Binding Modes of Aromatic Ligands to Mammalian Heme Peroxidases with Associated Functional Implications

The binding and structural studies of bovine lactoperoxidase with three aromatic ligands, acetylsalicylic acid (ASA), salicylhydoxamic acid (SHA), and benzylhydroxamic acid (BHA) show that all the three compounds bind to lactoperoxidase at the substrate binding site on the distal heme side. The binding of ASA occurs without perturbing the position of conserved heme water molecule W-1, whereas both SHA and BHA displace it by the hydroxyl group of their hydroxamic acid moieties. The acetyl group carbonyl oxygen atom of ASA forms a hydrogen bond with W-1, which in turn makes three other hydrogen-bonds, one each with heme iron, His-109 Nϵ2, and Gln-105 Nϵ2. In contrast, in the complexes of SHA and BHA, the OH group of hydroxamic acid moiety in both complexes interacts with heme iron directly with Fe-OH distances of 3.0 and 3.2Å respectively. The OH is also hydrogen bonded to His-109 Nϵ2 and Gln-105Nϵ2. The plane of benzene ring of ASA is inclined at 70.7° from the plane of heme moiety, whereas the aromatic planes of SHA and BHA are nearly parallel to the heme plane with inclinations of 15.7 and 6.2°, respectively. The mode of ASA binding provides the information about the mechanism of action of aromatic substrates, whereas the binding characteristics of SHA and BHA indicate the mode of inhibitor binding. The binding and structural studies of bovine lactoperoxidase with three aromatic ligands, acetylsalicylic acid (ASA), salicylhydoxamic acid (SHA), and benzylhydroxamic acid (BHA) show that all the three compounds bind to lactoperoxidase at the substrate binding site on the distal heme side. The binding of ASA occurs without perturbing the position of conserved heme water molecule W-1, whereas both SHA and BHA displace it by the hydroxyl group of their hydroxamic acid moieties. The acetyl group carbonyl oxygen atom of ASA forms a hydrogen bond with W-1, which in turn makes three other hydrogen-bonds, one each with heme iron, His-109 Nϵ2, and Gln-105 Nϵ2. In contrast, in the complexes of SHA and BHA, the OH group of hydroxamic acid moiety in both complexes interacts with heme iron directly with Fe-OH distances of 3.0 and 3.2Å respectively. The OH is also hydrogen bonded to His-109 Nϵ2 and Gln-105Nϵ2. The plane of benzene ring of ASA is inclined at 70.7° from the plane of heme moiety, whereas the aromatic planes of SHA and BHA are nearly parallel to the heme plane with inclinations of 15.7 and 6.2°, respectively. The mode of ASA binding provides the information about the mechanism of action of aromatic substrates, whereas the binding characteristics of SHA and BHA indicate the mode of inhibitor binding. Lactoperoxidase (LPO) 4The abbreviations used are: LPOlactoperoxidaseMPOmyeloperoxidaseEPOeosinophil peroxidaseASAacetylsalicylic acidSHAsalicylhydroxamic acidBHAbenzylhydroxamic acidABTS2,2′-azino-bis (3-ethylbenzthiazoline-sulfonic acid)r.m.s.d.root mean square deviation. 4The abbreviations used are: LPOlactoperoxidaseMPOmyeloperoxidaseEPOeosinophil peroxidaseASAacetylsalicylic acidSHAsalicylhydroxamic acidBHAbenzylhydroxamic acidABTS2,2′-azino-bis (3-ethylbenzthiazoline-sulfonic acid)r.m.s.d.root mean square deviation. (EC 1.11.1.7) is a member of the family of glycosylated mammalian heme-containing peroxidase enzymes which also includes myeloperoxidase (MPO), eosinophil peroxidase (EPO), and thyroid peroxidase. These enzymes also show functional similarities to non-homologous plant and fungal peroxidases because they follow a similar scheme of reaction (1Furtmüller P.G. Zederbauer M. Jantschko W. Helm J. Bogner M. Jakopitsch C. Obinger C. Arch. Biochem. 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The SHA and BHA directly with the heme iron, whereas ASA interacts through the heme water which in turn is to the heme These studies a the of substrate and inhibitor binding in the two mammalian bovine from The and the A.K. Singh N. Sharma S. Singh S.B. Kaur P. Bhushan A. Srinivasan A. Singh T.P. J. Mol. Biol. 2008; 376: 1060-1075Crossref PubMed Scopus (92) Google Scholar). The the of binding of and BHA to LPO in the the various The of the by in the by R. C. Singh T.P. Acta Biol. 1995; PubMed Scopus Google Scholar). 3.0 of acid in with of to the of hydrogen to the The at as a of the of The also from the of LPO complexes with and These crystal also used In the crystal the and ligand to at The and of in to a of The ligands and in the in to ligand of and of with of to of the all the three of LPO with and The to the of and BHA in the of LPO the of three complexes used the of and the with water and in the These crystal in the a with a of The of ASA in the by with basic the acetylsalicylic acid to The an with the ion in and the of at of the the of the hydroxamic acid in the of the complexes of LPO with SHA and BHA in the reaction with chloride J. Biol. Chem. Full Text PDF Google Scholar). acid a in both one each LPO complexes with and BHA at a on a at and The the three and W. 1997; PubMed Scopus Google Scholar). The of is in and the three complexes of LPO with and of of of group (1Furtmüller P.G. Zederbauer M. Jantschko W. Helm J. Bogner M. Jakopitsch C. Obinger C. Arch. Biochem. Biophys. 