Iron-Containing Enzymes: Versatile Catalysts of Hydroxylation Reactions in Nature - Hardcover

Hardcover

ISBN 10: 1849731810 ISBN 13: 9781849731812

Publisher: Royal Society of Chemistry, 2011

Synopsis

This book explains the mechanism and function of mononuclear iron containing enzymes. These important bioprocess intermediates have great industrial potential.

About this title

About the Author

The editors (SP de Visser and D Kumar) have been involved in studies of enzymatic systems such as heme enzymes like the cytochromes P450, peroxidases and catalases as well as on nonheme enzyme systems. These studies are at the forefront of chemistry and focus on short-lived species in enzymes and biomimetic complexes. Thus, for instance, the active oxidant of P450 enzymes is elusive and experimental studies gave conflicting results regarding what the active oxidant was. The studies of de Visser and Kumar and co-workers established what the active species of these enzymes is and ruled out many suggested alternative oxidants. They established a two-state-reactivity scenario for heme and nonheme iron(IV)-oxo oxidants that masquerades as multiple oxidants. Further studies established reaction mechanisms for the production of (unwanted) by-products and showed how these can be prevented. Recent collaborative work of de Visser and Kumar established a general trend for substrate hydroxylation reactions by the cytochromes P450 and explained the mechanism via a valence bond curve crossing model. De Visser and Kumar have published more than 100 scientific publications in high-impact journals and as a consequence are regular speakers and scientific meetings and conferences.

From the Back Cover

There are many mononuclear iron containing enzymes in nature that utilize molecular oxygen and transfer one or both oxygen atoms of O2 to substrates. These enzymes catalyze many processes including the biosynthesis of hormones, the metabolism of drugs, DNA and RNA base repair and, the biosynthesis of antibiotics. Therefore, mononuclear iron containing enzymes are important intermediates in bioprocesses and have great potential in the commercial biosynthesis of specific products since they often catalyze reactions regioselectively or stereospecifically. Understanding their mechanism and function is important and will assist in searches for commercial exploitation. In recent years, advances in experimental as well as theoretical methodologies have made it possible to study the mechanism and function of these enzymes and much information on their properties has been gained. This book highlighting recent developments in the field is, therefore, a timely addition to the literature and will interest a broad readership in the fields of biochemistry, inorganic chemistry and computational chemistry. The Editors, leaders in the field of nonheme and heme iron containing monoxygenases, have filled the book with topical review chapters by leaders in the various sub-disciplines.

Excerpt. © Reprinted by permission. All rights reserved.

Iron-Containing Enzymes

Versatile Catalysts of Hydroxylation Reactions in Nature

By Sam P de Visser, Devesh Kumar

The Royal Society of Chemistry

Copyright © 2011 Royal Society of Chemistry
All rights reserved.

ISBN: 978-1-84973-181-2

Contents

Chapter 1 Experimental and Computational Studies on the Catalytic Mechanism of Non-heme Iron Dioxygenases Sam P. de Visser, 1, 
Chapter 2 Non-heme Iron-Dependent Dioxygenases: Mechanism and Structure Timothy D. H. Bugg, 42, 
Chapter 3 Transient Iron Species in the Catalytic Mechanism of the Archetypal α-Ketoglutarate-Dependent Dioxygenase, TauD Denis A. Proshlyakov and Robert P. Hausinger, 67, 
Chapter 4 Density Functional Theory Studies on Non-heme Iron Enzymes Tomasz Borowski and Per E. M. Siegbahn, 88, 
Chapter 5 Theoretical Spectroscopies of Iron-Containing Enzymes and Biomimetics Shengfa Ye, Gemma J. Christian, Caiyun Geng and Frank Neese, 119, 
Chapter 6 Bioinspired Non-heme Iron Catalysts in C–H and C=C Oxidation Reactions Anna Company, Laura Gómez and Miquel Costas, 148, 
Chapter 7 Application of Magnetic Circular Dichroism, X-Ray Absorption Spectroscopy and Extended X-Ray Absorption Fine Structure in Determining Geometric and Electronic Structure of Non-heme Iron(IV)-oxo Enzymatic Intermediates and Related Synthetic Models Somdatta Ghosh Dey and Abhishek Dey, 209, 
Chapter 8 Structure, Mechanism and Function of Cytochrome P450 Enzymes Kirsty J. McLean, Hazel M. Girvan, Amy E. Mason, Adrian J. Dunford and Andrew W. Munro, 255, 
Chapter 9 Drug Metabolism by Cytochrome P450: A Tale of Multistate Reactivity Devesh Kumar, 281, 
Chapter 10 Oxidation of Unnatural Substrates by Engineered Cytochrome P450cam Saptaswa Sen, Soumen Kanti Manna and Shyamalava Mazumdar, 330, 
Chapter 11 QM/MM Studies of Cytochrome P450 Systems: Application to Drug Metabolism Richard Lonsdale, Jeremy N. Harvey and Adrian J. Mulholland, 366, 
Chapter 12 Mechanism and Function of Tryptophan and Indoleamine Dioxygenases Sarah J. Thackray, Igor E.mov, Emma Lloyd Raven and Christopher G. Mowat, 400, 
Subject Index, 427, 

