as Cu(SO4)) was added to the ultrafiltration device, and the solution was concentrated to 1 ml. A final concentration step adjusted the pure PHMcc to 1 mm. The protein was then flash frozen in cryotubes and stored in aliquots at –80 °C. The final Cu/protein ratio was in the region of 2.0–2.2:1. Copper and Protein Concentration—Protein concentration was determined using the A280 and an extinction coefficient (1 mg/ml) of 0.98 as previously described (10.Jaron S. Blackburn N.J. Biochemistry. 1999; 38: 15086-15096Crossref PubMed Scopus (70) Google Scholar). A280 measurements were recorded on a Shimadzu UV-265 spectrophotometer at ambient temperature. Copper concentrations were determined using a PerkinElmer Optima 2000 DV inductively coupled plasma optical emission spectrometer. HPLC Separation and Detection of Product and Substrate—Reverse-phase HPLC was performed with a Varian Pro Star solvent delivery module equipped with a Varian Pro Star model 410 autosampler (250-μl syringe, 100-μl sample loop), on a 250 × 4.6-mm Varian Microsorb-MV 100-5 C18 column. Substrate (dansyl-YVG; American Peptide Co.) and product (produced by the PHM-catalyzed reaction) were monitored by their dansyl fluorescence using a Waters 474 scanning fluorescence detector (λEx = 365 nm, λEm = 558 nm). Solvent A was 0.1% trifluoroacetic acid in water, and solvent B was 0.1% trifluoroacetic acid in acetonitrile). Product was separated from substrate via isocratic loading and elution at 25% B in A. Steady State Kinetic Measurements—The kinetics of the peroxide reaction were determined by following the rate of substrate consumption (or product formation) as a function of time. The reaction was performed in a water-jacketed glass reaction vessel, with stirring, in 100 mm MES buffer, pH 5.5, at 25 °C. All reagents except for hydrogen peroxide were added to the following final concentrations: dansyl-YVG (50–400 μm), Cu2+ as copper sulfate (5 μm), and PHM (2.5–5 μm). After the reagents were allowed to incubate for 2 min, the reaction was initiated by adding H2O2 from a 15 mm stock, to a final concentration of 0.5–4.0 mm. In a typical experiment using 1 mm H2O2, 330-μl aliquots were removed every 30 s, transferred to a 1.5-ml microcentrifuge tube containing 10 μl of 10% trifluoroacetic acid, and vortexed for 10 s. Substrate and product were separated by HPLC, and their concentrations were determined using a standard curve built from chromatograms of 10–200 μm samples of dansyl-YVG run under identical conditions. Kinetic constants were extracted from the raw data by fitting to the Michaelis-Menten equation using nonlinear regression in Sigmaplot 8.0. Measurement of the Dansyl-YVG Dissociation Constant KD—For the oxidized enzyme, an Amicon (5-ml) ultrafiltration device was first preincubated overnight with 300 μm Cu(II)-loaded PHM (2.0 copper/protein) in 100 mm MES, pH 5.5, containing 1 mm dansyl-YVG. The protein solution was then washed repeatedly with buffer until the substrate concentration in the filtrate had fallen to a low level as determined by measurement of dansyl fluorescence of the filtrate. This conditioning procedure ensured that all irreversible substrate and/or protein binding sites on the membrane were occupied. Next, dansyl-YVG was titrated into the PHM solution, and aliquots of the filtrate were extracted for analyses as follows. A septum was placed over the ultrafiltration cell, and the cell was pressurized by injecting 0.6 ml of air over the solution. After a small amount of filtrate had been collected, it was returned to the concentrator, and the process was repeated three times. On the fourth pressurization, 10 μl of filtrate was saved for analysis of the concentration of “free” substrate by fluorimetry (λEx = 365 nm; λEm = 558 nm). The remaining filtrate was returned to the concentrator along with the next titration aliquot (30 μl) of 10 mm substrate for a net volume gain of 20 μl. This procedure was repeated until a total substrate concentration of 1 mm was reached. For the reduced protein, the following modifications to the procedure were used. All solutions were first purged with argon and placed in an anaerobic chamber. Cu(II)-loaded PHM (2.0 copper/protein) in 100 mm MES, pH 5.5, was reduced with a 5-fold excess of buffered ascorbate and then titrated with dansyl-YVG in a conditioned ultrafiltration cell in the anaerobic chamber using an identical protocol. The data were analyzed by constructing plots of fractional binding, f = ([ST] – [SF])/[ET] versus free substrate SF (where the subscripts T and F represent total and free concentrations, respectively). These data were fit by nonlinear regression (Sigmaplot 8.0) to the equation, f=nSFKD+SF (Eq. 1) where KD is the dissociation constant for binding of dansyl-YVG to PHM, and n is the number of binding sites. Values of KD and n were refined in the fits. 18O Incorporation Experiments—18O-Labeling reactions were carried out similarly to those for kinetic analysis, except that all reactions were carried out in an anaerobic chamber. 