2006; 445: 199-213Crossref PubMed Scopus (279) Google Scholar) (1Furtmüller P.G. Zederbauer M. Jantschko W. Helm J. Bogner M. Jakopitsch C. Obinger C. Arch. Biochem. Biophys. 2006; 445: 199-213Crossref PubMed Scopus (279) Google Scholar) I.M. Moguilevsky N. Bollen A. van der Veen L.A. Otto C. Dekker H.L. Wever R. J. Biol. Chem. 1999; 274: 26794-26802Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) T.L. Edwards S.L. Wariishi H. Gold M.H. J. Biol. Chem. 1993; 268: 4429-4440Abstract Full Text PDF PubMed Google Scholar) oxygen in bond in bond in and all in the most in the in the in a The structures of the three complexes of LPO with ASA SHA and BHA the model of of bovine lactoperoxidase the to of with Acta Biol. 1997; PubMed Scopus Google Scholar) from the Acta Biol. 1994; PubMed Scopus Google Scholar). the of the and to of and of the used the of these in the The electron the heme in all the three The of heme in the of that with model of the and with M. Acta A. 1991; PubMed Scopus Google Scholar) and P. K. Acta D. PubMed Scopus Google Scholar) on a At the of these of the and to of and respectively. The at electron at four with two and one at two at two and one at and two at The electron also at the distal heme which the of and BHA and The electron one ion also in all the three These also electron in the The of all these in the of The at the of also electron the of in all the three The of water oxygen in water oxygen in and water oxygen in the of The with of and and respectively. The are in The and and in the with and respectively. The the four (i) and in with ASA SHA and BHA are in The of LPO these in the of of of is as that of catalyzing the oxidation of of at that the of the of lactoperoxidase by the substrate binding site by of heme iron with the The the peroxidase of The the and BHA complexes to as and respectively. These indicate that the peroxidase with SHA and BHA, but with ASA a The that all the three ligands in the one mode of binding ASA and a mode of binding SHA and The structures of the LPO complexes that SHA and BHA bind to heme iron ASA is at the position from the heme The oxidation of in the of ASA and to in the of SHA and BHA that SHA and BHA the binding of to heme iron, whereas ASA The of the is in The the of and on the of LPO to and respectively. the of and of the substrate binding site it of and that the of the substrate binding site is the binding of these The of the substrate binding site on the distal heme are In all the three the aromatic organic ligands on the distal heme side. from ASA is at a position that is from the heme iron the of SHA and The distances of the ligand from the heme iron and BHA are and respectively. The of ASA in the substrate binding site is also from of SHA and BHA as the plane of the aromatic ring of ASA is by 70.7° with to the plane of heme moiety, whereas of SHA and BHA are at 15.7 and 6.2°, respectively. In the of LPO water W-1, and in the substrate binding site on the distal side. from ASA to LPO at the distal by four conserved water and The two conserved water and and two water and are of an between LPO and The that both and of the heme moiety are The aromatic ring of ASA is also and plane is inclined at an of 70.7° with to the plane of the heme The heme iron is the site by The conserved water molecule the position of is at a of from the heme also forms two other hydrogen bonds with His-109 Nϵ2 at a of and Gln-105 Nϵ2 at a of The acetyl group oxygen atom of ASA is also to at a of The oxygen atom of acetyl group is to a water molecule that is to water which in turn is to the oxygen atom of ring C. The group oxygen and are to and Nϵ2 at distances of and The two are in the because of the of group in whereas such are in SHA and BHA because a similar moiety in SHA and BHA to with and Nϵ2. such are in MPO as well because the in MPO to the of LPO a in which the of that of in LPO, the substrate binding In the of the aromatic ring of ASA in the by and is well in a of The between the of the substrate binding site and the ligand in a by the substrate binding site The is to the of the heme moiety including the iron one of the and the conserved water molecule W-1, forms the on the and and makes the and and and forms the with and from the The is an to the binding site from the of the The and are similar in both LPO and The whereas is I.A. Singh A.K. Singh N. Sinha M. Singh S.B. Bhushan A. Kaur P. Srinivasan A. Sharma S. Singh T.P. J. Biol. Chem. 2009; 284: 14849-14856Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), substrate specificities in the two the and because is in the substrate the is as substrate The substrate in MPO is and van der distances between and of the substrate binding site on the distal bonds der in a from SHA to LPO in the substrate binding site on the distal side. The binding SHA to LPO is M. L. S. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). from it interacts with four and SHA a moiety to group of it is to with Nϵ2 and of it is well in the substrate binding In the heme moiety and four are The aromatic ring of SHA is inclined at from the plane of the The is to in the C.A. Fenna R.E. Biochemistry. 