X-ray Crystal Structure and Characterization of Halide-binding Sites of Human Myeloperoxidase at 1.8 Å Resolution

The x-ray crystal structure of human myeloperoxidase has been extended to 1.8 Å resolution, using x-ray data recorded at −180 °C (r = 0.197, freer = 0.239). Results confirm that the heme is covalently attached to the protein via two ester linkages between the carboxyl groups of Glu242 and Asp94 and modified methyl groups on pyrrole rings A and C of the heme as well as a sulfonium ion linkage between the sulfur atom of Met243and the β-carbon of the vinyl group on pyrrole ring A. In the native enzyme a bound chloride ion has been identified at the amino terminus of the helix containing the proximal His336. Determination of the x-ray crystal structure of a myeloperoxidase-bromide complex (r = 0.243, free r = 0.296) has shown that this chloride ion can be replaced by bromide. Bromide is also seen to bind, at partial occupancy, in the distal heme cavity, in close proximity to the distal His95, where it replaces the water molecule hydrogen bonded to Gln91. The bromide-binding site in the distal cavity appears to be the halide-binding site responsible for shifts in the Soret band of the absorption spectrum of myeloperoxidase. It is proposed that halide binding to this site inhibits the enzyme by effectively competing with H2O2 for access to the distal histidine, whereas in compound I, the same site may be the halide substrate-binding site. The x-ray crystal structure of human myeloperoxidase has been extended to 1.8 Å resolution, using x-ray data recorded at −180 °C (r = 0.197, freer = 0.239). Results confirm that the heme is covalently attached to the protein via two ester linkages between the carboxyl groups of Glu242 and Asp94 and modified methyl groups on pyrrole rings A and C of the heme as well as a sulfonium ion linkage between the sulfur atom of Met243and the β-carbon of the vinyl group on pyrrole ring A. In the native enzyme a bound chloride ion has been identified at the amino terminus of the helix containing the proximal His336. Determination of the x-ray crystal structure of a myeloperoxidase-bromide complex (r = 0.243, free r = 0.296) has shown that this chloride ion can be replaced by bromide. Bromide is also seen to bind, at partial occupancy, in the distal heme cavity, in close proximity to the distal His95, where it replaces the water molecule hydrogen bonded to Gln91. The bromide-binding site in the distal cavity appears to be the halide-binding site responsible for shifts in the Soret band of the absorption spectrum of myeloperoxidase. It is proposed that halide binding to this site inhibits the enzyme by effectively competing with H2O2 for access to the distal histidine, whereas in compound I, the same site may be the halide substrate-binding site. myeloperoxidase lactoperoxidase polyethylene glycol (average mass, 8 kDa) Myeloperoxidase (MPO, EC1.11.1.7)1 is a heme-containing enzyme found in mammalian neutrophils, where it catalyzes the hydrogen peroxide mediated peroxidation of halide ions (1.Agner K. Acta Chem. Scand. 1941; 2 (Suppl. 8): 1-62Google Scholar, 2.Harrison J.E. Schultz J. J. Biol. Chem. 1976; 251: 1371-1374Abstract Full Text PDF PubMed Google Scholar) and the pseudohalide thiocyanate (3.Wever R. Kast W.M. Kasinoedin J.H. Boelens R. Biochim. Biophys. Acta. 1982; 709: 212-219Crossref PubMed Scopus (95) Google Scholar, 4.Van Dalen C.J. Whitehouse M.W. Winterbourn C.C. Kettle A.J. Biochem. J. 1997; 327: 487-492Crossref PubMed Scopus (350) Google Scholar), according to the following reaction. H2O2+Cl−+H3O+=HOCl+2H2O REACTION 1Products of these reactions and their secondary metabolites are responsible for killing phagocytized bacteria and viruses (5.Klebanoff S.J. Science. 1970; 169: 1095-1097Crossref PubMed Scopus (228) Google Scholar, 6.Belding M.E. Klebanoff S.J. Ray C.G. Science. 1970; 167: 195-196Crossref PubMed Scopus (142) Google Scholar). MPO is one member of a gene family of mammalian peroxidases that also includes eosinophil peroxidase, lactoperoxidase (LPO), thyroid peroxidase, and prostaglandin H synthase (7.Merlie J.P. Fagan D. Mudd J. Needleman P. J. Biol. Chem. 1988; 263: 3550-3553Abstract Full Text PDF PubMed Google Scholar, 8.Kimura S. Ikeda-Saito M. Proteins Struct. Funct. Genet. 1988; 3: 113-120Crossref PubMed Scopus (139) Google Scholar, 9.Cals M.M. Maillart P. Brignon G. Anglade P. Dumas B.R. Eur. J. Biochem. 1991; 198: 733-739Crossref PubMed Scopus (115) Google Scholar). The mature enzyme is a 140-kDa dimer of identical halves, each consisting of two polypeptide chains of 108 and 466 amino acids resulting from post-translational excision of 6 amino acids from a single polypeptide precursor (10.Koeffler H.P. Ranyard J. Pertcheck M. Blood. 