Microperoxidase-8. Tuning of its catalysis and reactivity

In this thesis, microperoxidase-8, Fe ^III MP-8, and the manganese variant Mn ^III MP-8, were studied. Insight in i) the mechanism of oxygen exchange between the oxo group of porphyrin high-valent metal-oxo species and solvent, ii) their cytochrome P450 (P450) and peroxidase catalytic reactivity and iii) the formation of their catalytic reactive intermediates is provided.<h3>Determinants of the peroxidase and P450 chemistry for Fe ^III MP-8 and Mn ^III MP-8 (Chapter 1)</h3>In the Chapter 1 of this thesis, peroxidase and P450 enzymes were compared in order to understand the major differences between both types of enzymes explaining the differences in their chemistry. It was shown that peroxidases and P450s share similar intermediates in their catalytic cycles but that both types of enzymes were structurally different. Differences in the distal environment of the heme and in the nature of the axial ligand can be regarded as having an influence on their chemistry.<h4>1. Differences in the heme distal environment</h4>A comparison of the distal heme cavity for peroxidase and P450 enzymes shows that peroxidases do not have a clearly defined hydrophobic distal pocket allowing the binding of organic substrates near the heme iron centre. With the exception of hydroperoxides which bind to the heme iron centre and are subsequently reduced by the heme group to generate catalytic reactive intermediates, the organic substrate is proposed to bind on theδ-meso heme edge thereby allowing electrons transfer to the heme prosthetic group. The fact that organic substrates cannot bind to the distal part of the peroxidase active site near the oxo group of Compound I, explains why these enzymes do not support oxygen transfer reactions. Moreover the fact that only for peroxidase mutants where some "extra space" has been created on the distal part of the active site, monooxygenases reactions are observed but catalysed at a lower rate than for heme-thiolate enzymes, corroborates the assumption that substrate binding near the heme iron favours oxygen transfer. Evolutionary speaking, peroxidases are well designed to reduce small oxygen containing molecules, hydroperoxides, and to convert them into oxidative power which may be beneficial to the cell because of the formation of useful compounds such as polymers. P450s, however, have a distal active site pocket which allows substrate binding near the heme iron center. From an evolutionary point, P450s are well designed to convert harmful xenobiotics or modifying membrane lipid components in the cell. The fact that the substrate binds close to the site where the reactive catalytic intermediates are generated favours in-situ conversion and direct oxygen transfer to the substrate. Moreover this oxygen transfer can be mediated by the various catalytic intermediates of the reaction cycle preceding the Compound I analogue and not only by the Compound I analogue. The physical proximity of the substrate and the reactive intermediates favours a reaction of the catalytic intermediates with the substrate over a transformation of a given catalytic intermediate into the following catalytic intermediate of the reaction cycle of the enzyme.<h4>2. Differences in the nature of the proximal axial ligand</h4>Peroxidases and P450s differ by the nature of their proximal axial ligand. The negative charge of the cysteinate ligand of P450s is proposed to lower the oxidation potential of the Compound I analogue intermediate thereby favouring oxygen transfer to substrate over electron abstraction from substrate . The negative charge of the sulphur atom is somewhat reinforced by the involvement of a network of hydrogen bonds from the residues of the heme proximal active site to the sulphur of the thiolate group. Theoretical MO-calculations also have shown that the sulphur orbitals of the cysteinate mix with the a _2u cationic state of the porphyrin high-valent iron-oxo intermediate along the reaction pathway. This is not observed for the proximal histidine ligand of peroxidases. In peroxidases, the neutral histidine ligand is proposed to favour electron abstraction from substrate over oxygen transfer to substrate .