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| ISBN-13: | 9781849739115 |
|---|---|
| Publisher: | RSC |
| Publication date: | 10/27/2015 |
| Series: | Metallobiology , #4 |
| Pages: | 388 |
| Product dimensions: | 6.15(w) x 9.20(h) x (d) |
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Heme Peroxidases
By Emma Raven, Brian Dunford
The Royal Society of Chemistry
Copyright © 2016 The Royal Society of ChemistryAll rights reserved.
ISBN: 978-1-84973-911-5
CHAPTER 1
Self-processing of Peroxidases
PAUL R. ORTIZ DE MONTELLANO
Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco CA 94158-2517, USA
Email: ortiz@cgl.ucsf.edu
1.1 Introduction
The peroxidases, like other families of hemoproteins, include members that undergo post-expression, autocatalytic processing to generate the mature proteins. Hemoproteins have a potential for autocatalytic self-modification reactions beyond that available to most other proteins. This potential stems from the inherent high reactivity of the Compound I and Compound II intermediates generated during the catalytic cycle, the fact that the substrate specificity of peroxidases is broad and often involves outer sphere electron transfer reactions, and the reactivity of the radicals that can be formed by peroxidase reactions. In many instances, these autocatalytic modification reactions are incidental and impair or terminate peroxidase function, but in other instances evolution has optimized such reactions to tailor the catalytic functions of proteins for specific purposes. This maturation process can result in modification of specific protein side chains, remodeling of the prosthetic heme group, or covalent crosslinking of the heme to the protein through one or more bonds.
This chapter focuses on the mechanisms and potential roles of autocatalytic reactions that: (a) modify the protein structure of peroxidases, (b) result in simultaneous modification of both the heme and protein, or (c) only impact the prosthetic heme group. It does not, however, deal with protein or heme modifications that require the intervention of secondary enzymes or the general modifications of the protein or heme that result in loss of peroxidase activity. Specifically, this chapter does not discuss posttranslational modifications such as phosphorylation, the mechanisms of heme or protein degradation caused by excess H2O2, or, except for one exception, formation of the cysteine–vinyl link characteristic of cytochrome c. Nevertheless, it includes a discussion of site-specific peroxidase mutants that give rise to well-defined alterations in the protein or heme structure, as these help to understand the mechanisms and determinants of these reactions.
1.2 Protein Modifications
1.2.1 Lignin Peroxidases
Lignin peroxidase (LiP), a fungal enzyme, promotes the oxidative degradation of lignin, a complex polymeric structure that accounts for approximately 30% of the carbon in the biological sphere. LiP, like manganese peroxidase, catalyzes the one-electron oxidation of a diffusible mediator that can more easily penetrate the dense lignin matrix to promote its degradation. The diffusible mediator in the case of LiP is veratryl alcohol, which is oxidized to the veratryl radical cation. The radical cation can then oxidize lignin or other substrates, or can be further oxidized by removal of a second electron to the aldehyde (Scheme 1.1).
The crystal structure of LiP from Phanerochaete chrysosporium at a resolution of 1.7 Å revealed the unexpected presence of electron density that could be assigned to a hydroxyl group located on the Cβ atom of Trp171.4 Biochemical and crystallographic evidence showed that this protein modification was the result of an autocatalytic process that occurred when the newly synthesized protein reacted with H2O2. Thus, a crystal structure of the naïve protein without prior exposure to H2O2 had an intact Trp171 without electron density for the extra hydroxyl group, whereas it was present after the protein was exposed to H2O2. Tryptic digestion of the mature protein and isolation of the peptide containing Trp171 showed that it was modified in a manner consistent with introduction of a hydroxyl adjacent to the aromatic ring. This modification was only found in the peptide after reaction of LiP with H2O2. As molecular oxygen was not required for introduction of the Trp171 hydroxyl modification, it was postulated that the mechanism for its introduction involves two sequential one-electron oxidations, resulting in formation of an exocyclic conjugated imine. Michael addition of water to this intermediate produces the final modified amino acid (Scheme 1.2). Involvement of the normal catalytic mechanism in the oxidation of Trp171 is confirmed by the finding that when an alternative substrate is present, increased concentrations of H2O2 are required for full modification due to a competition between protein and substrate oxidation. The involvement of a Trp171 radical in the modification process is supported by spin trapping experiments with 2-methyl-2-nitrosopropane (MNP) in which the spin trap was shown by tryptic mapping to bind to the C6 atom of Trp171 and thus to prevent formation of the hydroxylated tryptophan residue.