1985; 65: 484-491Crossref PubMed Google Scholar, 11.Akin D.T. Kinkade Jr., J.M. J. Biol. Chem. 1986; 261: 8370-8375Abstract Full Text PDF PubMed Google Scholar). Each half molecule contains a covalently bound heme that exhibits unusual spectral properties. The Soret band at 428 nm in the ferric enzyme is considerably red-shifted compared with other heme proteins, and relatively strong absorption bands in the visible region are responsible for the characteristic green color of the enzyme (12.Wever R. Plat H. Biochim. Biophys. Acta. 1981; 661: 235-239Crossref PubMed Scopus (42) Google Scholar). Selective cleavage of a single disulfide bridge linking the two halves of MPO yields the hemi-enzyme that exhibits spectral and catalytic properties indistinguishable from those of the intact enzyme (13.Andrews P.C. Krinsky N.I. J. Biol. Chem. 1981; 256: 4211-4218Abstract Full Text PDF PubMed Google Scholar). The overall protein fold of MPO was first revealed by a 3-Å resolution crystal structure of the canine enzyme (14.Zeng J. Fenna R.E. J. Mol. Biol. 1992; 226: 185-207Crossref PubMed Scopus (271) Google Scholar). Apparently identical halves of the dimeric molecule are related by a noncrystallographic dyad axis and covalently linked by a single disulfide bridge at Cys153. The secondary structure is largely α-helical, with very little β-sheet. Each half molecule consists of a central core of five helices and a covalently attached heme. Four of these helices derive from the large polypeptide and the fifth from the small. The remainder of the large polypeptide folds into four separate domains and a single open loop that surround the central core. The small polypeptide wraps around the surface of the molecule with only its carboxyl-terminal helix penetrating the interior to form part of the central core. A very similar protein fold has been found for the catalytic domain of the membrane-bound enzyme prostaglandin H synthase, which shares 22% sequence identity with MPO (15.Picot D. Loll P.J. Garavito M. Nature. 1994; 367: 243-249Crossref PubMed Scopus (1154) Google Scholar). Structure determination has confirmed the presence of a calcium-binding site and three sites of asparagine-linked glycosylation (Asn189, Asn225, and Asn317) as well as the identities of the proximal His336 and distal His95. However, the relatively low resolution (3 Å) did not allow full characterization of the covalent linkages between the heme and the protein. Details of these interactions and full characterization of the heme by a resolution crystal structure of the human enzyme R. J. Biochem. Biophys. PubMed Scopus Google Scholar). The heme was identified as a of in which the methyl groups on pyrrole rings A and C been modified to allow of ester linkages with Glu242 and A covalent was identified as a sulfonium ion linkage between the sulfur atom of and the β-carbon of the vinyl group on pyrrole ring A. of the has been confirmed by of the heme by cleavage and P. J. Kinkade Jr., J.M. Biochem. Biophys. PubMed Scopus (42) Google Scholar). The between and hydrogen peroxide in the of compound I, in which two are as D. A. S. A. PubMed Scopus Google Scholar). In the of this form of the enzyme is of peroxidation of halide ions and thiocyanate of a of and The by which MPO catalyzes halide peroxidation is H2O2 and halide are also is with to and at is of the of with J.M. Eur. J. Biochem. PubMed Scopus Google Scholar, J.M. A.J. Biol. PubMed Scopus Google Scholar, Plat H. R. Biochim. Biophys. Acta. PubMed Scopus Google Scholar, P.C. Krinsky N.I. J. Biol. Chem. 1982; Full Text PDF PubMed Google Scholar). The of H2O2 with MPO to form compound is A group on the enzyme with a of to to be the distal histidine, be for H2O2 binding to whereas halide binding is at low this group is R. Biochim. Biophys. Acta. PubMed Scopus Google M. PubMed Scopus Google Scholar). the binding of to and of the Soret from 428 nm to and and of these spectral been to the of halide binding to the enzyme J.M. Eur. J. Biochem. PubMed Scopus Google Scholar, R. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). However, these at the of halide-binding sites on the enzyme and are separate sites for halide binding as a and as In this on the site region of MPO as revealed by a structure determination of the human enzyme to 1.8 Å resolution, using x-ray data recorded at −180 In structure determination of complex has revealed a of halide-binding and their to the catalytic of the enzyme is of human MPO C by a similar to those J. Mol. Biol. 1988; PubMed Scopus Google Scholar, Fenna R.E. PubMed Scopus Google Scholar). containing 2 and with containing the same of the with to at to containing the and for low data was as data recorded using a on a x-ray with A native low data to 1.8 Å resolution was recorded from a single in a of from a crystal The crystal to was at and of data recorded at and data with and S. of the Scholar). The to group with a single dimeric molecule of MPO as the from the crystal a = = = = compared with of a = = = = at R. J. Biochem. Biophys. PubMed Scopus Google Scholar), of the at and are in and of in a The for was the Å resolution structure of human MPO in the access R. J. Biochem. Biophys. PubMed Scopus Google Scholar). consists of protein and water The was for in the resolution to with from for and of Å and using the 1.8 Å resolution low data was using Scholar) and from and R. Acta 1991; Scopus Google Scholar). of and between of using in with the The resolution of x-ray data in the was from Å to and 1.8 A of the was not in to allow for of a free Nature. 