Formation and Cleavage of CâC Bonds by Enzymatic Oxidation

Review Cite This: Chem. Rev. 2018, 118, 6573−6655

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Formation and Cleavage of C−C Bonds by Enzymatic Oxidation− Reduction Reactions F. Peter Guengerich* and Francis K. Yoshimoto

Chem. Rev. 2018.118:6573-6655. Downloaded from pubs.acs.org by KAOHSIUNG MEDICAL UNIV on 08/09/18. For personal use only.

Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, United States Department of Chemistry, University of Texas-San Antonio, San Antonio, Texas 78249-0698, United States ABSTRACT: Many oxidation−reduction (redox) enzymes, particularly oxygenases, have roles in reactions that make and break C−C bonds. The list includes cytochrome P450 and other heme-based monooxygenases, heme-based dioxygenases, nonheme iron mono- and dioxygenases, flavoproteins, radical S-adenosylmethionine enzymes, copper enzymes, and peroxidases. Reactions involve steroids, intermediary metabolism, secondary natural products, drugs, and industrial and agricultural chemicals. Many C−C bonds are formed via either (i) coupling of diradicals or (ii) generation of unstable products that rearrange. C−C cleavage reactions involve several themes: (i) rearrangement of unstable oxidized products produced by the enzymes, (ii) oxidation and collapse of radicals or cations via rearrangement, (iii) oxygenation to yield products that are readily hydrolyzed by other enzymes, and (iv) activation of O2 in systems in which the binding of a substrate facilitates O2 activation. Many of the enzymes involve metals, but of these, iron is clearly predominant.

CONTENTS 1. Introduction 1.1. General Aspects of Oxygen Activation 2. C−C Bond Forming Reactions 2.1. General Considerations 2.2. P450 Reactions 2.2.1. P450 Mechanisms 2.2.2. P450 Diradical Coupling Reactions 2.2.3. P450 Rearrangements Involving Diradical Coupling 2.2.4. P450s 5A1 and 8A1 and Rearrangements of Prostaglandins 2.2.5. P450 and Other Natural Products: Coupling of Unstable Products 2.2.6. P450 Reactions in Drug Metabolism− Coupling of Unstable Products 2.2.7. P450 Nonredox Reactions 2.2.8. Cyclopropanation by Engineered P450 Derivatives 2.3. Flavoproteins 2.3.1. Berberine Bridge Enzyme 2.3.2. Other Flavoproteins 2.4. Prostaglandin Synthesis 2.5. Lipoxygenase-Coupled Reactions 2.5.1. Dicyclobutane Formation 2.5.2. P450 74 Rearrangements 2.6. Rieske Oxygenases RedG and PR4B68 and Prodiginine Synthesis 2.7. Radical SAM and Cobalamin Reactions 2.8. Vitamin K-Mediated Carboxylation 2.9. Lignin Synthesis © 2018 American Chemical Society

2.9.1. Structural Lignins 2.9.2. Dietary Lignins 2.10. MauG and Tryptophan Cross-Linking 3. C−C Bond Breaking Reactions 3.1. P450 Reactions 3.1.1. Steroids 3.1.2. P450 74 Cleavage Reactions 3.1.3. Simple Models, Industrial Chemicals, and Drugs 3.1.4. More Natural Products 3.2. Flavoproteins 3.2.1. Flavin Monooxygenase Reactions 3.2.2. Flavin-Based Pseudodioxygenases 3.2.3. Prenyl FMN 3.2.4. N5 Nucleophlic Reactions 3.3. Desaturation and Cleavage of Fatty Acids 3.4. Oxidations of Heme 3.4.1. Nonenzymatic Heme Degradation 3.4.2. Heme Oxygenases 3.4.3. Coproheme Decarboxylation 3.4.4. HemF 3.4.5. Mycobilin and Staphylobilin Formation 3.5. Radical SAM Reactions 3.5.1. Repair of Photodimers in DNA 3.5.2. Coproporphyrinogen Oxidase 3.5.3. Mycofactin-Biosynthetic Protein 3.6. β-Carotene Dioxygenases

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Received: January 12, 2018 Published: June 22, 2018 6573

DOI: 10.1021/acs.chemrev.8b00031 Chem. Rev. 2018, 118, 6573−6655

Chemical Reviews 3.7. Tryptophan and Indoleamine 2,3-Dioxygenases 3.8. Mononuclear Nonheme Iron Dioxygenases 3.8.1. Examples of Mononuclear Nonheme Iron Dioxygenases 3.8.2. Mechanisms of Mononuclear Nonheme Iron Dioxygenases 3.9. Sterol 4-Demethylation 3.10. Iron/α-KG (Fe/α-KG) Dioxygenases 3.10.1. Mechanisms of Iron/α-KG Dioxygenases 3.10.2. Examples of Iron/α-KG Dioxygenases 3.11. 1-Aminocyclopropane-1-carboxylic Acid Oxidase 3.12. Dioxygenases with Variable Metal Groups 3.12.1. Quercetinase 3.12.2. Acireductone Dioxygenase 3.13. Lignin Degradation 3.13.1. Lignin-Degrading Peroxidases 3.13.2. Laccases 4. Summary and Conclusions Author Information Corresponding Author ORCID Notes Biographies Acknowledgments Dedication Abbreviations References

Review

Iron has a key role in many of the reactions we will discuss and is the dominant metal ion in the prosthetic groups. (Note: we will refer to “prosthetic groups” as parts of catalysts that do not show up in the overall reaction stoichiometry (e.g., heme, flavins). In a strict sense, “cofactors” is a term meaning cosubstrates, which do show up in the overall reaction stoichiometry [e.g., NAD(P)+/ NAD(P)H].