The Trp171Phe and Trp171Ser mutants in which Trp171 is replaced by redox-inactive residues no longer oxidize veratryl alcohol, a physiological substrate. However, both mutants form Compound I normally with H2O2 and retain most of their ability to oxidize conventional peroxidase substrates, including ABTS (2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) and DFAD (4-[(3,5-difluoro-4-hydroxyphenyl)azo]benzenesulfonic acid). Similar results were found with a nonphenolic tetrameric lignin model, in that the wild-type enzyme oxidized this substrate whereas the Trp171Ser mutant did not. In contrast, a Glu146Ser or Glu146Gly mutation in the channel leading from the surface to the heme edge did not prevent oxidation of veratryl alcohol or the tetrameric lignin model. These results show that the oxidation of veratryl alcohol and lignin-like products is mediated at the Trp171 site, whereas more conventional peroxidase substrates appear to be oxidized preferentially at a second site, probably one involving the channel leading into the heme crevice. However, the specific role of Trp171 hydroxylation remains unclear. The finding that Trp171 hydroxylation in pristine LiP competes with veratryl alcohol oxidation suggests that the hydroxyl modification is not absolutely essential for veratryl alcohol oxidation, whereas the lack of activity of the Trp171Ser mutant towards a lignin model suggests Trp171 is critical for that function.
A different protein modification has been found for the LiP from Trame-topsis cervina, a protein that lacks the highly conserved tryptophan residue equivalent to Trp171 in the Phanerochaete chrysosporiumenzyme. Crystallographic, kinetic, and spectroscopic data indicate that the oxidation of veratryl alcohol or polymeric substrates by this enzyme depends critically on Tyr181, a surface-exposed tyrosine. An EPR analysis indicates that Tyr181 is oxidized during the catalytic cycle to a radical that is proposed to mediate substrate oxidation. Subsequent work revealed that pristine LiP showed a lag period in veratryl alcohol oxidation, whereas the enzyme that had already been involved in veratryl alcohol oxidation did not. Mass spectrometric analysis revealed that the mature enzyme had a veratryl alcohol moiety covalently attached to Tyr181. Although the exact nature of the covalent link remains unclear, it is likely to involve a crosslink between the tyrosine and veratryl alcohol aromatic rings. Exposure of the naïve enzyme to H2O2 in the absence of veratryl alcohol resulted in hydroxylation of Tyr181 to a catechol and loss of the ability to oxidize veratryl alcohol and 1,4-dimethoxybenzene (Scheme 1.3), although both the protein with the hydroxylated Tyr181 and that with the Tyr181-veratryl adduct were able to oxidize low potential substrates such as ferrocytochrome c. No covalent changes were observed with the Y181N mutant of the protein. Kinetic studies demonstrated that covalent binding of veratryl alcohol to Tyr181 stimulated oxidation of veratryl alcohol by Compound II by 4100-fold. Thus, formation of the covalently modified tyrosine in the case of the Trametopsis cervina enzyme has direct consequences on the catalytic performance of the enzyme.
1.2.2 KatG Peroxidases
The KatG catalase-peroxidases that are found in microorganisms have both a high catalase activity in the range of 5000–16 000 s-1 and a much lower, but still significant, peroxidase activity of 10–25 s-1. Mechanistic studies have been carried out with several of these enzymes and crystal structures are available for the KatG proteins from Burkholderia pseudomallei, Haloarcula marismortui, Synechocystis PCC 7942, and Mycobacterium tuberculosis. The crystal structure of a KatG excreted from the eukaryotic fungus Magnaporthe griseahas also been reported. The most unusual feature of all the KatG enzymes revealed by these crystal structures is the presence of a highly conserved Met-Tyr-Trp cross-linked tripeptide in the distal (substrate) side of the heme pocket (Figure 1.1). The presence of this cross-linked tripeptide is confirmed by mass spectrometric data on tryptic peptides from the B. pseudomallei, Synechocystis, M. tuberculosis, and Magnaporthe grisea proteins. In the case of M. tuberculosis KatG, the residues in the tripeptide are Met255-Tyr229-Trp107 (Figure 1.1). The tripeptide is located in the active site of the protein directly above the prosthetic heme iron atom.