1992; PubMed Scopus Google Scholar). water in the as at a of at in that hydrogen in the that to at 2 in following a of from the of in the hydrogen for water in the distal heme cavity in the Å resolution structure of human water in the distal cavity from the the of their be from the of and to the where by and the presence of protein not a was in the and in in the for data in the resolution The of the was using the M.W. J.M. Scholar). for the been with the access A crystal of human MPO was to consisting of 2 and bromide. for the crystal was in the same with as in a of at −180 data recorded as for the native enzyme and are in The of bound ions from a using as ions in the and of the structure was using as for the of the complex been with the access The consists of amino 2 2 2 chloride 6 and water The for in the to 1.8 Å is and the free is The from for and are Å and A for the molecule of in the and in in halves of the molecule has in the and in the for of the the of this appears to be by hydrogen between the and groups of of its with amino are at and and between and in each half of the for the and chains to be the for other protein at resolution according to the M.W. J.M. Scholar). the two halves of the MPO molecule A and in the are in the of chains on the surface of the In the A half of the molecule is in in the and for this is a little the is in the for protein in the A half compared with the half The amino is on the first in the small polypeptide as (14.Zeng J. Fenna R.E. J. Mol. Biol. 1992; 226: 185-207Crossref PubMed Scopus (271) Google Scholar). In the sequence of human from the are 2 and to W.M. A. S. S. H.P. M. G. PubMed Scopus Google Scholar, K. S. S. J. Biol. Chem. Full Text PDF PubMed Google Scholar), these are not in the The is very well for four polypeptide for two surface and in halves of the The amino terminus of the small polypeptide is in the A half well in the half where the chains of are in hydrogen with in a the carboxyl terminus of the large polypeptide is in the half well in the A half where the carboxyl group is hydrogen bonded to the of in a A of chains on the surface of the molecule in the A half and in the of in this is to for the of the chains of and The overall polypeptide fold for human MPO is the same as for the enzyme (14.Zeng J. Fenna R.E. J. Mol. Biol. 1992; 226: 185-207Crossref PubMed Scopus (271) Google Scholar) and in the However, the resolution of the has characterization of a of The calcium-binding site has The of and the of the whereas the other five carboxyl and and and carboxyl are of these from the loop of the large whereas the other two which is to the distal in the small The of of the the glycosylation site been The consists of the structure with a linked to the first between these chains in the two halves of the molecule of the dimer in Details of the and its interactions with the protein be The heme group is a of in which the methyl groups on pyrrole rings A and C been modified to allow of ester linkages with the carboxyl groups of Glu242 and In the β-carbon of the vinyl group on pyrrole ring A a covalent with the sulfur atom of to a sulfonium ion linkage The resulting on the sulfur atom not to be in interactions with the protein. The heme ring is considerably from pyrrole rings and are ring A to a ring C are the distal resulting in a structure for the heme. of these to the it is to the of the central atom with to a as is for heme-containing However, the is to the proximal Å a the of pyrrole rings and D. The of the proximal to the with to of whereas the of this is hydrogen bonded to the of The carboxyl groups of of the heme also interactions with the protein. The ring with the groups of and and also a hydrogen with a water The ring C with of the hydrogen with the and with a water whereas the other hydrogen with the carboxyl group of for these interactions and the covalent are in methyl vinyl methyl in a to the distal cavity via a with a surface pyrrole ring D. The pyrrole ring the of the whereas the surface consists of the and of and and to the of the A with water from this to the surface of the protein. The distal heme cavity is by the chains of His95, and with five water Each of these three chains is hydrogen bonded to a water whereas a water molecule is hydrogen bonded to the heme pyrrole ring C hydrogen between these water as in A fifth water molecule is hydrogen bonded to