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1.1. General Aspects of Oxygen Activation

Description of some fundamental aspects of oxygen activation is in order prior to detailed consideration of mechanisms. Molecular oxygen (O2) is in the triplet state and does not normally react with carbon molecules, which are in the singlet state. (The state refers to the quantum state of the molecules and the energy levels seen in an applied magnetic field.) Singlet oxygen does exist, even in some rare biological situations, but is 22 kcal mol−1 higher in energy and difficult to control in enzymatic reactions that require activated oxygen but also great regio- and stereoselectivity. So how does O2 undergo formally spin-forbidden reactions with biological molecules? Our understanding of redox enzymes has not always been so complete as today. Before the pioneering work of Mason,3−6 Hayaishi,7−13 and others in the 1950’s, the situation was still unclear. Indeed, Wieland was of the opinion that the addition of oxygen to carbon molecules was the result of activation of the carbons or hydrogens, followed by addition of oxygen from water, as exemplified by the fatty acid β-oxidation process (i.e., desaturation followed by hydration, see section 3.3 below).14 This is in contrast to the concept of activation of the oxygen molecule. For more on the theories of Wieland and the opposing ideas of Warburg, see refs 4, 11, 12, and 14−16. Even later, there were serious proposals of the activation of mobile species of O2 being generated and then moving to react with carbon atoms,17,18 and some of these have recently surfaced again.19,20

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1. INTRODUCTION The making and breaking of bonds is the basis of metabolism, enzymology, and of biochemistry as a whole. Reviewing all such reactions would not be possible in this article, and we have chosen to restrict the scope of the review to C−C bonds. C−C bonds are inherently difficult to break, if the atoms are not activated, and we will see that a fairly common approach in nature involves first oxidizing one of the carbons in the process. Thus, a rather common paradigm is to couple the breaking of C−C bonds to oxidation−reduction reactions (e.g., oxygenating one of the carbons). In part because of our own research interests (and the original submission invitation), we have restricted our review to the making and breaking of C−C bonds (not including C−O and C−N bonds) by redox processes. The processes are divided into (i) C−C bond-making reactions and (ii) C−C bondbreaking reactions. As we will see, many enzymatic reactions we will consider involve both C−C bond breaking and forming and may not be exclusively classified in one group or the other. At the outset, we should emphasize that we do not claim to be comprehensive in covering all C−C bond-forming and -breaking reactions catalyzed by redox-active enzymes, but we have attempted to provide many representative enzymes and examples. We have also restricted the definition of redox-active enzymes to those using prosthetic groups to do oxidation− reduction chemistry. For instance, even though there are aspects of pyridoxal chemistry that involve some formal oxidation and reduction processes to generate an electron sink in decarboxylation,1 pyridoxal is not generally considered a redox group and has not been included here. (However, see a recent addition to the literature.2)

Figure 1. Issue of reaction of triplet O2 with singlet (carbon) atoms (R). Two solutions are shown, one involving a metal complex M (usually Fe or Cu) and another with a π-stabilized radical (e.g., reduced flavin) that can react with O2. (The reactions are not intended to indicate stoichiometry.)

However, any reactive species such as a superoxide anion (O2− •), singlet oxygen, or hydroxyl radical would be extremely hard to control in delicate regioselective hydroxylations and other oxidations. One major solution to the spin-forbidden dilemma is to bind O2 to a transition metal, most commonly iron or copper, and then consider the electronic state of the metal−oxygen complex in its reactivity with organic molecules (Figure 1). Examples with iron are termed compound 0 (FeIII−O2̅), compound I (formally FeO3+), and compound II (FeOH3+). These species vary in their stability and ease of detection in enzymes and in biomimetic models. In some cases, these have been trapped and studied spectroscopically, but in many other cases their roles have been inferred but never proven. As we will see, this has led to some differences in opinion about catalytic mechanisms. 6574


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section 3.12).27 As we will discuss later, no flavin-dependent dioxygenases are known.

The other major approach to activating and harnessing O2 to do useful oxidation chemistry is to stabilize the superoxide anion (Figure 1). This stabilization can be done with the use of a prosthetic group in which the electronic charge associated with a radical (created in the 1-electron reduction of O2) is stabilized through an extended π system, as in the case of flavins and pterins. The stabilization leads to the reaction of the flavin semiquinone with superoxide to generate a 4a-hydroperoxy flavin, which is the active oxidant in many cases.21 (Flavin N5oxides are also now recognized as intermediates, possibly arising from initial N5-hydroperoxide products of the reaction of reduced flavins with O2.22) Some recent cases of oxygen activation without prosthetic groups or cofactors have been reported.23,24 These cases involve π-rich substrates that appear to act in the same way as flavins to stabilize O2 and lead to oxygenated species that break down to yield the observed products.25 Among the oxygenases, there is an important distinction between monooxygenases and dioxygenases.4 Monooxygenases incorporate only one of the two atoms of O2 into substrate(s). The other atom is reduced to the level of H2O (i.e., a 2-electron reduction of O2) (eq 1). These are called mixed-function oxidases and require the input of two electrons, which usually come from NAD(P)H as the donor4 or occasionally ascorbic acid. An accessory protein, generally a flavoprotein, is required to couple a 2-electron process [NAD(P)H hydride transfer]1 with a one-electron process (metal center, e.g., Fe and Cu) (eq 1). +

NAD(P)H + O2 + R → NAD(P) + H 2O + RO

2. C−C BOND FORMING REACTIONS C−C bonds have to be formed in order to generate both relatively simple and complex molecules. There are a number of ways of doing this, including the use of prosthetic groups (e.g., thiamine pyrophosphate).1 We have restricted the scope of this review to enzymes that normally work by using oxidation and reduction (redox enzymes). 2.1. General Considerations

