Crystal Structures of Chloroperoxidase with Its Bound Substrates and Complexed with Formate, Acetate, and Nitrate

Abstract

Chloroperoxidase (CPO) is a heme-thiolate enzyme that catalyzes hydrogen peroxide-dependent halogenation reactions. Structural data on substrate binding have not been available so far. CPO was therefore crystallized in the presence of iodide or bromide. One halide binding site was identified at the surface near a narrow channel that connects the surface with the heme. Two other halide binding sites were identified within and at the other end of this channel. Together, these sites suggest a pathway for access of halide anions to the active site. The structure of CPO complexed with its natural substrate cyclopentanedione was determined at a resolution of 1.8Å. This is the first example of a CPO structure with a bound organic substrate. In addition, structures of CPO bound with nitrate, acetate, and formate and of a ternary complex with dimethylsulfoxide (Me2SO) and cyanide were determined. These structures have implications for the mechanism of compound I formation. Before binding to the heme, the incoming hydrogen peroxide first interacts with Glu-183. The deprotonated Glu-183 abstracts a proton from hydrogen peroxide. The hydroperoxo-anion then binds at the heme, yielding compound 0. Glu-183 protonates the distal oxygen of compound 0, water is released, and compound I is formed. Chloroperoxidase (CPO) is a heme-thiolate enzyme that catalyzes hydrogen peroxide-dependent halogenation reactions. Structural data on substrate binding have not been available so far. CPO was therefore crystallized in the presence of iodide or bromide. One halide binding site was identified at the surface near a narrow channel that connects the surface with the heme. Two other halide binding sites were identified within and at the other end of this channel. Together, these sites suggest a pathway for access of halide anions to the active site. The structure of CPO complexed with its natural substrate cyclopentanedione was determined at a resolution of 1.8Å. This is the first example of a CPO structure with a bound organic substrate. In addition, structures of CPO bound with nitrate, acetate, and formate and of a ternary complex with dimethylsulfoxide (Me2SO) and cyanide were determined. These structures have implications for the mechanism of compound I formation. Before binding to the heme, the incoming hydrogen peroxide first interacts with Glu-183. The deprotonated Glu-183 abstracts a proton from hydrogen peroxide. The hydroperoxo-anion then binds at the heme, yielding compound 0. Glu-183 protonates the distal oxygen of compound 0, water is released, and compound I is formed. Chloroperoxidase (CPO) 2The abbreviations used are: CPO, chloroperoxidase; CPD, cyclopentanedione; PEG, polyethylene glycol. is a 42-kDa heme-containing glycoprotein that is secreted by the fungus Caldariomyces fumago. The enzyme catalyzes the hydrogen peroxide-dependent chlorination of cyclopentanedione during the biosynthesis of the antibiotic caldarioymcin (1Beckwith J.R. Hager L.P. J. Biol. Chem. 1963; 238: 3091-3094Abstract Full Text PDF PubMed Google Scholar, 2Beckwith J.R. Clark R. Hager L.P. J. Biol. Chem. 1963; 238: 3086-3090Abstract Full Text PDF PubMed Google Scholar). Additionally, CPO catalyzes the iodination and bromination of a wide range of substrates (3Hager L.P. Morris D.R. Brown F.S. Eberwein H. J. Biol. Chem. 1966; 241: 1769-1777Abstract Full Text PDF PubMed Google Scholar, 4Patterson E.L. Andres W.W. Mitscher L.A. Appl. Microbiol. 1967; 15: 528-530Crossref PubMed Google Scholar, 5Itoh N. Izumi Y. Yamada H. Biochemistry. 1987; 26: 282-289Crossref Scopus (69) Google Scholar). Besides performing H2O2-dependent halogenation reactions, the enzyme catalyzes dehydrogenation reactions (6Thomas J.A. Morris D.R. Hager L.P. J. Biol. Chem. 1970; 245: 3129-3134Abstract Full Text PDF PubMed Google Scholar). These reactions can be performed in a stereoselective manner by CPO (7Casella L. Poli S. Gullotti M. Selvaggini C. Beringhelli T. Marchesini A. Biochemistry. 