18O2 reactions were performed by first flushing the enzyme solution with argon for 15 min. The argon headspace was replaced with 18O2, and the reaction mixture was equilibrated for 2 min. H 182O2 experiments were identical to those completed with H 162O2. All reactions were quenched with trifluoroacetic acid (10% in water) and purified on HPLC prior to mass spectrometry measurements. Mass Spectrometry—Samples for mass spectrometry were purified on HPLC and then diluted 250 times for infusion with 50:50 acetonitrile/water in 0.1% formic acid. Samples were directly infused into the electrospray ionization source of a LCQ Deca XP Plus (Thermo, San Jose, CA) ion trap mass spectrometer at 3 μl/min. Typically, 100 profile scans were acquired (200-ms maximum injection time and three microscans) over a range of m/z 500–1500. The standard isotope distribution for substrate and product was calculated using MS-Isotope in Protein Prospector 4.0.5 (by P. Baker and K. Clausner; available on the World Wide Web at prospector.ucsf.edu/ucsfhtml4.0/msiso.htm). Measurement of Peroxide Concentration—Hydrogen peroxide concentrations were determined using a BIOXYTECH H2O2-560 quantitative peroxide formulation kit (OXIS International Inc.). 25 μl of quenched reaction mixture was diluted to 2 ml and mixed thoroughly. 100 μl of this dilution was then added to 1 ml of working reagent. The A560 was recorded for the samples and a series of standards, after incubating for 1 h at ambient temperature. Peroxide Degradation Monitored by Oxygen Production—Oxygen production was monitored using a Rank Brothers oxygen electrode at 25 °C. 100 mm MES (pH 5.5) was added to a stirred cell until a stable base line was achieved. The stirred cell was capped, and H2O2 was then added with a Hamilton syringe, through a small opening in the cap, to a final concentration in the cell of 1 mm. Reagents from the standard reaction including Cu2+, PHM, and substrate, were added consecutively, by Hamilton syringe, with oxygen levels monitored after each addition. Reaction Using Glucose and Glucose Oxidase (GO) to Generate H2O2— Experiments in which a glucose/GO system was used to generate H2O2 were performed in a Rank Brothers oxygen electrode at 25 °C. 100 mm MES buffer (pH 5.5) containing 50 mm d-glucose was added to the cell and allowed to equilibrate, and the response was adjusted to 21%. The base line was allowed to stabilize, the original buffer was removed, and fresh buffer at 100 or 21% oxygen saturation was added. The stirred cell was capped, and glucose oxidase was immediately added with a Hamilton syringe to a concentration of 43 μg/ml. Once all the oxygen was converted to hydrogen peroxide, substrate and PHM were added with a Hamilton syringe to final concentrations of 200 and 5 μm, respectively. Each reaction was performed in a total volume of 2 ml. The reaction was quenched at the desired time, by the addition of 70 μl 10% trifluoroacetic acid. The quenched reaction was immediately analyzed for H2O2, product, and substrate concentrations. Stoichiometry of Peroxide Consumption to Product Formation Using the Glucose/GO Reaction to Generate Hydrogen Peroxide—These experiments were performed in the same manner as the glucose/GO reactions above, with the exception that substrate was added to 400 μm, and only 21% oxygen saturation was used. The initial H2O2 concentration (formed by conversion of the dissolved oxygen to peroxide by glucose/GO) was assayed prior to the addition of substrate and PHM and again after the reaction was quenched with trifluoroacetic acid. EPR Spectroscopic Quantitation of the Reduction of the Copper Centers in PHMcc by Hydrogen Peroxide—EPR spectra were obtained from PHM samples with [Cu(II)] = 250 μm, on a Bruker E500-X-band EPR spectrometer equipped with a SuperX microwave bridge, a superhigh Q cavity, and a nitrogen flow cryostat (Helitran; Advance Research Systems). The following experimental conditions were used: temperature, 80 K; microwave frequency, 9.4 GHz; microwave power, 20 milliwatts; modulation frequency, 100 kHz; modulation amplitude, 10 G. EPR signals were quantified by double integration under nonsaturating conditions and by comparison with 100, 200, and 300 μm Cu(II) (EDTA) standards. Titration with hydrogen peroxide was performed by adding 10-μl aliquots of a 30% hydrogen peroxide stock solution to a 200-μl initial sample volume. The reaction cycle of PHM (Fig. 1B) involves a reductive phase in which the enzyme is reduced by ascorbate to a dicopper(I) reduced intermediate and an oxidative phase in which the reduced enzyme reacts with peptide substrate and dioxygen to generate the