Many of the C−C bond-forming reactions catalyzed by redox enzymes fall into one of two major classes, either coupling of diradical systems or the rearrangement of unstable enzyme products, either within or outside of the enzyme. The former are homolytic reactions, and the latter are heterolytic or “ionic.” Some of the diradical systems may involve one carbon radical and the equivalent of a second radical in the form of an iron−oxygen complex until it is used (e.g., some P450 reactions). The list of enzymes to be addressed for C−C bond formation includes P450s, other heme proteins, nonheme iron proteins, flavoproteins, radical S-adenosylmethionine (SAM) enzymes, and cobalamins. 2.2. P450 Reactions

Many of the reactions discussed here, both C−C bond forming and C−C bond breaking, are catalyzed by P450 enzymes. Humans have 57 P450 (CYP) genes and most mammals have 40−120, but P450s are also found in plants, most bacteria, fungi, and other forms of life and the total number of known sequences is now ≥206000 (http://www.uniprot.org/uniprot/?query= cytochrome+P450&sort=score). Some species of fungi have >300 P450s.28 Plants each contain hundreds of CYP genes (142− 412 among several plant sequences)29 and as many as 1476 in wheat (drnelson.uthsc.edu/cytochromeP450.html). Collectively, the P450s catalyze ≥95% of the reactions known to occur via redox chemistry;30 this value is driven by the literature showing roles of P450s in not only natural product biochemistry but also the dominant role of P450s in the oxidation of drugs, drug candidates, and industrial and agricultural chemicals. 2.2.1. P450 Mechanisms. It is useful to review general aspects of P450 catalysis before considering individual applications in both C−C bond forming and C−C bond breaking reactions (Figure 2). The cycle shown in Figure 2 is for a microsomal P450 utilizing the diflavin NADPH-P450 reductase as a source of electrons. In many cases, this reductant is replaced by a flavoprotein/ ferredoxin system, particularly in mitochondria and bacteria. There are some other cases involving alternate electron sources, and a few P450s even use H2O2 as an active oxygen source, circumventing the need for electrons.31 The typical reaction cycle contains at least nine discrete electronic steps (Figure 2). It should be noted that at least several of these are accompanied by conformational protein changes,35 but complete roles of such changes are still undefined. An overall point to make is that, although substrate binding may facilitate oxygen activation in some cases,36 the activation of molecular oxygen proceeds by a pathway independent of the substrate. This pattern is shared by flavin-based monooxygenases21 and differs from many dioxygenases, which have iron liganded to both parts of the substrate and to the oxygen, leading to oxygen activation.37,38

(1)

Dioxygenases incorporate both atoms of O2 into organic substrates (eqs 2, 3) R + O2 → RO2

(2)

R + R′ + O2 → RO + R′O

(3)

Distinguishing between a dioxygenase that inserts two atoms of oxygen together (eq 2) from a monooxygenase that does two monooxygenation steps (eq 1) sequentially may not be easy, especially if cofactor requirements have not been rigorously characterized. The most definitive way is with the use of 18O labeling, particularly with a mixture of 18O2 and 16O2 gas. In a dioxygenase (eq 2), the product will contain either two atoms of 18 O or two atoms of 16O but not a mixture of 18O and 16O atoms. Mass spectrometry provides a simple answer, as exemplified in the work of Samuelsson with prostaglandin E. 26 If a monooxygenase is catalyzing two sequential steps (eq 1) then the first product will contain a mixture of 18O and 16O. The second product (RO2) will contain a mixture of 18O−18O, 18 O−16O, and 16O−16O, which again can be readily distinguished by mass spectrometry.26 Some dioxygenases utilize the stoichiometry shown in eq 3, where the two atoms of oxygen are incorporated but into separate products. An example is the case of iron/α-ketoglutarate (α-KG)-dependent dioxygenases, which will be discussed later (section 3.10). One oxygen is incorporated in the substrate (R), and the other is used to oxidize α-KG to succinate (with the O label appearing in the carboxylic acid group of succinate). Monooxygenases generally utilize heme, nonheme iron, copper, or flavins as prosthetic groups. Dioxygenases generally use heme or nonheme iron; copper-based dioxygenases are rare (e.g., quercetinase, which we will deal with later in section 3.12.1). A few of the enzymes to be discussed use other metals (e.g., nickel) (some quercetinases and acireductone dioxygenase, 6575


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the form of FeIVO with the additional charge and radical (•+) in the porphyrin ring.52−54 This entity is termed compound I, based on electronic similarity to the entity first characterized in peroxidase chemistry.55 Compound I is highly unstable in P450s, in contrast to some peroxidases and catalase, and to date has only been characterized in the case of a few bacterial P450s.56−58 We will also see elements of this type of chemistry with some other iron-based enzymes (heme oxygenase, dioxygenases). Compound I abstracts a hydrogen atom in step 7 (generating compound II, formally FeIV−OH). The resulting pair of radicals can usually be considered as a caged reaction (i.e., FeOH3+ plus R•), and the Fe−O bond splits homolytically for “oxygen rebound” to the incipient substrate radical (R•), yielding the product ROH (step 8), which is released in step 9 to complete the basic cycle. As we will discuss later, compound I has a high redox potential and can also abstract an electron from an amine or even some hydrocarbons. Compound I is generally considered to be the active oxoiron (ferryl) species in most P450 reactions.54 Compound I has been trapped through rapid-freeze quenching by reacting a P450 enzyme with a stoichiometric amount of m-chloroperbenzoic acid (m-CPBA), an oxygen surrogate, and has been subsequently characterized by spectroscopic techniques (i.e., Mössbauer, EPR).56 The resulting ferryl species has been clearly shown to hydroxylate alkanes.33 As an electrophilic oxygen species, compound I abstracts hydrogen atoms of C−H bonds and, in an analogous manner, can abstract a hydrogen atom from an O− H bond to yield an oxygen radical intermediate. One of the precursors to compound I is formally an iron(III) bound to peroxide (Fe3+-O2− in Figure 2), which is known as ferric peroxide (compound 0, vide supra) and has been proposed to be an active iron species that performs some of the various other reactions that P450s catalyze (e.g., epoxidation and C−C bond cleavage).59 Compound I has electrophilic properties while ferric peroxide (unprotonated) is nucleophilic. Two points about this catalytic cycle are relevant here. One is related to both C−C bond-forming and -breaking. In some cases, step 8 (Figure 2) appears to be slow enough to allow the R• (substrate radical) to undergo some rearrangement, moving the site of the radical on the molecule.60 In some cases, the FeOH3+ species, which is still a very reactive molecule, can also abstract a hydrogen atom (e.g., in desaturation reactions).45,54,61,62 In the case of the C−C bond-forming reactions, as we will see, a dominant aspect is the formation of diradical systems that can couple together (to form new C−C bonds). Another major issue that will be discussed regarding several C−C bond-breaking reactions is whether they use the FeO3+ species (compound I) or Fe3+O2̅ (compound 0). These are both unstable species and, except in a few cases,34,56,57 cannot be generated in the laboratory. Even if compound 0 were generated directly,34 it could proceed to yield compound I before reacting (Figure 2). We will see that both compound I and compound 0 mechanisms have been proposed for some of the P450 C−C bond-cleavage reactions (e.g., P450s 19A1, 17A1, 51A1, and 1A2). Mechanistically, these are quite different in that compound I is an electrophile and compound 0 is a nucleophile, as already mentioned. Distinguishing between the mechanisms has not been trivial in some cases. 2.2.2. P450 Diradical Coupling Reactions. Many of the P450-dependent C−C bond forming reactions involve diradicals. Direct evidence for their existence has been difficult to obtain even when such rationalizations seem very reasonable (e.g., there is little if any EPR evidence for their existence in almost all of the