<h3>Compound I oxo exchange: implications for oxygen transfer (Chapters 2 and 3)</h3>In the field of heme-based catalysis, labelling studies are used to discriminate between peroxidase-type of chemistry and P450-type of chemistry. Tracing of the oxygen donor atoms in the product allows to differentiate between true oxygen transfer , where the oxygen atom inserted in the substrate originates from the primary oxygen donor, P450 chemistry, and apparent oxygen transfer where the oxygen atom inserted in the substrate originates from the solvent, peroxidase chemistry. However, heme-enzymes and the corresponding models were shown to exchange the oxo group of their Compound I intermediate, or Compound I analogue, with bulk solvent. Chapters 2 and 3 of this thesis are oriented on the mechanistic aspects of the exchangeability of the oxo group of Compound I with bulk solvent. In Chapter 2, HRP and the labelled oxygen donor are left incubating during an exchange step of variable time length before the substrate, aniline, is added. The conversion of aniline results in the insertion of one oxygen atom in the substrate by para -hydroxylation. The para -hydroxylated aniline formed during the conversion step subsequent to the exchange step was analysed by MS. This reveals an increasing percentage of the incorporation of oxygen atoms originating from the solvent when the duration of the exchange step is increased. When catalase was added between the two steps of the experiment, no product was formed. This shows that the heme catalyst induces oxygen exchange between bulk water and H ₂ O ₂ , to form H ₂ O ₂ containing oxygen atoms issued from the solvent. A mechanism explaining this oxygen exchange for an axially coordinated heme has been suggested including the reversibility of the formation of Compound I for HRP, but also for Fe ^III MP-8, hemin and hematin. Actually, water becomes a substrate for the porphyrin high-valent iron-oxo intermediate and competes with the organic substrate, when present, for being oxidised. In the study presented in Chapter 2, substrate oxidation and water oxidation were decoupled thereby emphasising the water oxidation step. Moreover this study indirectly suggests that oxidised heme-based systems can catalyse the formation of peroxide bonds at the (modest) apparent rate of≈1 s ^-1 . This is a relevant outcome of this study, since the design of molecules containing a peroxide bond is of fundamental importance for oxidation chemistry and is of particularly interest for the chemical industry.In Chapter 3, the same argumentation as proposed in Chapter 2 is supported by additional exchange experiments with iron and manganese water-soluble porphyrins. This chapter is an attempt to unify the views on porphyrin high-valent metal-oxo/solvent exchange processes. Direct oxygen exchange of the oxo group of Compound I, for a solvent oxygen atom is not found to account for the regeneration of H ₂ O ₂ during the exchange step. Also a mechanism such as oxo/hydroxo tautomerism where the transfer of two protons from the trans water axial ligand to the oxygen of the oxo group results in an exchange of the oxo oxygen cannot explain Fe ^III MP-8 supported oxygen exchange. The fact that Fe ^III MP-8 has an axial histidine ligand prevents solvent binding trans to the oxo group, which would be a prerequisite for proton transfer resulting in oxygen exchange between both axial ligands. Obviously this cannot explain oxygen exchange catalysed by axially ligated heme or metalloporphyrins like Fe ^III MP-8. The reversible formation of Compound I, proposed in Chapter 2, explains the results in a clear way.<h3>Catalytic reactivity of Fe ^III MP-8 and Mn ^III MP-8 (Chapter 4 and 5)</h3>The heme-peptide model, Fe ^III MP-8, shows peroxidase activity and can also be used as a heme-enzyme model for studying P450 chemistry based on the fact that, upon addition of ascorbate, the reactive species which are responsible for peroxidase chemistry are scavenged. As a consequence Fe ^III MP-8 can be active in two modes: the peroxidase mode where the catalysis is dominated by porphyrin high-valent iron-oxo intermediates and the P450 mode where the porphyrin high-valent iron-oxo intermediates are (partially) scavenged and catalysis is mainly performed by intermediates appearing prior to Compound I in the reaction cycle. The switch between the two modes is provided by the addition of ascorbate to the system in the case of P450 chemistry, which acts as scavenger for porphyrin high-valent iron-oxo intermediates.In Chapter 4 the reactivity of Fe ^III MP-8 for H ₂ O ₂ supported O- and N-dealkylation, a peroxidase/P450 type of reaction, has been investigated. In the peroxidase mode, i.e. without ascorbate addition, the rate of conversion of the substrates is correlated with their quantum mechanically calculated first ionisation potential. This indicates that their conversion proceeds via an initial electron abstraction from the substrate. This is corroborated by the observation of a large amount of polymerisation products together with the formation of small amounts of N-dealkylated products. In contrast however O-dealkylation was not observed. This observation that O-alkylated substrates are not dealkylated in the peroxidase mode can be related to the fact that their first ionisation potential is too low. In the P450 mode, i.e. in the presence of ascorbate, O- and N-alkylated substrates are both converted and a correlation with the calculated first ionisation potential of the substrates no longer exists. This