CHAPTER 1

Experimental and Computational Studies on the Catalytic Mechanism of Non-heme Iron Dioxygenases

SAM P. DE VISSER

Manchester Interdisciplinary Biocentre and School of Chemical Engineering and Analytical Science, University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom

1.1 Introduction

Dioxygen is essential for human life: it is involved in various bioprocesses such as cellular respiration, the biosyntheses of hormones as well as in defense mechanisms. Since molecular oxygen is needed in most parts of the human body, there are specially designed metalloenzymes with the task of transporting molecular oxygen from the lungs to the source where it is needed. In addition, the body has created metalloenzymes that utilize molecular oxygen for the biotransformation of compounds both from a biodegradation and biosynthesis point of view. These enzymes are highly versatile, efficient and are known to react stereospecifically and/or regioselectively with substrates. As a consequence, metalloenzymes are of interest to the biotechnology industry, where they are used in bioprocesses, including the synthesis of pharmaceuticals and fine-chemicals.

In nature two types of oxygen-using metalloenzymes have been identified, namely, the monoxygenases and the dioxygenases. The former binds molecular oxygen and transfers one atom of O2 to a substrate, while the other atom is reduced to water. By contrast, in the dioxygenases both atoms of molecular oxygen are relayed to one or two substrate(s). In addition to this the metalloenzymes differ in the way that the metal is bound to the protein backbone. Thus, the enzymes are distinguished as either a heme or a non-heme enzyme. This chapter will focus on non-heme iron-containing dioxygenases, while later chapters (Chapters 8–11) are dedicated to heme-based monoxygenases.

Mononuclear non-heme iron-containing enzymes carry out a large variety of oxygenation reactions in nature with functions that vary from biosynthesis to biodegradation. In many cases the malfunction of these enzymes is correlated to a disease, so that it is important to understand the mechanistic details of dioxygen activation and oxygenation reactions by non-heme iron-containing enzymes. Most mononuclear non-heme iron-containing enzymes utilize molecular oxygen and transfer either one (monoxygenases) or two (dioxygenases) oxygen atoms to one or two substrates. However, there are also systems that oxidize molecular oxygen to two water molecules, thereby reducing the substrate, for instance via a ring-closure reaction (as in isopenicillin N synthase). Later we will give several examples of each of these processes. Despite the large versatility of non-heme enzymes, they often share a structural similarity where the metal (iron) is bound to the protein backbone via two or three protein ligands. A common motif observed in mononuclear non-heme iron enzymes is where the metal is bound to two histidine and a carboxylic acid side chain of the protein via a 2-His/1-carboxylic acid (Asp/Glu) facial triad. An example of this from the prolyl-4-hydroxylase active site is given in Figure 1.1. Generally, dioxygen binding is followed by the generation of a high-valent iron(IV)-oxo species, which has been identified in several enzymes and biomimetic systems as an active oxidant able to abstract hydrogen atoms of strong C–H bonds. Thus, in the α-ketoglutarate dependent dioxygenases the formation of an iron(IV)-oxo species occurs concomitant with decarboxylation of α-ketoglutarate to give succinate (Succ). Many excellent reviews have appeared in recent years that describe the structural biology, kinetics, crystal structures and protein folding, and spectroscopic properties of enzyme intermediates. In this chapter we give an overview of the mechanism and chemical properties of intermediates in the catalytic cycle of non-heme enzymes, while more detail will follow of several enzymatic systems in subsequent chapters, e.g. Chapters 2–4.

1.2 α-Ketoglutarate Dependent Dioxygenases (αKDD) and Halogenases (αKDH)

An extensively studied group of non-heme iron-containing enzymes utilizes α-ketoglutarate as a cofactor and act as a substrate hydroxylase (as in the α-ketoglutarate dependent dioxygenases, αKDD) or halogenase (α-ketoglutarate dependent halogenases, aKDH). In many cases these mononuclear non-heme iron enzymes utilize α-ketoglutarate (sometimes called 2-oxoglutarate) as a cofactor to generate a high-valent iron(IV)-oxo species. These α-ketoglutarate dependent enzymes are very versatile and catalyze many different reactions in biosystems, ranging from substrate hydroxylation, substrate halogenation, desaturation to ring-closure processes. The most common reaction, however, is concomitant decarboxylation of α-ketoglutarate combined with substrate monoxygenation to give hydroxylated products as performed by the α-ketoglutarate dependent dioxygenases (aKDD). This is an important function in biosystems, hence αKDD have been found to be involved in the biosynthesis of the vancomycin, fosfomycin and carbapenem group of antibiotics, as well as in DNA and RNA base repair mechanisms in mammals. These enzymes are also involved in crosslinking of collagen, oxygen sensing and responses to hypoxia. Close similarity in function and catalytic mechanism is found between the αKDD with the α-ketoglutarate dependent halogenases (αKDH) that combine decarboxylation of α-ketoglutarate with substrate halogenation.