Figure 2. General catalytic cycle for P450 reactions. RH indicates a substrate. The electrons are provided by either NADPH-P450 reductase (microsomal P450s, as shown) or a ferredoxin reductase/ferredoxin system (mitochondrial and some bacterial P450s). For some other electron delivery modes, see Guengerich and Munro.31 Note the Fe3+− O2− (ferric peroxide, compound 0) and FeO3+ (compound I) forms discussed in the text. The electron transfers from the reductase are simplifications in that the course of electron flow is probably from FMNH2/FADH• to FMNH•/FADH• in the first reduction (step 2) and (assuming that the reductase contributes the second electron to the P450) from FMNH•/FAD• to FMNH•/FAD in the second reduction step (4). In many bacterial systems and in mammalian mitochondria, the electrons are donated by ferredoxins (e.g., adrenodoxin and putidaredoxin). In some cases with mammalian P450s, the second electron (step 4) is donated by cytochrome b5 (b5).32 In the literature, there exists different nomenclature for the same iron intermediates in this P450 catalytic cycle.33,34 For clarity, throughout the text of this manuscript compound I is referred to interchangeably with FeO3+, and ferric peroxide is referred to interchangeably with FeIII−O2− or compound 0 (protonated form FeIII−OOH).

The catalytic cycle (Figure 2) begins with binding of the substrate (RH) in step 1, near the iron but not attached to it. Step 2 is a one-electron reduction by either the diflavin reductase or a ferredoxin, which in turn has been reduced by a flavoprotein. The rate of reduction may or may not be facilitated by substrate binding.36,39,40 The possibility exists that substrate binding can occur after reduction, and this has been shown.41 Further, a population of ferrous P450 is found in bacterial or liver cells, apparently in the absence of substrates.42 Regardless of the route to the substrate-bound ferrous enzyme, it reacts with O2 to form a ferrous-O2 complex (step 3), electronically analogous to oxyhemoglobin but much less stable. The stability varies considerably,36,43,44 and for several P450s, this species has not been detected yet.45 The next step (step 4) involves the input of another electron. This electron can be provided by the flavoprotein reductase. In some cases, this second electron can come from another hemoprotein, cytochrome b5 (b5),46,47 as originally proposed by Hildebrandt and Estabrook,48 although electron transfer has been ruled out in a number of the cases in which stimulation of catalysis by b5 is observed.32,49−51 Following the input of the second electron, the next step (step 5) is protonation (of Fe3+O2−) to yield an iron hydroperoxide complex (formally FeIII−OOH) (one electron is transferred from the FeIII and one from O2− to form the Fe−O bond, leaving the iron in a formal 3+ (III) state. This complex loses water (step 6) to generate a formal FeO3+ complex, generally thought to be in 6576


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derived by a number of C−C and other coupling reactions, yielding entities that can be further decorated by various enzymes (Figure 3). The biological advantages of making these alkaloids in the plants are, in general, unknown.29 Many are probably for protection, although in many cases little is known about the natural enemies of the organism.

examples we will cover), presumably due to short half-lives, and such reactions are generally insensitive to the addition of radical scavengers. It is possible to propose mechanisms with actual diradicals in some cases. In others, the mechanisms have been written with a combination of a carbon radical plus FeOH3+, an entity capable of pulling another hydrogen atom from a C−H bond.

Figure 5. Products of more coupling reactions in the synthesis of isoquinoline alkaloids from (S)-reticuline by plant P450s.65

Details of the C−C coupling reactions are usually not known. Some of the reactions probably involve C• diradical coupling, as proposed in one step of morphine synthesis (Figure 4). Some other examples are shown in Figure 5, and roles of two specific P450s with the enantiomers of reticuline are shown in Figure 6. 2.2.2.2. Morphine Biosynthesis. The synthesis of morphine begins with tyrosine. Morphine is synthesized in mammals as well as in plants, in very low concentrations, and most of the

Figure 3. Coupling reactions involved in benzylisoquinoline alkaloid biosynthesis.29,63 The colors of the rings (black, red, and blue) trace the origins in 1-benzylisoquinoline.