1996; PubMed Scopus Google Scholar), whereas the aromatic ring of ASA is at 70.7° in the a of the binding of SHA to LPO, four water W-1, and are by the The position of conserved heme water molecule is by hydroxyl group of hydroxamic acid moiety, which makes a with the heme SHA the a is in the of an water molecule is which is to both and Nϵ2 of In case the heme iron is from the plane the site by The OH group of the hydroxamic acid moiety is to heme iron at a of 3.0 The OH group is also to His-109 Nϵ2 and Gln-105 Nϵ2 with distances of and respectively. forms hydrogen bond with a water The benzene ring of SHA is in the of and forms a of with of ASA BHA is also in the substrate binding site on the distal The binding the binding of BHA to LPO is M. L. S. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). in the of the conserved water W-1, and are by BHA The BHA the as as benzene ring is the substrate a the water molecule in the of is present in the of and Nϵ2. that the position and of BHA are from of SHA in the The heme iron in case is from the plane of heme The benzene ring of BHA is parallel to the plane of ring with an of The between the oxygen atom of the hydroxyl group of BHA and heme iron is in the of it forms three hydrogen bonds with Gln-105 Nϵ2, His-109 Nϵ2, and a water The of between BHA and LPO are between LPO and ASA and the oxidation of a of aromatic electron through the of The of three complexes of LPO with and BHA indicate a between the binding of and BHA with implications the ligands to as substrates The of the of LPO with ASA that ASA interacts with heme iron the conserved heme water molecule the complexes of BHA with peroxidase A. Schuller D.J. K. Smith A.T. M. Biochemistry. PubMed Scopus Google Scholar) and peroxidase H. Y. Fukuyama K. FEBS Lett. 1997; PubMed Scopus Google Scholar) and that of SHA with A. peroxidase K. H. K. Fukuyama K. S. S. H. T. Biochemistry. 1999; PubMed Scopus Google Scholar) also with heme iron an water However, the complexes of SHA with LPO MPO C.A. Fenna R.E. Biochemistry. 1996; PubMed Scopus Google Scholar), and peroxidase E.L. Biochemistry. PubMed Scopus Google Scholar) as well as the of BHA with LPO show that the ligands directly with the heme The of with the heme iron the heme water molecule the modes of substrate whereas a with the heme iron in the of from ASA forms one hydrogen bond with conserved heme water molecule W-1, which in turn interacts with heme iron, Gln-105 and The oxygen atom of the acetyl group is to conserved water molecule which is to the oxygen atom of the of D. In to these two from and on one it forms two other hydrogen bonds with and Nϵ2 of from an forms a van der with distances the of the aromatic ring of ASA and of the the position of ASA is by the from all in the substrate binding the other SHA forms at hydrogen bonds with the heme moiety and through hydroxamic acid However, it a moiety to with and Nϵ2 as a from the side. SHA is the in the with heme iron, and also forms a similar of van der but in the of the of the van der are with the of the heme In the of BHA forms hydrogen bonds with the heme moiety, and which a with with that of SHA In a nearly similar of van der distances are but a of are by a of BHA is from the by with to SHA it is that ASA interacts with all the in the substrate binding site and the position of heme water molecule and is with to the site of the whereas SHA and BHA directly with the heme iron, the binding and the These binding of and SHA and BHA M. L. S. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar) to in with the structural as by the of SHA and BHA with The structures of three complexes of LPO with aromatic substrate, and two aromatic SHA and BHA indicate the in their modes of binding as a substrate and as an The aromatic substrate molecule is as the conserved heme water molecule W-1, whereas an inhibitor the heme water molecule and interacts directly with the heme a ligand to as a substrate of LPO, it to a moiety with the hydrogen bond and to with of the at the distal heme and moiety such as hydroxyl group with a group at the position of the aromatic ring that it is to hydrogen bonds with and Nϵ2. The are important to the in the aromatic ligands with to the heme moiety and In the of with and Nϵ2, the aromatic ligands the and displace the conserved heme water molecule W-1, in the with the heme a hydrogen molecule is to bind at the site of such ligands as ASA as a substrate and SHA and BHA as are the modes of binding of substrate and to it is important to the binding of LPO from that of The in the two enzymes both and are The in the of the substrate binding in two enzymes is by the differences in the of the in LPO and in a of these characteristics the in LPO are and Nϵ2, whereas the in MPO is the of the striking differences in the of the in the substrates by the two LPO hydrogen bond and whereas MPO moieties. In contrast, are differences in the modes of binding of and both enzymes by similar it that LPO and MPO show substrate but they