Structural properties of peroxidases

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Abstract

Peroxidases are heme proteins which are able to catalyze the oxidization of a large variety of substrates through the reaction with hydrogen peroxide. The specific biological function, the reduction potential of the iron and the nature of the substrates which can be oxidized, are strongly determined by the structural features of the protein matrix around the prosthetic group. In particular, two main features are considered to be responsible of the specificity of the biological function: the strong anionic character of the fifth, proximal ligand to the iron, which is able to stabilize high oxidation states, and the hydrophilic nature of the residues in the distal pocket. Beside the correct reduction potential for the oxidation reaction, the specificity towards different substrates also depends on the protein structural arrangement which can determine specific binding sites for substrates and mediators. Particularly, in the case of MnP, the Mn2+ binding site has been individuated in the X-ray structure. NMR studies were previously reported which provided an iron-manganese distance consistent with that from the X-ray structure. This information can help in defining the possible pathway for the electron transfer from the Mn2+ ion to the iron. On the contrary, in the case of LiP no information is available on the possible binding site of veratryl alcohol as well as of other aromatic substrates. This article reviews these structural properties of peroxidases with particular emphasis to their implications in the catalytic process. Finally, the calcium ions have been located in the structure of LiP and MnP: their structural relevance will be discussed on the light of the possible role in determining the optimal arrangement of residues in the distal cavity for the enzymatic reaction.

Introduction

Peroxidases are heme proteins which utilize hydrogen peroxide to catalyze the oxidation of a wide variety of organic and inorganic substrates (Everse and Everse, 1991). They have molecular weights ranging from 35 000 to 100 000 (from 251 to 726 residues) and contain a heme b (with the exception of myeloperoxidase which contains a chlorin (Ikeda-Saito and Prince, 1985; Sibbet and Hurst, 1984)) where, in the resting state, is present an iron ion in the oxidation state +3. The iron is 5 coordinated to the 4 pyrrole nitrogens of the heme and to a nitrogen of an axial histidine (the so-called proximal histidine, with the exception of chloroperoxidase which contains a thiolate ligand (Cramer et al., 1978)). The sixth coordination position is free, thus determining a high spin S=5/2 state for the iron.

The catalytic process occurs through a multi-step reaction which involves, first, the reaction of the active site with hydrogen peroxide. This produces its reduction to water and the oxidation of the protein by two electrons. The latter state of the protein is called Compound I, and contains an oxyferryl (Fe(IV)=O) center and an organic cation radical which can be located either on the heme or on a protein residue, depending on the isoenzyme. Then, Compound I oxidizes one substrate molecule (S) to give a substrate radical and Compound II, where the organic cation radical is reduced to its resting state. Finally, Compound II is reduced by a second substrate molecule to the resting iron(III) state (Everse and Everse, 1991)heme[Fe(III)](PX)+H2O2→heme[O=Fe(IV)−R+⋅](CompI)+H2Oheme[O=Fe(IV)−R+⋅](CompI)+S→heme[O=Fe(IV)](CompII)+Sheme[O=Fe(IV)](CompII)+S→heme[Fe(III)](PX)+S

Several peroxidases have been isolated, sequenced and characterized. They have been classified essentially in three classes, depending on the organism (Class I, intracellular prokaryotic peroxidases, Class II extracelluar fungal peroxidases and Class III secratory plant peroxidases; Welinder, 1992). It has been proposed, from an extensive comparison among the amino acid sequences, that heme peroxidases from plants, fungi and bacteria are evolutionary related (Welinder and Gajhede, 1993; Welinder, 1992).

In the past few years, several X-ray structures of peroxidases from different sources were reported which added to the X-ray structure of cytochrome-c peroxidase (CcP), reported more than 15 years ago (Poulos et al., 1980; Finzel et al., 1984). The latter structure has been the structural basis for the analysis of the properties and function of all the other peroxidases until these recent X-ray structures became available. Now the structures of lignin peroxidase (LiP; Poulos et al., 1993; Piontek et al., 1993), Arthromyces ramosus peroxidase (Kunishima et al., 1994), Coprinus cinereus peroxidase (CiP; Petersen et al., 1994), manganese peroxidase (MnP; Sundaramoorthy et al., 1994), and pea ascorbate peroxidase (APX; Patterson and Poulos, 1995) are available.

From a comparison among them, it was noted that, despite the low level of sequence homology (often <20%) the overall folding and the organization of the secondary structure is conserved (Li and Poulos, 1994; Poulos et al., 1995; Welinder and Gajhede, 1993).