2.2.2.1. Benzyl Isoquinoline Alkaloids. The benzyl isoquinoline alkaloids are a group of >2500 known structures found in nature (mainly in plants), some with medicinal properties that have been known since ancient times.63 These compounds are

Figure 4. Proposed C−C diradical coupling in the synthesis of salutaridine from reticuline by plant and mammalian P450s.64 6577


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2.2.2.3. P450 3A4 and Raloxifene Dimer. Another coupling is the formation of a dimer of the drug raloxifene by P450 3A4 (Figure 7).62,72 Because of the coupling of the phenolic rings, the reaction can easily be rationalized by the coupling of two one-electron oxidation species generated from the parent drug raloxifene. The authors concluded that the active site of P450 3A4 is sufficiently large enough to accommodate two molecules of (P450 3A4) substrate, a view supported by X-ray crystallography.73,74 P450 3A4 can also form a dimer from 17β-estradiol,62,75 but this is an O-linked product and not within the scope of this review. 2.2.2.4. P450 121 and Dicyclotyrosine. Mycobacterium tuberculosis causes more deaths each year than any other known pathogen, and there is wide interest in finding better drugs to kill the organism. M. tuberculosis has 20 P450 genes, and the functions of only some are known.76 A knockout strain with deletion of rv2276, the gene coding for P450 121, showed the physiological significance of this P450 in the bacterium and suggests it as a drug target. An X-ray crystal structure of M. tuberculosis P450 121 has been published.77 A key to understanding the function of the enzyme came with the identification of cyclization of tyrosine as a reaction catalyzed by the enzyme (Figure 8).78 The mechanism involves a compound I intermediate (Figure 2) and, most probably, some type of diradical cyclization (Figure 8).79 More detailed spectroscopic characterization of the enzyme has been reported,80 but the exact biological role of the cyclized product is yet unknown. 2.2.2.5. P450 158A2 and Flaviolin Coupling. P450 158A2 is one of 18 P450s found in the soil bacterium Streptomyces coelicolor.81 Defining the functions of the P450s of this complex prokaryotic organism has been challenging. The organism uses a type III polyketide synthase to make hydroxylated naphthalene molecules.82 Some of these polymerize to melanins to contribute to virulence83 and are also UV-protective.82 One compound in this naphthalene group, flaviolin, was found to be oxidized to dimers and trimers by S. coelicolor P450 158A2.84 A crystal structure of the ferric protein contained two molecules of flaviolin, and a diradical pathway for synthesis of dimers is proposed (Figure 9).84 Further, X-ray crystallography structures implicated water molecules and the hydroxyl groups of flaviolin in assisting in the

Figure 6. C−C bond coupling with reticuline by plant P450s 80G2 and 719B1.29,63,66

reaction steps are the same.64,67−69 A key step is the C−C bond coupling (C12−C13) of two phenyl rings in the conversion of (R)-reticuline to generate the important intermediate salutaridine (Figures 4 and 6), which goes on to form thebaine, having an additional ether linkage of the two rings. The regiochemistry of the reaction with (R)-reticuline is extremely important (in generating salutaridine), in that alternate coupling products include (+)-pallidine, (−)-isoboldine, and (−)-corytuberine.64 A P450 in opioid poppies catalyzes the coupling to give salutaridine (Figure 6);70 Zenk and his associates also identified a porcine liver P450 enzyme that formed salutaridine.69,71 Human P450s 2D6 and 3A4 can oxidize (R)-reticuline to multiple C−C cyclized products, including salutaridine.64 Subsequent studies showed that human P450s 3A4 and 3A5 can catalyze the O6demethylation of thebaine (derived from salutaridine) and that P450 2D6 catalyzes the O3-demethylation of codeine, and thus all of the steps in morphine biosynthesis are now established in humans.67 However, the extent of contribution of this pathway to endogenous control of pain remains to be determined. A mechanism for the oxidative (C12−C13) coupling of (R)reticuline to form salutaridine has been proposed (Figure 4).64 As in most P450-catalyzed C−C bond-forming reactions, the mechanism involves generation of a diradical system, followed by coupling of two radicals.64

Figure 7. P450 3A4 raloxifene dimerization by P450 3A4.62,72 6578


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Figure 8. C−C diradical coupling in the synthesis of dicyclotyrosine by P450 121, supported by peracetic acid in a model system.79,80 PCET: protoncoupled electron transfer.

Figure 9. Proposed C−C diradical coupling in the synthesis of flaviolin by S. coelicolor P450 158A2.84,85

oxygen activation process.85 A structure of the Fe2+O2 complex (with two flaviolins bound) was reported.85 The closely related S. coelicolor P450 158A1 catalyzes similar flaviolin dimerization reactions but does not form the trimer seen with P450 158A2.86 Two flaviolin molecules are also seen in the X-ray crystal structure of P450 158A1 but are in different positions than in P450 158A2.86 A lysine group (Lys-90) in P450 158A1 is important in disallowing the binding of the third flaviolin needed to form trimers; this residue is substituted with an isoleucine (Ile87) in P450 158A2.87 The conclusion was reached that the (diradical) coupling reactions occur within the enzyme, in that P450 158A1 generates only two of the three diflaviolin products that P450 158A2 does, and the molar ratios differ compared with the 158A2 products. Thus, each enzyme seems to maintain its

own stereo- and regioselectivity.86 This result would not be expected if diradical coupling was thermodynamically controlled and occurred outside of the active site.

