The Hydrogen Peroxide Reactivity of Peptidylglycine Monooxygenase Supports a Cu(II)-Superoxo Catalytic Intermediate

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 the two coppers are an and the is this (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 electron the CuH ligand His108, a water molecule, and the which reduced the to a Jaron and Blackburn S. Blackburn N.J. Biochemistry. 1999; 38: 15086-15096Crossref PubMed Scopus (70) Google Scholar) that O2 react at CuH and that the to CuM a for the electron and a of is with of the a in the can be to with no of catalytic activity J. R. Mains R.E. Eipper B.A. Biochemistry. PubMed Scopus Google Scholar), whereas in the enzyme oxygen reduction and substrate hydroxylation tightly in the of slow out where of into be J.P. J.P. J. Biol. Chem. 278: PubMed Scopus Google Scholar). These and J.P. J.P. J. Biol. Chem. 278: PubMed Scopus Google Scholar, Blackburn N.J. J.P. J. Am. Chem. Soc. PubMed Scopus Google Scholar, Merkler D.J. J.P. J. Am. Chem. Soc. 2004; 126: PubMed Scopus Google Scholar) to peroxide a intermediate in both PHM and and to that the reactive oxygen species is a which a hydrogen from substrate to the electron or hydroperoxo species is an should be to product by the oxidized enzyme with hydrogen peroxide and substrate in the oxidative phase of the mechanism in has been to in other M. J. Biochem. PubMed Scopus Google Scholar, R. J. Am. Chem. Soc. PubMed Scopus Google Scholar), monooxygenase J. Biol. Chem. PubMed Google Scholar), and J. Biol. Chem. 278: PubMed Scopus Google Scholar). Peroxide have the that when labeled peroxide is used the of the oxygen the is transferred to the J. Biol. Chem. 278: PubMed Scopus Google Scholar). In this have investigated the reactivity of hydrogen peroxide with the oxidized of the PHM catalytic and that product is in the However, when 18O-labeled peroxide was used the of of the with We also that peroxide reactivity is eliminated by a mutation at the CuH center, which should not be involved in the peroxide The have to the that Cu(II)-peroxo or species are not involved in the reaction and an mechanism species and and were from or and were used further hydrogen peroxide and oxygen were from at and and of and and and the and were and 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, S. Mains R.E. Eipper B.A. Blackburn N.J. Biochemistry. PubMed Scopus Google Scholar) a with a In was a of were was to and the was for The was at and with in of The was to through a and to a at a rate of The enzyme at and to this of a of The enzyme was further by on a a with PHM from was in and with of the The was by a in PHM close to of for of from to in was in a to which copper was at on with the of copper was The was in an from to was by three to bound of Cu(II) was to the and the was to step the to The was in and in at The was in the of and was the and an of S. Blackburn N.J. Biochemistry. 1999; 38: 15086-15096Crossref PubMed Scopus (70) Google Scholar). were on a at were a and of and was with a solvent with a on a and product by the were by a was in and solvent was in was from substrate via and at in A. of the peroxide reaction were by the rate of substrate consumption product a of The reaction was in a reaction with in at for hydrogen peroxide were to the dansyl-YVG copper and PHM the were to for the reaction was by H2O2 from a to a of In a H2O2, were transferred to a of and for and product were by HPLC, and were a from of of dansyl-YVG under identical conditions. were from the data by to the in of the the oxidized enzyme, an was first with PHM in MES, The was with the substrate in the to a by of of the that substrate binding on the were dansyl-YVG was into the PHM and of the were for was the and the was by of the a of been was to the and the was three the of was for of the of substrate by The was to the with the of substrate for a of was a substrate of was the reduced the to the were were first with and in an anaerobic PHM in MES, was reduced with a of and with dansyl-YVG in a in the anaerobic an identical The data were by of substrate the and and These data were by to the where is the for binding of dansyl-YVG to PHM, and is the of binding of and were in the 18O were carried out to for that were carried out in an anaerobic were by first the enzyme with for The was with 18O2, and the reaction was for H were identical to with H were with in and on to for were on