suggests that O- and N-dealkylations in the P450 mode proceed via a non-radical type of mechanism and thus through other intermediates than Compound I and Compound II, since ascorbate is a scavenger of Compound I and II. As an alternative reactive species the non-radical type PorFe ^III -(hydro)peroxo intermediate, may be the species involved in the P450 mode of Fe ^III MP-8 supported O- and N-dealkylation. The PorFe ^III (hydro)peroxo intermediate is known as Compound 0 and appears before Compound I in the catalytic cycle. Mechanisms for Compound 0 supported O- and N-dealkylations are discussed in detail.The aim of the investigations described in Chapter 5 was to gain information on the nature of the different reactive species involved in MP-8 supported catalysis comparing again peroxidase and P450 reactions. For this purpose the manganese variant of Fe(III)MP-8, Mn(III)MP-8 was synthesised. Iron and manganese porphyrin complexes have a similar chemistry but differ in their reaction kinetics providing information concerning which type of intermediate is involved in catalysis. The conversion of guaiacol (2-methoxyphenol), ortho -dianisidine (3,3'-dimethoxybenzidine) and aniline catalysed by Fe ^III MP-8 and Mn ^III MP-8 was studied. The first two substrates are models for the peroxidase reactivity of heme-enzymes and the third one is a model for the P450 reactivity of heme-enzymes. The pH-dependence of the rate of conversion, k _cat , of each substrate was studied for Fe ^III MP-8 and Mn ^III MP-8 supported conversions, using H ₂ O ₂ as oxygen donor. For the peroxidase mode it was shown that the optimal pH for Mn ^III MP-8 supported conversions is pH 11, about 2 units higher than for Fe ^III MP-8 which has an optimal pH of 9. This can be correlated to the lower reduction potential of the Mn ^III MP-8/Mn ^II MP-8 transition when compared to the iron complex. The iron atom is proposed to better stabilise the deprotonated coordinated hydroperoxide molecule than the manganese centre due to its higher electron withdrawing effect on the proximal oxygen of the (hydro)peroxo group. For the cytochrome P450 mode, i.e. in the presence of ascorbate, it was found that Mn ^III MP-8 was not able to catalyse the para -hydroxylation of aniline whereas it was possible for Fe ^III MP-8 under the same conditions. These results are in line with the conclusions of Chapter 4 and indicate that the MP-8 supported cytochrome P450 chemistry proceeds via a PorFe ^III -hydroperoxo intermediate. This also explains the absence of aniline hydroxylation activity for Mn ^III MP-8 based catalysis because the Mn ^III -(hydro)peroxo intermediate is much less reactive toward electrophilic hydroxylation than the corresponding iron intermediate. As a consequence the hydroxylation of aniline by Fe ^III MP-8 may be performed by Compound 0. However, as stressed also in Chapter 4, Compound I and II, typical of the peroxidase mode, may also play a role in the P450 mode since ascorbate competes with aniline for oxidation by Compound I and II.<h3>Characterisation of peroxidase and P450 intermediates (Chapter 6 and 7)</h3>One of the major drawbacks of MP-8 is the high inactivation rate observed for the catalyst under operational conditions in both the P450 and the peroxidase mode. In an effort aiming at understanding the reasons for the fast inactivation of Fe ^III MP-8, the fate of the catalyst was studied under turnover conditions in the absence of substrate. Chapter 6 presents the characterisation of a modified Fe ^III MP-8 with a hydroxylated His18. The modified catalyst was isolated under operational conditions in the presence of H ₂ O ₂ . The structure of the native and the modified Fe ^III MP-8 were compared by HPLC, UV/visible, ESI-MS ² and ¹ H-NMR. Analysis showed the formation of a product more hydrophilic than Fe ^III MP-8, with an intact heme ring and having an extra oxygen inserted on the peptide. ESI-MS ² and ¹ H-NMR suggest the extra oxygen atom to be inserted on the Nδ1 of the imidazole ring of the His18. The formation of the modified intermediate is inhibited by ascorbate and labelling studies have shown that the inserted oxygen originates from the solvent. This suggests the modified Fe ^III MP-8 to derive from a Compound I analogue of Fe ^III MP-8. The fact that the solvent-assisted hydroxylation occurs on the proximal histidine ligand suggests the second oxidation equivalent of the analogue of Fe ^III MP-8 Compound I to be majorly delocalised on the His18 axial ligand by mesomerism.The characterisation and the analysis of the kinetics of formation of the heme-intermediates competent in MP-8-supported peroxidase and P450 catalysis is the subject of Chapter 7. The kinetics for the formation of Compound 0, Compound I analogue and Compound II were compared for Fe ^III MP-8 and Mn ^III MP-8. Analysis reveals a one order of magnitude higher rate of formation