1994; 33: 6377-6386Crossref PubMed Scopus (77) Google Scholar). CPO also functions as a catalase, facilitating the decomposition of hydrogen peroxide to oxygen and water (6Thomas J.A. Morris D.R. Hager L.P. J. Biol. Chem. 1970; 245: 3129-3134Abstract Full Text PDF PubMed Google Scholar). Furthermore, CPO catalyzes P450-like oxygen insertion reactions (8Griffin B. Everse J. Everse K.E. Grisham M.B. Peroxidases in Chemistry and Biology. II. CRC Press, Boca Raton, FL1991: 85-137Google Scholar). Of particular interest for commercial applications is the enantioselective formation of epoxides from alkenes (9Allain E.J. Hager L.P. Deng L. Jacobsen E.N. J. Am. Chem. Soc. 1993; 115: 4415-4416Crossref Scopus (229) Google Scholar, 10Colonna S. Gaggero N. Casella L. Carrea G. Pasta P. Tetrahedron-Asymmetry. 1993; 4: 1325-1330Crossref Scopus (74) Google Scholar), because CPO utilizes H2O2, whereas P450 enzymes depend on both molecular oxygen and electron transfer proteins. The capability of CPO to perform these diverse reactions makes it one of the most versatile of all known heme proteins. Structurally, CPO is unique, but it shares features with both peroxidases and P450 enzymes (11Sundaramoorthy M. Terner J. Poulos T.L. Structure. 1995; 3: 1367-1377Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar). As in cytochrome P450 enzymes, the proximal heme ligand of CPO is a cysteine (Cys-29), but similar to peroxidases, the distal side of the heme in CPO is polar. However, unlike other peroxidases, the normally conserved distal arginine is lacking in CPO and the catalytic acid base of CPO is a glutamic acid (Glu-183) and not a histidine. When Glu-183 is mutated to a histidine, the chlorination activity is reduced by 85%, which indicates the importance of this acidic residue for the catalytic activity of CPO (12Yi X. Conesa A. Punt P.J. Hager L.P. J. Biol. Chem. 2003; 278: 13855-13859Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). CPO is reactive at low pH with an optimum at pH 2.8 (1Beckwith J.R. Hager L.P. J. Biol. Chem. 1963; 238: 3091-3094Abstract Full Text PDF PubMed Google Scholar), which is likely due to the high redox potential required to oxidize a chloride anion, and therefore a glutamic acid residue instead of a histidine is used as a catalytic acid base. The crystal structure of CPO in the ferric ground state was determined more than 10 years ago by the group of Thomas Poulos (11Sundaramoorthy M. Terner J. Poulos T.L. Structure. 1995; 3: 1367-1377Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar). On the distal side of the heme, a water molecule is located 3.4 Å away from the iron. This water is displaced by the incoming H2O2. It is generally assumed that Glu-183 abstracts a proton from the hydrogen peroxide bound to heme, yielding a short-lived peroxo-anion species (compound 0) (13Wagenknecht H.A. Woggon W.D. Chem. Biol. 1997; 4: 367-372Abstract Full Text PDF PubMed Scopus (85) Google Scholar). In the next step Glu-183 is expected to protonate the terminal oxygen to facilitate the heterolytic cleavage of the O-O bond. Subsequently, water is released and compound I, the oxoferryl(IV) porphyrin π cation radical, is formed. Compound I can oxidize a chloride anion and has been proposed to form an Fe(III)-hypohalite intermediate (14Libby R.D. Thomas J.A. Kaiser L.W. Hager L.P. J. Biol. Chem. 1982; 257: 5030-5037Abstract Full Text PDF PubMed Google Scholar). Such a species has been detected spectroscopically at pH 4.4 (13Wagenknecht H.A. Woggon W.D. Chem. Biol. 1997; 4: 367-372Abstract Full Text PDF PubMed Scopus (85) Google Scholar). However, it has not yet been entirely resolved whether this hypohalite intermediate directly chlorinates the organic substrate (15Libby R.D. Beachy T.M. Phipps A.K. J. Biol. Chem. 1996; 271: 21820-21827Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), or if HOCl is released upon protonation of the intermediate, and free HOCl or Cl2 perform the chlorination reactions (16Griffin B.W. Biochem. Biophys. Res. Commun. 1983; 116: 873-879Crossref PubMed Scopus (23) Google Scholar). Because CPO shows only low substrate specificity and chlorinates a wide range of substrates non-stereospecifically (17Ramakrishnan K. Oppenhuizen M.E. Saunders S. Fisher J. Biochemistry. 