hydroxylated product. As depicted in Fig. 1, many of the proposed mechanisms have suggested a CuM(II)-peroxide or hydroperoxide as the hydroxylating species, formed from the reaction of PHM-Cu(I)2 with O2 and subsequent transfer of one electron from each Cu(I) center to dioxygen. If such an intermediate exists, then it should also be generated directly from the reaction of hydrogen peroxide with the oxidized (dicopper(II)) enzyme in the presence of substrate. Measurement of Peptidyl-α-hydroxyglycine Product Using HPLC— To test whether this peroxide shunt chemistry occurred in PHMcc, we measured the reaction of the oxidized enzyme with dansyl-YVG and hydrogen peroxide. Initial concentrations of reagents were 200 μm substrate, 1 mm peroxide, and 5 μm PHM in 100 mm MES buffer, pH 5.5. Aliquots were sampled at 30-s time intervals, and product was separated from substrate by HPLC, using the fluorescence of the dansyl group for detection (Fig. 2a). Under these conditions, all of the substrate was converted into product within 2–3 min. Further, since the substrate was present in 40-fold excess over the enzyme, at least 40 enzyme turnovers occurred, implying that the reaction was catalytic. Control experiments where peroxide was incubated with substrate and 5 μm Cu2+ in the absence of PHM or in the presence of PHM that had been heated at 90 °C for 30 min gave no product (data not shown), demonstrating that the reaction was enzymatic. Fig. 2 shows rate data for the peroxide reactivity over a range of H2O2 concentrations (Fig. 2c) or dansyl-YVG concentrations (Fig. 2d). Kinetic parameters extracted from these plots are listed in Table 1. Table 1 also lists kinetic parameters for the ascorbate/O2-dependent reaction (Fig. 2b). These data show that although the rate of the peroxide reaction is much slower than the ascorbate/O2 reaction under the standard reaction conditions of 1 mm H2O2 and 200 μm dansyl-YVG, this is due primarily to a large increase in the value of Km for the dansyl-YVG substrate, which is 2 orders of magnitude larger than for the ascorbate reaction. A modest decrease in kcat from 9.2 to 5 s–1 is observed for the peroxide reaction.TABLE 1Comparison of kinetic and thermodynamic parameters for the PHM-catalyzed hydroxylation of dansyl-Tyr-Val-Gly by the ascorbate/dioxygen and peroxide pathwaysKm,H2O2Km,dansyl-YVGkcatKDμmμms-1μmAscorbate/O25.1 ± 0.69.2 ± 0.4Peroxide reaction2780 ± 260800 ± 4005 ± 3Reduced PHM22.5 ± 2.7Oxidized PHM145 ± 16 Open table in a new tab The large increase in Km for the peroxide pathway suggests that the dansyl-YVG binds to a different form of the enzyme than in the ascorbate/O2-dependent pathway, consistent with reactivity occurring within the oxidized rather than the reduced form of the enzyme. To gain further insight into this possibility, we determined the dissociation constant for dansyl-YVG to PHM in its Cu(I) and Cu(II) forms. PHM was titrated with aliquots of dansyl-YVG in an ultrafiltration cell at 23 °C, and a small volume of filtrate was extracted for measurement of the free substrate concentration from its fluorescence signal. Plots of fractional formation of enzyme-dansyl-YVG versus free substrate were fit to theoretical curves for substrate binding. The results are shown in Fig. 3. The KD values were 22.5 and 145 μm for the Cu(I) and Cu(II) forms of PHM, respectively. The KD for Cu(I)-PHM is in the same range as Km for the ascorbate/O2-dependent pathway, consistent with the dansyl-YVG binding to the Cu(I) form as predicted by the general mechanism of Fig. 1. The KD for Cu(II)-PHM is about 7 times larger than the reduced KD and indicates that structural factors in the oxidized enzyme weaken the binding. Thus, the increased Km measured for the peroxide pathway, although larger than KD for Cu(II)-PHM, is more consistent with substrate binding to the Cu(II) than the Cu(I) form of the enzyme. We also examined the possibility that hydrogen peroxide does not interact directly with the dicopper(II) enzyme but instead reduces it to the dicopper(I) form and that product forms by reaction of the dicopper(I) form with dioxygen and substrate. Here peroxide would be fulfilling the same role as ascorbate. As a test for this reductive peroxide pathway, we studied the reaction of PHM with perox" @default.
• W2013752604 created "2016-06-24" @default.
• W2013752604 creator A5007506110 @default.
• W2013752604 creator A5034871882 @default.
• W2013752604 creator A5039807387 @default.
• W2013752604 creator A5057493602 @default.
• W2013752604 creator A5077274330 @default.
• W2013752604 date "2006-02-01" @default.
• W2013752604 modified "2023-10-14" @default.
• W2013752604 title "The Hydrogen Peroxide Reactivity of Peptidylglycine Monooxygenase Supports a Cu(II)-Superoxo Catalytic Intermediate" @default.
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