The α-ketoacid dioxygenases and halogenases undergo a catalytic cycle that shows many similarities; therefore, both are given in Figure 1.2. As shown in Figure 1.2, the metal is bound to two histidine and a carboxylic acid (Asp/ Glu) side chain of the protein in the dioxygenases, while in the halogenases the carboxylic acid ligand is replaced by a halide anion. The other three ligand sites of the metal are vacant in the resting state but upon cofactor α-ketoglutarate (αKG) binding two of these ligand sites are occupied, namely, with the keto-group of αKG trans to the carboxylic acid or halide ligand, while the carboxylic acid group of αKG binds trans to a histidine group (structure A in Figure 1.2). Upon substrate (SubH) binding the water ligand is displaced from the iron center (B) and replaced by molecular oxygen to form structure C which is a ferric-superoxo structure. The terminal oxygen atom of the superoxo group attacks the α-keto position of αKG to form a bicyclic ring-structure (D). Subsequently, the dioxygen bond breaks and carbon dioxide is released from the complex to give succinate and a high-valent iron(IV)-oxo complex (E). This intermediate abstracts a hydrogen atom from the substrate to give the ferric-hydroxo complex (F). At this stage the mechanism for the dioxygenases and halogenases diverges, whereby the hydroxyl group rebounds to the Sub• radical to form hydroxylated products in the α-ketoacid dioxygenases, whereas the halide atom is abstracted in the α-ketoacid halogenases to form halogenated products. In the final step products are released from the metal center and replaced by new reactant molecules to make the catalytic cycle complete. This catalytic cycle has been corroborated with both experimental and theoretical studies and in the next few sections we will highlight key findings of specific αKDD and αKDH enzymes.

1.2.1 Taurine/α-Ketoglutarate Dioxygenase (TauD)

Probably the most extensively studied mononuclear non-heme iron dioxygenase is taurine/α-ketoglutarate dioxygenase (TauD) since it is available in large amounts, highly soluble, and relatively stable. Thanks to this, it was the first enzyme where a high-valent iron(IV)-oxo species was characterized with resonance Raman, Mössbauer and X-ray absorption spectroscopy. As a consequence TauD has become the template for mononuclear nonheme iron enzymes and has prompted many additional studies. Here we give a brief overview of recent advances in the field and focus on the consensus mechanism of substrate hydroxylation. A more detailed elaboration of the mechanism and function of TauD enzymes follows in Chapters 2 and 3.

There are several crystal structures available of TauD enzymes, namely, as the protein databank files (pdb) 1GQW, 1GY9, and 1OS7. The latter two structures are substrates (αKG and taurine) bound complexes at 3.0 and 1.9 Å resolution, respectively. Figure 1.3 shows extracts from the 1OS7 pdb file, which is a tetramer. The dimer interface contains two conserved helices of secondary structure that are hydrophobic in nature. The tertiary structure that includes this dimer interface is described by an antiparallel four helical bundle. Each monomer contains a non-heme iron active center that is bound to the protein via a 2-His/1-Asp ligand motif through interactions with His99, Asp101 and His255. Substrate αKG binds as a bidentate ligand with the carboxylic acid group trans to His99 and the keto-group trans to Asp101. The terminal carboxylic acid tail of αKG is locked in a salt-bridge with Arg266 that keeps it in a tight and constraint conformation. The final ligand position of the metal is vacant in the protein databank file (pdb) but will be occupied by molecular oxygen in a later stage in the catalytic cycle. Taurine does not bind directly to the metal but is held in position through many hydrogen bonding interactions in a substrate binding pocket nearby the active site. Key hydrogen bonding interactions of the sulfate group of taurine are with the phenolate group of Tyr73, with the guanidinium group of Arg270, with the imidazole ring of His70 and with several crystal water molecules. Site-directed mutants of His70Ala or Arg270Lys failed to bind taurine and implicated the importance of these residues for substrate anchoring through hydrogen bonding interactions. Two phenylalanine rings (of Phe159 and Phe206) surround the substrate binding pocket; the former has been identified with substrate binding and orientation.