1983; 22: 3271-3277Crossref PubMed Scopus (28) Google Scholar), it might indeed be that free HOCl or Cl2 perform these reactions in solution. In contrast, it has been reported that glycals (18Liu K.K.C. Wong C.H. J. Org. Chem. 1992; 57: 3748-3750Crossref Scopus (30) Google Scholar) and steroids (19Neidleman S.L. Diassi P.A. Junta B. Palmere R.M. Pan S.C. Tetrahedron Lett. 1966; 44: 5337-5342Crossref PubMed Scopus (23) Google Scholar, 20Levine S.D. Neidleman S.L. Oberc M. Tetrahedron. 1968; 24: 2979-2984Crossref Scopus (14) Google Scholar) are stereospecifically halogenated, indicating that there must be an interaction between the organic substrate and CPO. Binding of organic substrates at the active site is also supported by the observation that epoxidation and sulfoxidation reactions (21Colonna S. Gaggero N. Manfredi A. Casella L. Gullotti M. Carrea G. Pasta P. Biochemistry. 1990; 29: 10465-10468Crossref PubMed Scopus (152) Google Scholar) are catalyzed stereospecifically. Binding of the substrate cyclopentanedione (CPD) to CPO has been shown by NMR titration experiments (22Wang X. Goff H.M. Biochim. Biophys. Acta. 1997; 1339: 88-96Crossref PubMed Scopus (9) Google Scholar). The structures of CPO bound with small diatomic or triatomic ligands, e.g. carbon monoxide, nitric oxide, cyanide, and thiocyanate have been determined, but so far the soaking of organic substrates into CPO crystals has been unsuccessful (23Sundaramoorthy M. Terner J. Poulos T.L. Chem. Biol. 1998; 5: 461-473Abstract Full Text PDF PubMed Scopus (143) Google Scholar). Therefore information on the structural basis of substrate binding and specificity has been limited. Here we present crystal structures of CPO bound to its substrate CPD and of a ternary complex of the enzyme with cyanide and Me2SO. We co-crystallized CPO with either bromide or iodide and could identify binding which suggest a pathway for halide access to the heme. are the structures of CPO complexed with acetate, nitrate, and which have been shown to to the enzyme M. K. Hager L.P. Biochemistry. PubMed Scopus Google Scholar). The implications of these structures for the mechanism of compound I formation are with was and as S. Hager L.P. Lett. Scopus (30) Google Scholar). were by the at of CPO in pH was with of the solution. The for the iodide crystals and at pH similar to (11Sundaramoorthy M. Terner J. Poulos T.L. Structure. 1995; 3: 1367-1377Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar). crystals within a CPO crystals were in transfer into and at pH crystals were in a similar as the iodide crystals in a and at pH The for the bromide crystals of with and with and CPD was in Me2SO. in were into a soaking CPD, and at pH and were for at were then into the soaking with for and in ternary complex was by first soaking the crystals in and at pH for at were then to a CPD, and at pH for 10 at were into a CPD, and at pH and and for the complex and at pH The and at pH The CPO formate crystals were in a and at pH The complex was by soaking CPO crystals in the presence of in a and at pH for 10 at were into a of with either or and and then in and data of crystals were at with a or at at and were with the J. Appl. 1993; 26: Scopus Google Scholar) were determined by molecular with J. Biol. 57: PubMed Scopus Google Scholar) the CPO structure (11Sundaramoorthy M. Terner J. Poulos T.L. Structure. 1995; 3: 1367-1377Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar) as a The was used for of the A. PubMed Scopus Google Scholar). was with P. J. M. T. Biol. 1998; PubMed Scopus Google Scholar) and at with A. E.J. Biol. PubMed Scopus Google Scholar). were with A. Morris R. Biol. PubMed Scopus Google Scholar). were with P.J. J. Appl. 24: Google Scholar), R.M. Biol. PubMed Scopus Google Scholar), and L. data and of data of of of I, heme I, heme heme heme heme heme heme formate heme formate heme in a catalyzes and iodination reactions. CPO has been crystallized in the presence of but the high chloride and resolution of the data chloride were in the (11Sundaramoorthy M. Terner J. Poulos T.L. Structure. 