and for with in acid. were directly into the of a at were and three a of The for substrate and product was in P. and on the at of Peroxide peroxide were a peroxide of reaction was to and of this was to of The was for the and a of for at Peroxide by was a oxygen at was to a a was The was and H2O2 was with a through a in the to a in the of from the reaction PHM, and were by with oxygen and to in which a was used to H2O2 were in a oxygen at was to the and to and the was to The was to the was and at or oxygen was The was and glucose was with a to a of the oxygen was to hydrogen peroxide, substrate and PHM were with a to of and reaction was in a of The reaction was at the by the of acid. The reaction was for H2O2, and substrate of Peroxide to the to were in the the with the that substrate was to and oxygen was The H2O2 by of the oxygen to peroxide by was to the of substrate and PHM and the reaction was with acid. of the Reduction of the in by were from PHM with on a with a a and a The conditions were were by under conditions and by with and Cu(II) with hydrogen peroxide was by of a hydrogen peroxide to a The reaction of PHM (Fig. a reductive phase in which the enzyme is reduced by to a dicopper(I) reduced intermediate and an oxidative phase in which the reduced enzyme with substrate and dioxygen to the in of the have a or the from the reaction of with O2 and of one electron from Cu(I) center to If an intermediate should also be directly from the reaction of hydrogen peroxide with the oxidized enzyme in the presence of substrate. of whether this peroxide chemistry in PHMcc, measured the reaction of the oxidized enzyme with dansyl-YVG and hydrogen of were peroxide, and PHM in were at and product was from substrate by HPLC, the of the for (Fig. Under of the substrate was into product the substrate was in the enzyme, at least enzyme that the reaction was where peroxide was with substrate and in the of PHM or in the presence of PHM that been at for no product not that the reaction was shows rate data for the peroxide reactivity a of H2O2 (Fig. or dansyl-YVG (Fig. from are in 1. also for the reaction (Fig. These data that the rate of the peroxide reaction is much the reaction under the reaction conditions of H2O2 and this is to a large in the of for the dansyl-YVG which is of for the in from to is for the peroxide of and for the hydroxylation of by the and peroxide in a The large in for the peroxide that the dansyl-YVG to a of the enzyme in the with reactivity the oxidized the reduced of the further into this the for dansyl-YVG to PHM in Cu(I) and Cu(II) PHM was with of dansyl-YVG in an at and a of was for of the substrate from of formation of substrate were to theoretical for substrate The are in The were and for the Cu(I) and Cu(II) forms of PHM, The for is in the for the with the dansyl-YVG binding to the Cu(I) by the mechanism of 1. The for is about the reduced and that in the oxidized enzyme the the measured for the peroxide for is with substrate binding to the Cu(II) the Cu(I) of the We also the that hydrogen peroxide not directly with the dicopper(II) enzyme but to the dicopper(I) and that product forms by reaction of the dicopper(I) with dioxygen and substrate. peroxide be the a for this reductive peroxide the reaction of PHM with peroxide and substrate under anaerobic conditions. the anaerobic H2O2 were with to oxygen and with anaerobic were in an anaerobic Under reaction were to be identical to measured under aerobic conditions not which to out a reductive for However, that of the PHM catalytic reaction are to the of peroxide to dioxygen and that be by the of anaerobic conditions the reaction to be to the of on the PHM peroxide reactivity It has been Biochem. J. 1994; PubMed Scopus Google Scholar) that the for is by the of the reductant. this with the dansyl-YVG reductant. The measured was with for These data further that peroxide is not a reductive this were the not be to in the was used to determine the oxygen in the In was that when PHM by the the oxygen into the of the α-hydroxyglycine product is derived from oxygen D.J. Kulathila R. Consalvo A.P. Biochemistry. PubMed Scopus Google Scholar). If the peroxide a peroxide the oxygen should be derived from peroxide J. Biol. Chem. 278: PubMed Scopus Google Scholar). carried out and under anaerobic or aerobic conditions. The are in and 2. When was the product to the with of reacted under anaerobic