The enzyme is divided in two different structural domains, enveloping the heme moiety. It has been proposed that the two domains originate from an early gene duplication event (Welinder and Gajhede, 1993). The structures of peroxidases are constituted by 10–11 α-helices, linked by loops and turns, while β-structures are essentially absent or are a minor component. Some glycine and proline residues are conserved, which determine the correct backbone bending, and a buried salt bridge, involving residues Aspl07 and Argl32 (LiP numbering) are conserved in all peroxidases. The latter fixes the long loop connecting the two structural domains. A comparison between the backbone structures of CcP (Class I) and LiP (Class II) is reported in Fig. 1.

Class I peroxidases do not contain any disulphide bridge; they do not contain any carbohydrate and any calcium ions. On the contrary, Cys bridges are present in Class II and III, even if differently located; all the Cys residues present in the proteins (around 8–10) form disulphide bridges which provide an high degree of rigidity to the protein. These two classes contain also two calcium ions whose binding sites have been located in the crystal structure (see later). Finally, Class II and III are glycosylated on the protein surface. Tyrosine residues are absent in fungal peroxidases (Class II), except for one residue in one isoenzyme of LiP. This makes the reduction potential of the protein matrix for this class higher than in the other and could explain why Compound I is not able to oxidize the protein itself (Welinder, 1992). However, despite these differences within the peroxidase families, the proteins present a similar folding.

In order to appreciate the high level of structure similarity among peroxidases, the structural features of the proteins of the peroxidase family were compared with those of another family of heme enzymes that also are dominated by α-helices, the cytochromes-P450 (Li and Poulos, 1994). It was found that peroxidases exhibit greater topological similarity than P450's. Li and Poulos (Li and Poulos, 1994) found that the Cα's in helical regions in LiP and CcP (15% sequence homology) have a r.m.s. deviation (rmsd) of 1.6 Å while the analogous comparison between two cyt-P450 proteins, with 17% of sequence homology, have 2.8 Å of rmsd. The reason for this larger variability in the structures of the Cyt-P450 family with respect to peroxidases was attributed to the more complex behavior of Cyt-P450 in substrate binding. In the latter proteins the substrate, which is hydroxylated during the enzymatic reaction, must bind in the heme active cavity and must have the correct orientation to react with the oxygen atom of the ferryl species. Peroxidases do not have these restrictions since the reaction often involve long range electron transfer from the substrate through the protein to the heme. As a result, peroxidases do not require major structural changes to adapt to different substrates.

Among the fungal peroxidases, the X-ray structures are available for LiP (Poulos et al., 1993; Piontek et al., 1993) and MnP (Sundaramoorthy et al., 1994) from the white rot fungus Phanerochaete chrysosporium, and for peroxidases from Arthromyces ramosus (Kunishima et al., 1994) and from Coprinus cinereus (Petersen et al., 1994). They show different potential glycosylation sites on the surface, of which some are in common for these proteins, even if in the X-ray structures often only one or two sites show indication of the presence of ordered carbohydrates.

Section snippets

The heme environment

Despite the low homology among peroxidases, iron coordination and most of the residues in the active site are completely conserved in the peroxidases sequenced up to now (Welinder and Gajhede, 1993). Fig. 2 shows the heme active site in LiP with the most relevant residues. The iron ion is invariantly 5 coordinated by the axial histidine. This residue is characterized by an anion character, due to the presence of a strong H-bond between Nε2 of the His ring and an Asp residue (Asp 238 in LiP),

Substrate binding sites

Peroxidases can catalyze the oxidation of a large variety of substrates which range from a protein, Cyt-c, as in the case of CcP, to small aromatic molecules for plant peroxidases, to large polymers, as lignin, and inorganic ions, as Mn2+, in the case of some lignolitic fungal peroxidases.

The specificity towards different substrates depends on several factors. Among them is the suitable reduction potential of the active species (iron ion and center of the cation radical) with respect to those

Structural role of the calcium ions

Class II and Class III peroxidases are characterized by the presence of two calcium ions, which essentially have a structural role (Welinder, 1992). Their binding sites have been located in the X-ray structures solved for the proteins of these two classes and have been found to be highly conserved in all fungal and plant peroxidases. One binding site is located in the proximal side domain and is formed by eight oxygen atoms from backbone and sidechain of residues, in LiP, Ser177, Asp194,

Acknowledgements

I warmly thank Prof. Ivano Bertini for helpful discussion. The financial support of the EC Biotechnology Program BIO2-CT94-2052 (DGI2SSMA) and of the Comitato Biotecnologie, CNR (Italy) is acknowledged.

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