and also to The distal is hydrogen bonded to which is between its and the heme from the and the Å) that it is hydrogen bonded to the and is not to the heme The of at the of the distal cavity, is hydrogen bonded to as well as to the of the ester between Glu242 and the heme pyrrole ring A methyl The of is in hydrogen bonded to the of The group of is hydrogen bonded to a bridge with the carboxyl group of as well as a hydrogen with the of shown in a of hydrogen water and the of from the distal to the surface of the at a from the to the distal The of the distal is hydrogen bonded to a water which is from the distal cavity water by the water is also hydrogen bonded to of a bridge between the two The of is hydrogen bonded to a water which is the first in a of four water linked by hydrogen and to the surface of the protein. Each of the five water is hydrogen bonded to The first is a carboxyl of whereas the other four are the of the seen was at a water molecule hydrogen bonded to the groups of and in halves of the is close to the amino terminus of a bound in this with the partial with the helix the in the of 2 chloride the chloride was in the at full and a of was at this chloride site. The site is of at the amino terminus of the helix containing the proximal of the small and two of polypeptide are in a and by two hydrogen The chloride has three and water in The water molecule is hydrogen bonded to the of and the group of are two in the of the chloride the with its ring at a of Å from the whereas is from the ion with its Å from the the binding properties of this was to a the x-ray data recorded from a crystal in at A revealed four separate ions bound to each half of the The of the at and in the two halves of the molecule at the site full by at this site and in this of the on that of the native revealed protein ions at surface sites in each half of the at and at water molecule and at and at water molecule these water replaced by in the their to and of these ions are well from the heme group at of and Å from the heme the the groups of and with a water molecule the whereas at the group of and the group of are in is of binding to the heme of and in the distal heme cavity at the of water molecule which is hydrogen bonded to the of Bromide ions in the at this to of in each half of the of the and native small shifts of the chains of the distal and the heme ester linked Glu242 The of which is only Å from in the native Å from the and the also Å from the bound are on the of the of in the as from a and may not be of water and by and Å from the also The distal cavity is Å from the by in the native interactions between the and water and the and the of The heme atom to the is the bridge between pyrrole rings A and at a of and the heme is at a of not to be between the and the group of the distal The of this the to the at a of Å and as is in ion with proximal helix chloride proximal helix distal cavity in a The of heme binding revealed by may be to other of this gene family the Asp94 and at (14.Zeng J. Fenna R.E. J. Mol. Biol. 1992; 226: 185-207Crossref PubMed Scopus (271) Google Scholar). It is that other mammalian peroxidases as eosinophil peroxidase, and thyroid also ester linkages to those of that the sulfonium ion linkage is a to It has been proposed by A.J. M. A. R. J. Chem. 1997; Scopus Google Scholar, A. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) that the sulfonium ion linkage a between the sulfur atom and the of the vinyl by with the in the of groups in However, a linkage is with the resolution crystal which that the the vinyl it has been found that the of two ester between the heme and polypeptide of is a hydrogen of to and of ester with to Asp94 and Glu242 of MPO J.M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). confirmed the presence of two heme ester linkages in and MPO and also their presence in eosinophil A. R. J. Biol. Chem. 1997; Scopus (42) Google Scholar). The Soret bands of the visible absorption of mammalian peroxidases are red-shifted with to those seen in other heme proteins, and these spectral shifts been to the of the protein on the spectral properties of the heme (12.Wever R. Plat H. Biochim. Biophys. Acta. 1981; 661: 235-239Crossref PubMed Scopus (42) Google Scholar). In this it is that a of human MPO exhibits a Soret band at considerably from the band of the native enzyme in its form A. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. R. J. Biol. Chem. 1997; Scopus (42) Google Scholar, A. Fenna R. A. Biochem. Biophys. 1994; PubMed Scopus Google Scholar). a Glu242 of one of the heme ester exhibits a Soret band at and Asp94 to the other ester also has a Soret band at nm A. Fenna R. A. Biochem. Biophys. 