conditions to and for the of 18O in the labeled that the oxygen was derived from peroxide from However, when reacted under aerobic of the product was labeled with and the with solvent be out from the this that the oxygen in the product was derived in from where was reacted with substrate in the presence of both the and in the These for a the oxygen at the is derived from peroxide but that a for this oxygen to with oxygen from for reaction of peroxide with the dicopper(II) enzyme to an intermediate that is in with a species that can subsequently with or peroxide is a and forms the dicopper(I) which itself product through the the that product formation should not should be under anaerobic to However, nonenzymatic peroxide oxygen to the reaction under anaerobic conditions. this measured the of peroxide to product in product during the reaction of dansyl-YVG with hydrogen of 18O in in a of the Peroxide peroxide with product for a of It is that peroxide consumption product formation by that peroxide was to oxygen and water in a nonenzymatic reaction was in the presence of the of the PHM catalytic that PHM catalysis a species of nonenzymatic peroxide further and The presence of oxygen from the of peroxide the rate of the reaction not under O2 is by peroxide in the reaction of PHM with under 18O2, the product still peroxide a to from nonenzymatic peroxide of to hydrogen peroxide for the PHM peroxide reaction dansyl-YVG PHM hydrogen peroxide, and for the reaction carried out in the presence of glucose glucose reaction glucose reaction glucose and glucose in a We therefore to product formation under conditions where the nonenzymatic reaction was or We used the reaction of glucose in the presence of glucose and to a of anaerobic hydrogen reaction was to proceed in a oxygen with no of the oxygen been (Fig. PHM and dansyl-YVG were into the and the rate of was by the that oxygen by peroxide was to hydrogen peroxide by the reaction into J.P. Biochemistry. 1999; 38: PubMed Scopus Google Scholar). shows oxygen to the The was to oxygen the when the was and the was to the the peroxide oxygen been was to be and provided that no further oxygen was into the shows the of product formation from the with the rate of product formation did not when peroxide was strictly peroxide consumption was strictly to product with the of peroxide to product to that peroxide with PHM to product by a that not on reduction to the dicopper(I) and reaction of the with the of O2 in the is the mechanism must formation of a catalytic intermediate from Cu(II) and peroxide the PHM catalytic The of the under aerobic conditions that a species must be a intermediate. In for the peroxide and the to have a peroxide must the Cu(II) to Cu(I) the PHM at one of the copper this measured the of peroxide to the copper centers of PHM at spectroscopy in The to of the oxidized These that peroxide can the Cu(II) centers in The of reduction did not with of and a of the copper in the is likely that an between Cu(II)-peroxo and which must be for in mechanism for peroxide and peroxide requires no additional to complete the monooxygenase a were the reactive α-hydroxyglycine and water the for electron from the other copper center that the be in with at whereas the peroxide reactivity was measured the of a CuH to of activity S. Mains R.E. Eipper B.A. Blackburn N.J. Biochemistry. PubMed Scopus Google Scholar), to product via the peroxide The was with peroxide and or The reaction was to proceed for to the by the to of substrate into The that no We also the peroxide reactivity of CuM has been to copper at the CuH (11.Blackburn N.J. Rhames F.C. Ralle M. Jaron S. J. Biol. Inorg. Chem. 2000; 5: 341-353Crossref PubMed Scopus (98) Google Scholar). The was also to that the peroxide reactivity is not at not proceed when CuH is We have that PHM is to the hydroxylation of peptidylglycine substrates from the oxidized enzyme and hydrogen peroxide the of When peroxide labeled with 18O was reacted with PHM and substrate in the presence of of the was into the of the was not to solvent of 18O when both the peroxide and the oxygen were labeled with 18O or when labeled peroxide was reacted under anaerobic conditions. the peroxide must an intermediate that is of with We that peroxide is in a nonenzymatic to dioxygen and that the is when PHM is substrate that PHM catalysis a species of nonenzymatic H2O2 However, were to the