Autocatalytic formation of this tripeptide is clearly demonstrated by the fact that heterologous expression of Mycobacterium tuberculosis KatG in Escherichia coliyields a naïve protein in which the three relevant residues, as shown by tryptic digestion, mass spectrometry, and UV-vis spectrometry, are not cross-linked. However, incubation of the protein with six equivalents of H2O2 triggers a rapid process in which the cross-linked tripeptide is quantitatively formed. Expression of the Met255Ile mutant of M. tuberculosis KatG, in which the methionine of the cross-linked peptide is missing, gives rise to a protein with only the Tyr-Trp crosslink, as well as a two- electron oxidized form proposed to be an intermediate in the formation of the cross-linked dipeptide. These results are consistent with earlier findings that mutants of B. pseudomalleiKatG that lacked the appropriate tryptophan (Trp111) or tyrosine (Tyr238) did not form the fully cross-linked tripeptide, but in addition specifically showed that its formation is due to an autocatalytic process. These results strongly support a crosslinking mechanism in which autocatalytic one-electron oxidation of both the Tyr and Trp aromatic side-chains produces the corresponding radicals that, in turn, undergo radical combination to form a carbon–carbon bond (Scheme 1.4). After proton tautomerizations to regenerate the two aromatic rings, two electrons are removed in a second catalytic turnover, producing an iminoquinone to which the methionine adds as a nucleophile in a Michael reaction. A final proton tautomerization step then yields the mature cross- linked tripeptide. This same mechanism is applicable to the formation of the cross-linked tripeptide in all the KatG enzymes.
In the absence of the intact cross-linked tripeptide, the catalase activity of the KatG enzymes is greatly attenuated (Table 1.1). In contrast, the peroxidase activity is modestly increased, presumably because the Compound I intermediate is not being drained as effectively by the much faster catalase reaction. The table shows that the cross-linked tripeptide is important for the catalase, but not peroxidase, function of the enzyme, although autocatalytic formation of the tripeptide depends on the peroxidase activity.
The exact role of the Met-Tyr-Trp tripeptide in KatG catalytic activity continues to be investigated. It has been proposed that reaction of KatG with H2O2 generates Compound II at the heme center together with the tripeptide radical. Reaction of this intermediate with a second molecule of H2O2 produces the ferrous dioxygen (or ferric superoxide, Compound III) complex, still with the tripeptide radical. Electron transfer from the dioxygen complex (nominally Fe+3O2•-) to the tripeptide radical then regenerates the starting enzyme and oxygen, effectively completing the conversion of H2O2 to molecular oxygen. This mechanism is supported by detection of the ferrous dioxy heme/tripeptide radical intermediate and demonstration that this intermediate cycles to produce molecular oxygen. The intact tripeptide may also help rescue catalase activity by reducing the catalase-inactive Compound II to the ferric state. The detailed catalytic mechanism of KatG enzymes has been reviewed.
1.3 Heme–Protein Crosslinking
1.3.1 Mammalian Peroxidase Ester Links
The mammalian peroxidases, as exemplified by lactoperoxidase (LPO), myeloperoxidase (MPO), eosinophil peroxidase (EPO), and thyroid peroxidase (TPO), are distinguished from the plant and fungal peroxidases by the presence of covalent bonds that attach the heme group to the protein. The crystal structures of caprine, buffalo, and bovine LPO and of canine and human MPO (Figure 1.2) show that the 1- and 5-methyls of the heme are attached through ester bonds to the carboxylic acid side-chains of Asp or Glu residues. The presence of the ester bonds is supported by the observation of appropriate carbonyl vibrations by difference Fourier transform infrared spectroscopy. Their presence has also been confirmed in LPO by NMR and mass spectrometric studies of proteolytically generated peptides. In caprine LPO, the 1-methyl forms an ester bond with Glu252 and the 5-methyl with Asp108, while in human MPO the corresponding bonds are to Glu242 and Asp94. In accord with these assignments, mutation of either Glu242 to a Gln or Asp94 to a Val in human MPO results in loss of some of the ester carbonyl resonance bands. However, a recent crystallographic study of MPO isolated from human leukocytes showed low electron density for Glu242, suggesting that it had high mobility and therefore that the ester bond to this residue might have relatively low occupancy. Nevertheless, electron density on the 1-methyl group was consistent with covalent attachment of a hydroxyl group to the methyl. It is therefore possible that under some conditions the Glu242 ester bond is only fully formed in a subpopulation of the MPO molecules.
(Continues...)
Excerpted from Heme Peroxidases by Emma Raven, Brian Dunford. Copyright © 2016 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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Table of Contents
Self-processing peroxidases;Cytochrome c peroxidase and cytochrome c complexes;
Understanding the reactivity and interactions of peroxidases with substrates;
Structural and functional properties of heme containing peroxidases;
Heme Peroxidase Kinetics;
Multiheme peroxidases;
Catalase-peroxidase (KatG) structure and function;
Mechanistic aspects of catalase-peroxidase;
Models complexes of heme peroxidases;
Mult-functional hemoglobin dehaloperoxidases;
The Peroxidase and Cyclooxygenase Activity of Prostaglandin H Synthase;
Myeloperoxidase: Structure and Function of the Green Peroxidase of Neutrophils;
Peroxidases as Potential Industrial Biocatalysts;
Structural and Reactivity of the Dyedye-decolorizing peroxidases (DyP) Family