1995; 3: 1367-1377Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar). We to identify halide binding sites by of the of and This was not with because the of this is at CPO was crystallized in the presence of either or resolution data of a crystal was shows a at the data was at the electron a binding site was identified at the surface of CPO and This binding site has of for bromide and for iodide and likely as a site for halide The anion binds in a on the surface and is by a hydrogen with the of and by in a water This site is to a narrow channel the surface with the heme. In addition, there is a channel with a the surface and active site but bound in to this channel. Å away from the site a binding site is located within the narrow channel The anions bound at this not directly with the but instead in a water which might the low for this site for iodide and for The channel has a of binding site for iodide is present at the end of the channel the heme This site is located Å from halide binding site and Å from the heme iron. The group of a hydrogen with the iodide The iodide also hydrogen with water which are present in the of the channel The other of are with of Å to the side of and These sites suggest a pathway for halide access to the heme the narrow channel. binding sites of were in the wide we of halide anions the channel to the heme. crystals with high of bromide or iodide can be used to for The anions in the at the surface M. Biol. PubMed Scopus Google Scholar). in CPO, halide binding are located at the surface of the these the anions water from the The halide anions form hydrogen with side and of the and form with The iodide and bromide at binding site also with a CPO These binding sites on the surface are far away from the to the heme and are to be of catalytic We a resolution data from an crystal at the The high resolution structure into the of water at the active site the heme, the channel is by side with the of Glu-183 and narrow water is present in the of this channel. The water are on a within this not at The side of and are located of the heme and the active site from The between and is water molecule is Å away from both side and is at this by a hydrogen with Glu-183. The between the and this water is of CPO with has been proposed to the chlorination of CPD during biosynthesis of (1Beckwith J.R. Hager L.P. J. Biol. Chem. 1963; 238: 3091-3094Abstract Full Text PDF PubMed Google Scholar, 2Beckwith J.R. Clark R. Hager L.P. J. Biol. Chem. 1963; 238: 3086-3090Abstract Full Text PDF PubMed Google Scholar). NMR titration experiments that CPO binds CPD with only low (22Wang X. Goff H.M. Biochim. Biophys. Acta. 1997; 1339: 88-96Crossref PubMed Scopus (9) Google Scholar), which might organic substrates into CPO crystals was unsuccessful (23Sundaramoorthy M. Terner J. Poulos T.L. Chem. Biol. 1998; 5: 461-473Abstract Full Text PDF PubMed Scopus (143) Google Scholar). Because CPD binds only crystals were in high of CPD data was to a resolution of CPD binds the heme and is between and with a of Å to both side and The substrate the heme with one oxygen of CPD the of the The between this oxygen and Glu-183 is The oxygen of CPD hydrogen with water one of which in a hydrogen with whereas the in a of water a small is in the upon substrate the side of Å away from the active site for The of the bound substrate with to the chlorination for the which halogenated, is in an It away from the active and the between the carbon and the is the molecule binds to the active which not formation of the hypohalite intermediate from hydrogen peroxide and because the between the CPD oxygen and is only 3.4 the structure the of NMR titration experiments (22Wang X. Goff H.M. Biochim. Biophys. Acta. 1997; 1339: 88-96Crossref PubMed Scopus (9) Google Scholar). CPD binds with only low but iodide was present the between CPO and CPD When the iodide and the structure it that CPD with the binding because the between the oxygen and the iodide is only The of CPD binding in the presence of is similar to the in the of (22Wang X. Goff H.M. Biochim. Biophys. Acta. 1997; 1339: 88-96Crossref PubMed