of PorFe ^III -OOH (k = 1.3×10 ⁶ M ^-1 .s ^-1 ) when compared to the rate of formation of PorMn ^III -OOH (k = 1.1×10 ⁵ M ^-1 .s ^-1 ). The overall rate of the reaction for both complexes with H ₂ O ₂ , increases with higher pH-values. The corresponding pK _a values which were found to explain this pH-dependency are in complete agreement with the optimal values for Fe ^III MP-8 (pH 9.2) and Mn ^III MP-8 (pH 11.0) supported catalysis (Chapter 5) and were found to correspond to the deprotonation of metal-bound water for Fe ^III MP-8. This explains how heme-peptide models which lack a distal histidine, acting as an acid/base catalyst for the deprotonation of H ₂ O ₂ and for the subsequent cleavage of the O-O peroxide bond, facilitate the deprotonation of H ₂ O ₂ . For MP-8, the peroxide deprotonation may proceeds through a concerted mechanism which results in the replacement of the hydroxyl ligand by a hydroperoxo ligand. It is not sure, however, how MP-8 catalyses the cleavage of the O-O peroxide bond, since no residue or group able to deliver protons to the distal oxygen in order to facilitate bond cleavage are present on the distal site. An eventual participation of the Nδ1 of the imidazole ring of the His18 of a second molecule of MP-8 as proton donor cannot be excluded. The manganese Compound 0 is proposed to be heterolytically cleaved into a Compound I type of intermediate, Mn ^IV MP-8=O(R ^·+ ), where the second oxidation equivalent seems to be localised on the peptide part of the molecule. This is suggested by the analysis of the transient UV/visible and EPR spectra of oxidised Mn ^III MP-8. Based on the kinetic analysis and on the results of Chapter 6, iron Compound 0 is also proposed to be heterolytically cleaved into a similar Compound I type of intermediate, Fe ^IV MP-8=O(R ^·+ ), with a cleavage rate analogue to the one of the manganese complex (k≈150 s ^-1 ).To summarise, in peroxidases, the hydroperoxo intermediate is rapidly converted, during the reaction cycle, into Compound I catalysed by the residues of the distal heme pocket. In P450s the corresponding hydroperoxo or peroxo intermediates may react with the substrate bound in the active site before they are converted into a Compound I analogue. In other words, P450 chemistry is not only based on the catalytic reactivity of Compound I but also on the reactivity of Compound 0 and the deprotonated form of Compound 0 respectively the PorFe ^III -hydroperoxo and the PorFe ^III -peroxo intermediates. Whereas the isoelectronic analogue of Compound I can be seen as an intermediate in electrophilic reactions such as Compound 0, the deprotonated Compound 0 is proposed to be active in both electrophilic and nucleophilic reactions.As a consequence, one might conclude that designing a biomimic for the P450 chemistry requires two majors conditions: i) the presence of an open active site and ii) the stabilisation of the catalytic reactive intermediates preceding the formation of the porphyrin high-valent iron-oxo intermediate in the reaction cycle. Fe ^III MP-8 and Mn ^III MP-8 resemble an open peroxidase active site having no distal environment. It has been shown that for both MP-8 models the co-ordination of a molecule of hydrogen peroxide on the metal is facilitated by bound water, providing the pH in the medium is high (Chapter 7). This questions the role played by the distal histidine ligand in peroxidases. Generally the hydroperoxide substrate is proposed to be deprotonated by the distal histidine ligand before it displaces metal bound water. But the preliminary deprotonation of the bound water by the distal histidine, followed by concerted deprotonation of the hydroperoxide and displacement of the hydroxyl ligand might be considered as a relevant alternative mechanism. In order to mimic P450 catalysis, the porphyrin iron-hydroperoxo and the porphyrin iron-peroxo intermediates should be stabilised with respect to the porphyrin high-valent metal-oxo intermediate. This is rendered possible by partially scavenging the porphyrin high-valent metal-oxo intermediates species analogue of Compound I using ascorbate, regenerating the native MP-8. The reductant competes with the substrate for the oxidation by the porphyrin high-valent metal-oxo intermediate and does almost not affect porphyrin iron-(hydro)peroxo-based intermediates. Therefore in the presence of ascorbate, i.e. in the P450 mode, MP-8 can be considered as a model for P450 chemistry and without ascorbate, i.e. in the peroxidase mode, MP-8 can be considered as a model for peroxidase chemistry.As a conclusion, the present work has contributed to the better understanding of the chemistry of metalloporphyrin hydroperoxo and metalloporphyrin peroxo intermediates both relevant active species of the P450 chemistry. Furthermore the peroxidase mimic, MP-8, can efficiently be used as a model for the P450 chemistry