Figure 10. P450 245A1 and the synthesis of indolocarbazole alkaloids.62,88,89 6579


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Figure 11. C−C coupling by bacterial P450 107B1 (HmtS) in the biosynthesis of himastatin.76,90,91

Figure 12. Proposed mechanism for migration of an aryl group in flavanone by P450 93C2.92−94

Figure 13. Oxidation of (R)-littorine to (S)-hyoscyamine aldehyde by Hyoscamus niger (nightshade plant) P450 80F1.95,96

2.2.2.6. P450 245A1 and Indolocarbazole Alkaloid Biosynthesis. Another example of diaryl coupling is the intramolecular cross-linking of two indole moieties in the biosynthesis of indolocarbazole alkaloids by P450 245A1 (Figure 10).62,88,89 The nature of the indole ring argues for a mechanism involving oneelectron abstractions and coupling of the two rings (Figure 10). 2.2.2.7. P450 107B and Himastatin Biosynthesis. Another example of apparent diradical coupling is seen with P450 107B in the synthesis of the natural product himastatin (Figure 11). Again, diaryl coupling is seen, probably the result of the proximity of two radicals. 2.2.3. P450 Rearrangements Involving Diradical Coupling. In some cases, bonds are broken and new ones are formed after rearrangement with the postulated intermediacy of radicals. 2.2.3.1. P450 93C2 and Rearrangement of Flavanone. P450 93C2 oxidizes flavanone, resulting in 3-hydroxyflavanone and 2hydroxyisoflavanone (Figure 12). The mechanism can be

rationalized by the formation of a carbon radical and an unusual migration of a phenoxy group to the adjacent carbon.92−94 2.2.3.2. Oxidation of Littorine by P450 80F1. Another migration occurs in the oxidation of (R)-littorine, in which a carboalkyl ester migrates to the adjacent carbon. The mechanism can be rationalized in the context of a radical intermediate (Figure 13).95,96 2.2.4. P450s 5A1 and 8A1 and Rearrangements of Prostaglandins. These mammalian reactions can be considered as both C−C bond-forming and -breaking but will be discussed here. The endoperoxide prostaglandin H2 (see section 2.4) is unstable and reacts with ferric hemeproteins. These reactions are different than those generally catalyzed by P450s, in that the “active oxygen” is supplied by the substrate and the roles of the enzymes are to direct the rearrangements. 6580


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site and then, due to chemical instability, rearrange to form stable products. Two examples with natural products are presented

Figure 14. Reactions of P450s 5A1 and 8A1 with prostaglandin H2 to form thromboxane A2 (TXA2) and prostacyclin (PGI2).97,98

The major reactions are catalyzed by P450s 5A1 and 8A1, thromboxane synthase and prostacyclin synthase, respectively (Figure 14). In general, thromboxane A2 is considered harmful in that it activates blood platelet aggregation and is a vasoconstrictor.

Figure 15. Conversion of prostaglandin H2 (PGH2) to thromboxane A2 (TxA2) and to hydroxyheptatrienoic acid (HHT) and malondialdehyde by P450s.98,101

Figure 16. P450-catalyzed coupling of indoles to yield dimeric products. (A) Indigo and indirubin,102 (B) oxazole product,102 (C) product of 4OBn indole,103 Bn: benzyl.

Prostacyclin (PGI2) is generally considered a good actor in that it inhibits blood platelet aggregation and is a vasodilator.99 Mechanisms for the rearrangements are shown in Figure 14.97,98,100 In addition to forming thromboxane A2, a side reaction is the conversion to hydroxyheptatrienoic acid (HHT) and malondialdehyde (Figure 15).98 This reaction can be catalyzed by a physiologically irrelevant bacterial P45098 or by mammalian hepatic P450s (1A2, 3A4).101 The physiological significance of these latter reactions is not clear. 2.2.5. P450 and Other Natural Products: Coupling of Unstable Products. In some cases, diradical coupling is not involved. Products are generated that appear to leave the active

below. (More will be seen with synthetic substrates later, section 2.2.6.) 2.2.5.1. Mammalian P450s and Indole Oxidation. The 3hydroxylation of indole yields indoxyl, which is an inherently unstable molecule. Indoxyl and substituted indoxyls form colored dimers, including indigo, indirubin, and other products (Figure 16). 3-Hydroxylation of indoles is catalyzed by several P450s, particularly human P450s 2A6 and 2E1,102,103 and was first detected by the blue color (indigo) that accumulates in bacterial cultures of these enzymes when heterologously expressed in bacteria.102,104,105 The (nonenzymatic) coupling is enhanced in the presence of oxygen and is base-catalyzed.106 6581


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Figure 17. P450 reactions involved in trichothecene biosynthesis in Trichoderma species.107

Figure 18. Ring formation reaction (to metabolite M14) catalyzed by P450s with the drug candidate YH3945, 1-{3-[3-(4-cyanobenzyl]-3H-imidazol-4yl]propyl}-3-(6-methoxypyridin-3-yl)-1-(2-trifluoromethylbenzyl)thiourea. The first two reactions are C-hydroxylation and loss of the cyanobenzyl group through N-dealkylation.108

Figure 19. Dimer formation via Diels−Alder reactions of products. (A) Dimerization of thiophene S-oxide formed from ticlodipine by P450 2C19 or 2D6.109 (B) Dimerization of an S-oxide formed from 2-phenylthiophene by P450 1A1 and conjugation with glutathione (GSH).110

2.2.5.2. P450s and Trichothecene Biosynthesis. P450s are also involved in key steps in the biosynthesis of trichothecenes, mycotoxins produced in several fungi (Figure 17).107 Key steps include an allylic hydroxylation and epoxidation to yield the unstable molecule isotrichodiol, which rearranges nonenzymatically to a product that is then hydroxylated to yield trichodermol. 2.2.6. P450 Reactions in Drug Metabolism−Coupling of Unstable Products. As with natural products (vide inf ra), drug metabolism by P450s can result in the formation of unstable molecules that couple by chemical means. A few examples are presented.