Scopus (9) Google Scholar), indicating that chloride not at this site and the that this binding site is for CPD to at a more we to the binding site by soaking the crystals first in cyanide, which to the and then transfer the crystals into a solution. data were to a resolution of cyanide molecule bound at the heme is in the electron The diatomic ligand binds in a with an of The between the cyanide carbon and the is Å This from the reported of Å for a complex (23Sundaramoorthy M. Terner J. Poulos T.L. Chem. Biol. 1998; 5: 461-473Abstract Full Text PDF PubMed Scopus (143) Google Scholar). The is in with reported R. Biochem. Biophys. 2003; PubMed Scopus Google Scholar, M. T. R. Biochemistry. PubMed Scopus Google Scholar). However, CPD was in the was at the iodide binding site This to which was used to the CPD solution. The in the soaking was It has been that is also a substrate of CPO J. Neidleman S.L. G. M. Biochem. Biophys. Res. Commun. 1983; 116: PubMed Scopus Google Scholar). CPO with and in the of and When the is with bromide or the and of either or are J. Neidleman S.L. G. M. Biochem. Biophys. Res. Commun. 1983; 116: PubMed Scopus Google Scholar). The oxygen and the group of form a hydrogen The molecule is not in a for the to with the compound I intermediate to form because the away from the active site. the between and is for a interaction with a reactive oxygen it is likely that binds at this site it the channel to the heme. The between the group of and the of the cyanide a halogenation of by the hypohalite CPO with and acetate, and formate all to the active site of CPO. The have been determined The reported at pH are for nitrate, for acetate, and for M. K. Hager L.P. Biochemistry. PubMed Scopus Google Scholar). low both and at the distal site of the heme and are between and The and In its first the and also the are located in a similar to the heme but not the because that with This residue also to in a the heme in its The between the by the heme and the molecule is The between the oxygen and the is only Å with Å in the heme and the can The of in the high resolution crystal structure are with data that the complex has a state M. K. Hager L.P. Biochemistry. PubMed Scopus Google Scholar). the of Å between the oxygen and heme with the observation that the complex a high state M. K. Hager L.P. Biochemistry. PubMed Scopus Google Scholar). In the the side of Å away from the active similar to the structure but unlike the The oxygen of both and the first of the the as the ferric water and form hydrogen with Glu-183. In the structure an molecule is bound the ligand which was as a This hydrogen with both and Glu-183 to be to form a hydrogen with the CPO shows a and formate at pH with pH M. K. Hager L.P. Biochemistry. PubMed Scopus Google Scholar). pH and formate are deprotonated and on both the catalytic residue and the are pH acidic than glutamic acid are and the can form hydrogen with the deprotonated the is in to pH The structures of CPO complexed with formate were determined in the presence of either or The formate binds in a to the heme in the structure This is in to the in the formate a to the heme and the proximal oxygen of formate directly interacts with the P. J. Biochemistry. 44: PubMed Scopus Google Scholar). In to the acetate, nitrate, and CPO the oxygen of formate not the of the ferric water However, binding of formate not for the water molecule to The formate molecule hydrogen with Glu-183 and an molecule bound at a similar as in the of formate to CPO a low complex in M. K. Hager L.P. Biochemistry. PubMed Scopus Google Scholar), which that formate binds as the ligand of the heme in structure the between the carbon and is for a the of on formate we of CPO in the presence of either formate or and with both formate and in a at pH to the used for of formate in a from to as expected for the formation of a low complex M. K. Hager L.P. Biochemistry. PubMed Scopus Google Scholar). of a small of the to However, both formate and were the was at indicating that in and at of has on formate binding to CPO. of on formate complex formation in the we also determined the structure of the