Spectroscopic studies on αKG addition to TauD under anaerobic conditions produced an absorption band at around 530 nm, which was attributed to αKG binding to the metal. Addition of taurine to the system increased the spectrum by about 28% and gave an absorption band at 520 nm, while substrate analogues did not give these spectral changes. From stopped-flow UV visible spectroscopy experiments Hausinger et al. measured formation rate constants of ca. 40 s-1 of these αKG–Fe(II)–TauD and taurine–αKG–Fe(II)– TauD chromophores, while a taurine binding rate constant of > 140 s-1 was determined, i.e. for the conversion of A into B in Figure 1.2. In addition, these studies provided evidence that B is able to react with molecular oxygen with a rate constant of 42 s-1, whereas A is inactive in a reaction with molecular oxygen. This is important for the enzyme and would prevent uncoupling, whereby αKG is decomposed to succinate and CO2 without concomitant substrate hydroxylation. Mössbauer spectroscopy studies on αKG and taurine binding provided evidence of a change in metal coordination from five-coordinated to six-coordinated since large Mössbauer parameter changes were observed: the isomer shift δ decreased from 1.27 to 1.16 ± 0.05 mm s-1 and the quadrupole splitting ΔEQ dropped from 3.06 to 2.76 ± 0.05 mm s-1.

The next step in the catalytic cycle of TauD includes binding of molecular oxygen. It was shown that TauD consumes equal amounts of αKG, molecular oxygen and taurine. Using 18O2 labeling it was shown that both succinate and hydroxylated taurine carry one atom that originates from molecular oxygen. In the absence of taurine, molecular oxygen reacted with complex A to form a yellow chromophore with absorbance band at 408 nm with a formation rate constant of 0.25 s-1 and a decay rate constant of 0.5 min-1 to give a greenish brown chromophore. Resonance Raman and nanoscale capillary LC/MS/MS studies on the latter provided convincing evidence of an Fe(III)catecholate species formed through self-hydroxylation of Tyr73 in the absence of taurine. With αKG as co-substrate self-hydroxylation was explained via an iron(IV)-oxo oxidant where the oxygen atom exchanges with solvent, while with succinate as a co-substrate probably an iron(III)-hydroperoxo intermediate is formed that rearranges to an iron(V)-oxohydroxo complex. These self-hydroxylation reactions may protect the enzyme from more destructive and nonselective oxidations in the absence of substrate. The absorbance in the greenish brown chromophore is due to a charge-transfer transition from catecholate to iron(III). Thus, self-hydroxylation is explained by the formation of an iron(IV)-oxo species (E in Figure 1.2) that in the absence of substrate hydroxylates a nearby amino acid residue (Tyr73). The detection of a distinct EPR spectrum in the reaction mechanism is explained through the formation of a long-lived Fe(III)OH--Tyr• intermediate in this reaction mechanism.

Stopped-flow absorption spectra provided unambiguous evidence for the accumulation of two intermediates after dioxygen binding. The first of these develops after 20–25 ms, has an absorbance band at 318 nm and decays after about 600 ms. This intermediate also has a strong absorbance feature at 520 nm, which decreases strongly during decay. Mössbauer spectroscopy showed this species to be an Fe(IV) center with an isomer shift δ = 0.31 mm s-1 and quadrupole splitting ΔEQ = –0.88 mm s-1, while the absence of an EPR signal implicates a quintet spin ground state. Subsequent resonance Raman studies of Hausinger et al. on 16O2 and 18O2 binding to TauD unequivocally assigned this first intermediate as an iron(IV)-oxo species. Recently, using density functional theory (DFT) calculations this difference spectrum was reproduced and all peaks were assigned to a characteristic vibration. Thus, the iron-oxo stretch vibration vFeO is located at 821 cm-1 and redshifts by 34 cm-1 upon replacement of 16O2 by 18O2 – hence a negative peak in the difference spectrum at 787 cm-1. The vibration at 859 cm-1 is the OOC–C vibration in the succinate group that carries one oxygen atom originating from molecular oxygen, but the down-shift is only 11 cm-1. Two bending vibrations in the carboxylic acid group of succinate are located at 555 and 583 cm-1, the former one is out-of-plane and the latter one in-plane. These two vibrations give a small down-shift of about 6 cm-1 upon replacement of 16O2 by 18O2, but the intensity of the peaks in the spectrum is different in the 16O2 as compared to the 18O2 spectrum, and as a consequence appears as a positive and negative peak.

(Continues...)

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Edition details

• Publisher: Royal Society of Chemistry
• Publication date: 2011
• ISBN 10: 1849731810
• ISBN 13: 9781849731812
• Binding: Hardcover
• Language: English
• Edition number: 1
• Number of pages: 462
• Editor: Samuel P De Visser