2.2.6.1. YH3945. An example of the formation of a new ring structure is shown with the drug candidate YH3945 in Figure 18.108 This path commences with N-demethylation and hydroxylation of a methylene adjacent to a thiourea moiety followed by an O-demethylation step, which facilitates some of the subsequent transformation with the conjugation in the pyridine ring, ultimately generating a new ring. 2.2.6.2. Thiophene S-Oxide Coupling. Another case of C−C bond coupling is seen with thiophene S-oxides (Figure 19). SOxygenation is associated with covalent binding to glutathione (GSH) and proteins (Figure 19B), possibly related to toxicity. Both ticlopidine and the simple model compound 2-phenyl6582


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how flavins are used to catalyze many other types of

thiophene have been shown to form S-oxides, which can undergo nonenzymatic Diels−Alder condensation to form dimeric products (Figure 19). These Diels−Alder reactions should show second-order kinetics, and whether they occur at low concentrations in cells is unknown. 2.2.7. P450 Nonredox Reactions. Essentially all P450 reactions involve either iron redox chemistry or at least the use of iron to make rearrangements of molecules (e.g., prostaglandins

reactions,124,125 including C−C bond-making reactions.

Figure 20. A nonredox rearrangement to yield C−C coupling catalyzed by S. coelicolor P450 154A1.114

or hydroperoxides) via high-valent iron intermediates (e.g., P450s 5A1, 8A1).111 Only a few P450s have been reported to catalyze nonredox chemistry, and two of those cases involve hydrolyses.112,113 The other case, involving S. coelicolor P450 154A1, is one of C−C bond-breaking and reformation to synthesize an unusual Paternò-Büchi-like oxetane product (Figure 20).114 2.2.8. Cyclopropanation by Engineered P450 Derivatives. Arnold and associates have engineered bacterial P450s to do some unusual reactions, utilizing the approach of directed

Figure 22. Mechanism of berberine bridge enzyme (BBE).126−128 R indicates the phosphoribitol side chain.

2.3.1. Berberine Bridge Enzyme. One unusual reaction, the formation of the protoberberine ring system (Figure 3), is catalyzed by a flavoprotein termed berberine bridge enzyme (BBE). The crystal structure of Eschscholzia californica (California poppy) BBE has been published, as well as the rates of the oxidative and reductive half-reactions.126 The FAD prosthetic group has an unusual bicovalent linkage to the protein (Figure 22). The enzymatic mechanism is postulated to involve an unusual oxidation of an N-methyl group concurrent with coupling to a neighboring aryl moiety (Figure 22), followed by rearrangement to give (S)-scoulerine and reoxidation of the reduced FAD.126 More examples of BBE-related flavoproteins are now known, including the enzyme involved in nicotine formation (Figure 23).129 Other flavoproteins in plants show sequence similarity and catalyze dehydrogenations of benzyl isoquinoline alkaloids but do not appear to catalyze such C−C bond formations.130,131 2.3.1.2. Cannabidiolic Acid Synthase. BBE is not the only flavoprotein that can use its oxidation/reduction capability to catalyze C−C bond coupling reactions (Figure 23). For example, tetrahydrocannabinolic acid synthase catalyzes an oxidation to form tetrahydrocannabinolic acid, and cannabidiolic acid synthase uses the same substrate (cannabigerolic acid) to form cannabidiolic acid.132,133 In the process, the flavin is reduced; to recycle the flavin, the enzyme reacts with O2 and produces H2O2 (Figure 23A). Other examples involve the synthesis of nicotine (Figure 23B) and its homologue anabasine (Figure 23C). In the latter, two molecules of CO2 are released, a new C−C bond is formed and H2O2 is produced.

Figure 21. Cyclopropanation reactions catalyzed by unnatural P450 derivatives (P411).118

evolution (or, perhaps more properly termed “molecular breeding”).115,116 Of particular interest, bacterial P450 102A1 (P450BM‑3) has been developed to insert carbenes into olefins, thus accomplishing cyclopropanation reactions (Figure 21).116−119 The same strategy can be applied to generate cyclopropane in peptides, beginning with the readily available dehydroalanine.120 The mechanism of this reaction is not very similar to that of the usual P450s, in that the enzyme is acting as a carbene transfer reagent. The heme-binding cysteine is not needed, and a serine residue is more useful.121 The engineered P450 is termed P411 due to its altered spectral properties,118 and other proteins and metals can also be utilized in developing catalysts such as these. 2.3. Flavoproteins

Flavins contain an isoalloxazine ring system that facilitates both electron transfer and reaction with O2 (Figure 1).1,21 Because they (along with pterins) can function in both one- and twoelectron transfer reactions, they serve as biological “transformers” in coupling two-electron chemistry [e.g., hydride transfers from NAD(P)H] with one-electron accepting metal centers (e.g., iron). The roles of 4a-hydroperoxides in monooxygenase reactions have been known for some time1,21,122,123 and will be discussed later under C−C cleavage reactions. Recently, a number of examples have been reported of 6583