1994; PubMed Scopus Google Scholar, R. G. A. R. A. R. J. Chem. Scopus Google Scholar). of that the covalent linkages to the heme in MPO to the shifts of the Soret band in the visible absorption is to that the covalent linkages to the heme are in the catalytic of mammalian The catalytic of was shown to be on the of covalently bound heme in the of in which in covalent heme been also catalytic properties from those of the native enzyme J.M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). the the Glu242 MPO was of the peroxidation of chloride and catalytic for the single A. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. R. J. Biol. Chem. 1997; Scopus (42) Google Scholar, R. G. A. R. A. R. J. Chem. Scopus Google Scholar). In and the of the distal is hydrogen bonded to the of and it has been proposed that this the in which the is free to a from the peroxide and R. in and Scholar). In MPO the of is hydrogen bonded to a water which is linked by to a of four water a of hydrogen to the surface of the a also to from the distal and that the is free to a from of a bound chloride ion to for the at the amino terminus of the proximal helix in the native enzyme is by that this site can also be by bromide. However, that the spectral that halide binding to MPO from halide binding to the site that in the distal A of this the close proximity of this site to the heme is with the for a bound to the in the spectral the halide to Å from the distal is with data that halide binding is by the of a group on the enzyme with a of to which has been to be the distal R. Biochim. Biophys. Acta. PubMed Scopus Google Scholar, M. PubMed Scopus Google Scholar). low halide binding be by between the and the halide the of halide binding in the distal cavity is with the for chloride and binding to MPO Plat H. R. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). the for chloride is on the of whereas for it is found that of MPO in at ion at at this site that the be that not water in this However, in the native enzyme for which the chloride of 2 at is half the of chloride ion was not in the distal the same data that the proximal site is by chloride in the native structure and by in the at the halide are with its as the site responsible for spectral that in MPO the distal cavity bromide-binding site to the site for of MPO by is with to J.M. Eur. J. Biochem. PubMed Scopus Google Scholar, J.M. A.J. Biol. PubMed Scopus Google Scholar, Plat H. R. Biochim. Biophys. Acta. PubMed Scopus Google Scholar, P.C. Krinsky N.I. J. Biol. Chem. 1982; Full Text PDF PubMed Google Scholar), and the distal cavity bromide-binding site is only Å from the of the distal close to with be with H2O2 from binding to MPO Fenna R.E. PubMed Scopus Google Scholar) has that the first in compound the of a hydrogen between H2O2 and the of the distal histidine, to of a to the The presence of a halide in of water molecule in the distal cavity with the of the distal to form a hydrogen with a from that are separate sites on MPO for halide binding as and as J.E. Schultz J. J. Biol. Chem. 1976; 251: 1371-1374Abstract Full Text PDF PubMed Google Scholar, R. Kast W.M. Kasinoedin J.H. Boelens R. Biochim. Biophys. Acta. 1982; 709: 212-219Crossref PubMed Scopus (95) Google Scholar, Plat H. R. Biochim. Biophys. Acta. PubMed Scopus Google Scholar, P.C. Krinsky N.I. J. Biol. Chem. 1982; Full Text PDF PubMed Google Scholar), that is only a single site J.M. Eur. J. Biochem. PubMed Scopus Google Scholar, R. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). of their from the the two surface bromide-binding sites are to be in be to the proximal helix site and the distal cavity site. A peroxidation at the proximal helix halide-binding site a free to the free with compound of which is also on the proximal of the heme J.E. J.M. J. PubMed Scopus Google H. A. J. Biol. Chem. 1976; Full Text PDF Google Scholar). the MPO proximal helix halide-binding site includes two been of of amino free in the spectrum of MPO compound A between a catalytic halide binding in the distal heme cavity as to a site is the of the atom in the of the reaction. In the distal cavity, the of compound I, from H2O2 is the whereas at a site a water molecule be the the of this atom in the of MPO chloride peroxidation has not been this a for this site in the catalytic of MPO be a as of the proximal helix with the helix appears of halide peroxidation by MPO not a halide substrate-binding site on the it is only for to with compound for peroxidation to In this it has been that the complex of MPO may be a for compound I, and one has that halide ions close to the heme in MPO Ikeda-Saito M. Biochim. Biophys. Acta. 1991; PubMed Scopus Google Scholar). In compound of MPO the distal cavity bromide-binding site be to and also be the site at which as on a to of that a halide ion bound at this site be Å from the heme and Å from the heme bridge between pyrrole rings A and to the heme and of the into the of the reaction. In a the distal a in first in a from hydrogen peroxide to of the and in the halide for to the heme of compound and with the for with the of human