nonenzymatic peroxide by out the reaction in the presence of glucose and glucose oxidase, which oxygen to peroxide and thus provided a strictly anaerobic this were to that the reaction was tightly and that rate did not when oxygen was from the of the product with from both peroxide and dioxygen peroxide to the Cu(II) centers to In this be the and a dicopper(I) intermediate that be for reaction with dioxygen in the for binding of O2 to the complex has not been for is at and Merkler D.J. J.P. J. Am. Chem. Soc. 2004; 126: PubMed Scopus Google Scholar). the PHM reaction is to be with O2 binding to the complex Merkler D.J. Blackburn N.J. J.P. Biochemistry. PubMed Scopus Google Scholar), the with but is that for dansyl-YVG from that for this that for the reductive should when the O2 in the to undetectable measured by the O2 in the no in the rate of the that peroxide be the of and must be reactive oxygen species the PHM active The in in the peroxide reaction and in the for dansyl-YVG binding to the Cu(I) and Cu(II) forms of PHM that the peroxide reactivity the oxidized The that oxygen is to into product from hydrogen peroxide and oxidized enzyme that an intermediate the reaction that is to The likely for this intermediate be a for Cu(II)-peroxo to be a must be in with require that the electron from CuH to CuM be in the a of have been to the of a from CuH to substrate (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), S. Blackburn N.J. Biochemistry. 1999; 38: 15086-15096Crossref PubMed Scopus (70) Google Scholar), or solvent J.P. J.P. J. Biol. Chem. 278: PubMed Scopus Google Scholar). on the electron to be has also been by and J.P. J.P. J. Biol. Chem. 278: PubMed Scopus Google Scholar). of the of a Cu(II)-peroxo be the formation of from the reaction of that a CuH center with peroxide and the Cu(II)-peroxo species not require additional for We that the CuH was to product formation from peroxide and oxidized has been to be active PHM in the S. Mains R.E. Eipper B.A. Blackburn N.J. Biochemistry. PubMed Scopus Google Scholar), and has been that the in activity is to from the CuH The complete of product formation that the peroxide in additional by the of peroxide to the CuH a also the CuM which have causes of copper binding at the center (11.Blackburn N.J. Rhames F.C. Ralle M. Jaron S. J. Biol. Inorg. Chem. 2000; 5: 341-353Crossref PubMed Scopus (98) Google Scholar). If this was active in the peroxide that peroxide binding and reactivity at was in the peroxide These data provide that electron from CuH to CuM is still in the peroxide and that a be the catalytic intermediate. mechanism for the peroxide reactivity that is in with of data is in We that hydrogen peroxide at the copper and a species that is to but also be The can react further the PHM or be to the The likely to react with the be the which the a a species to or in with a mechanism for oxygen with The that from the active into the a in the nonenzymatic of peroxide and the is during Our data to a to that of and J.P. J.P. J. Biol. Chem. 278: PubMed Scopus Google Scholar, Merkler D.J. J.P. J. Am. Chem. Soc. 2004; 126: PubMed Scopus Google Scholar), that the is the reduced oxygen intermediate. The mechanism that the peroxide and in a which can be a is by the measured for the two The large in rate is to the in substrate between oxidized and reduced CuM centers to which the substrate must in the peroxide and EXAFS has that tightly to to (11.Blackburn N.J. Rhames F.C. Ralle M. Jaron S. J. Biol. Inorg. Chem. 2000; 5: 341-353Crossref PubMed Scopus (98) Google Scholar). substrate via to on the that the CuM ligands and but also forms hydrogen to and on the that the the of the that must binding to the oxidized enzyme by two Our also provide in support of the of and the of hydrogen from peptidylglycine substrates for and (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). by was to be and to proceed with a activation the hydroperoxo of the intermediate a for the with bound in the equatorial and additional equatorial and a axial The redox of the copper centers in PHM are but the mechanism in that peroxide should the CuH to We used to the reaction of oxidized PHM with hydrogen peroxide at We a of about which be derived from CuH reduction or These that a of of the CuH centers can be reduced by peroxide, but this be to support the that be to the reaction chemistry of peroxide with the copper centers in with