complex with a In this structure the formate binds in the first is similar to the one in the in the this formate hydrogen to a water and Glu-183. In the the formate molecule binds in a the heme. In this the formate is a ligand of the heme because the between formate oxygen and is only In to the which the formation of a low crystal structure indicates a state of the formate complex similar to the complex This could be a because the data were at whereas the were at It has been that can the heme state A. C. G. J. Biol. Chem. 5: PubMed Scopus Google Scholar, C. A. G. J. Biochem. PubMed Scopus Google Scholar). We determined the crystal structures of CPO complexed with its substrates and with small ligands, nitrate, acetate, and binding of CPD and at the active site. The side of Å away from the active site to for the binding of In all structures and the of CPO with diatomic or triatomic (23Sundaramoorthy M. Terner J. Poulos T.L. Chem. Biol. 1998; 5: 461-473Abstract Full Text PDF PubMed Scopus (143) Google Scholar), the catalytic Glu-183 not The side of Glu-183 is because it hydrogen to a water which also interacts with the group of the heme, and a hydrogen with The group of Glu-183 is by with heme. for the of the Glu-183 side is that the have with the in of this It has been proposed that and as a proton facilitating cleavage of (11Sundaramoorthy M. Terner J. Poulos T.L. Structure. 1995; 3: 1367-1377Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar, H.A. Woggon W.D. Chem. Biol. 1997; 4: 367-372Abstract Full Text PDF PubMed Scopus (85) Google Scholar). one that hydrogen the substrate of CPO, binds at the heme in a similar as cyanide, Glu-183 a proton from the proximal oxygen bound to the to the of the cyanide because the is of a peroxide complex a of Å between the proximal oxygen and M. J. Am. Chem. Soc. Scopus Google Scholar). In this the between the proximal oxygen and deprotonated oxygen of Glu-183 be which is to for proton by Glu-183. we that Glu-183 is not of a required for heterolytic O-O cleavage of a hydrogen peroxide on the structures we a mechanism for binding to the heme in CPO We suggest that the deprotonated Glu-183 first abstracts a proton from the incoming hydrogen peroxide. The hydroperoxo-anion then binds at the heme yielding the compound 0. In the next Glu-183 protonates the distal oxygen of compound and the O-O is water is released, and compound I similar to the mechanism for peroxidases T.L. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, C. L. Casella L. R. L. 3: PubMed Scopus Google Scholar, J. J. Am. Chem. Soc. PubMed Scopus Google Scholar). of compound was reported during the of with from experiments at low or J. Am. Chem. Soc. 1992; Scopus Google Scholar). However, and could not the compound intermediate at with a M. K. S. S. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), for an in has in been reported M. T.M. J.R. J. Am. Chem. Soc. 2003; PubMed Scopus Google Scholar). in the compound species might be due to the that the substrate hydrogen peroxide the required for and that the heterolytic O-O cleavage is catalyzed by the compound Compound I with a halide anion to form the hypohalite on the and we suggest that access of to the heme is likely to the narrow channel the heme with the we the wide channel. binding sites were identified at the surface to the narrow within the and at the other end of the narrow channel the heme. The binding site the heme is also a binding site for other the substrate acetate, and glycol. This binding site might as an intermediate the substrate on the to the heme. Binding at this is because hydrogen with these of substrates could also with this residue might the of halogenation and reactions. We that the of these reactions might be by of to an residue and located on the side of the wide could be to or CPO has been during the by and Here we present crystal structures of CPO complexed with small and its the first of bound organic and the implications for compound and compound I formation. the it is that structure of the short-lived to a of the catalytic mechanism of this We S. for for of the and the of the and for during data