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example shown in Figure 24, a flavoprotein delivers electrons to a radical SAM-based transferase, an iron sulfur cluster protein involved in the conversion of tRNA N1-methyl guanosine to wyosine, an etheno derivative.134,135 In this case, the source of the additional carbons is pyruvate.135 2.3.2.1. UDP Galactose Pyranose Mutase. Another “new” role of a flavin involves the protein UDP galactose pyranose mutase (UGM) (Figure 25).136 A reduced flavin acts as a nucleophile (N5 atom), and the enzyme uses its redox capacity to process through an intermediate in which a UDP sugar hydroxyl acts as a nucleophile, generating a rearrangement leading to a new sugar ring. 2.3.2.2. Uridine Methylation in tRNA. Flavoproteins also work with other proteins in other ways, as exemplified by the post-transcriptional conversion of uridine to thymidine in tRNA, specifically m5U54 (Figure 26A).135 Covalent bonding of the RNA to Cys-223 of the enzyme is required (Figure 26B). The one-carbon unit is transferred from N5,N10-methylenetetrahydrofolate to the N5 atom of the reduced flavin of TrmFO. The carbon attached to the N5 atom of the flavin is in an electrophilic state and can be attacked by the double bond of the uracil moiety to form a covalent intermediate. Collapse of the intermediate, followed by the addition of a hydride ion, yields the thymine moiety and the oxidized flavin (Figure 26B). The flavin needs to be reduced again to reinitiate the process. TrmFO flavoproteins are found in many bacteria, but the only one for which a three-dimensional structure is known is the Thermus thermophilus enzyme.137 One aspect of redox enzymes that we are not discussing in depth is electron transfer through the amino acids of proteins. However, tyrosine and tryptophan radicals can be involved in some of the hemeprotein and flavin reactions, and in many protein systems, tyrosine can play a functional role as a redox intermediate.138 For instance, the role of a tyrosine radical cation (Tyr-343) in T. thermophilus TrmFO has been studied.139 This tyrosine is very close to the flavin, in an apolar environment, and shielded from the cleft forming the substrate binding site by the isoalloxazine ring. Tyr-343 has been identified as the electron donor responsible for quenching the excited state of FAD.139 2.3.2.3. OxaD, NotB, and Epoxidation. Flavins catalyze epoxidation reactions, although not as commonly as P450s (e.g.,

Figure 23. More BBE-like flavoprotein-catalyzed C−C bond coupling reactions.129 (A) Cannabigerolic acid (CGBA) is oxidized by tetrahydrocannabinolic acid (THCA) synthase (THCS) to THCA and by cannabidiolic acid (CBDA) synthase (CBDAS) to CBDA.132,133 (B) Synthesis of nicotine. (C) Synthesis of anabasine.129

Figure 24. Formation of the modified base wyosine in m1G37-tRNA by Tyw1.134,135 Ado-Met: S-adenosylmethionine (SAM) (see also Figure 40, vide inf ra).

2.3.2. Other Flavoproteins. Flavins can also act in an auxiliary role. Flavoproteins deliver electrons to P450s, either directly or indirectly (via iron−sulfur proteins) (Figure 2). In the

Figure 25. Rearrangement of UDP-galactopyranose to UDP-galactofuranose catalyzed by the flavoprotein UGM.136 R indicates the phosphoribitol side chain. 6584


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Figure 26. Reductive methylation of tRNA and rRNA by flavin methyltransferases.135 R indicates the phosphoribitol side chain. (A) Overall reaction catalyzed by TrmFO and RlmFO. (B) Proposed mechanism. N5,N10−CH2−THF: N5,N10-methylene tetrahydrofolate. The N5-substituted flavin is derived by transfer from a methylene (or other one-carbon unit) from the substituted folate.

Figure 27. Rearrangement of notoamide E following epoxidation by the flavoprotein NotB.141

squalene epoxidation140). In the case of the biotransformation of notoamide S, the enzyme OxaD forms an epoxide on a 5-

main targets of almost all nonsteroidal anti-inflammatory drugs. Several structures of mammalian prostaglandin synthases are now known.142,143 The enzymes are rather rigid, a property that has facilitated modeling studies and drug discovery efforts. The conversion of arachidonic acid to prostaglandin H2 involves two functions of the enzyme, the cyclooxygenase and the peroxidase functions. The prosthetic group is heme, and the enzyme behaves as a dioxygenase in the cyclooxygenase step. The cyclooxygenase function is of relevance in terms of this review, in that an internal C−C bond is formed. The peroxidase function must also be discussed briefly in light of its role in activation of the system.143 It is of note that the enzyme containing Mnsubstituted protophyrin IX (PPIX) has cyclooxygenase activity but not the peroxidase function.142,144 The catalytic mechanism involves initial abstraction of the 13pro-(S) hydrogen atom of arachidonic acid (Figure 29).142 The resulting radical reacts with O2 to form a peroxy radical, which then reacts with the 8,9-double bond to form a 9,11endoperoxide and move the radical to carbon 8. C−C bond formation them places the radical at carbon 13. Isomerization and reaction with another molecule of O2 yields the 15-peroxy radical, which abstracts a hydrogen atom to form prostaglandin G2 (Figure 29). The hydroperoxide is reduced to the alcohol prostaglandin H2 via the peroxidase function of the enzyme.142 Further transformations of prostaglandin H2 do not involve C−C bond breaking and formation except for the transformations to thromboxane and prostacyclin, which are catalyzed by P450s and are discussed elsewhere (section 2.2.3). The coupling of the C−C bond-making cyclooxygenation reaction with the peroxidase function is complex and will only be treated briefly here. However, it is an important part of the action of the enzyme mechanism and merits some discussion (Figure

Figure 28. Prostaglandins synthesized from prostaglandin H 2 (PGH2).142 PG: prostaglandin; TX: thromboxane. See also Figure 14.

hydroxytryptophan entity (Figure 27). Rearrangement leads to the transfer of a pentenyl unit to form a new C−C bond.141 2.4. Prostaglandin Synthesis

Arachidonic acid is converted to prostaglandin G 2 by prostaglandin synthases, or cyclooxygenases. Prostaglandin G2 is reduced to prostaglandin H2, which then serves as a source of a number of prostaglandins (Figure 28).142 Mammals, including humans, have two prostaglandin synthases (PTGS1 and PTGS2, sometimes referred to as COX-1 and COX-2, not to be confused with cytochrome oxidase, a more critical enzyme for life). These enzymes are the 6585


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Figure 29. Mechanism of oxidation of arachidonic acid to prostaglandin (PG) G2.142

Figure 30. Coupling of cyclooxygenation and peroxidation functions in the prostaglandin synthase reaction.143 AA: arachidonic acid.

Figure 31. Enzymatic synthesis of a bicyclobutane fatty acid by a cyanobacterial hemoprotein-lipoxygenase fusion protein.147

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30). The heme iron uses chemistry similar to other peroxidases and P450s, plus a tyrosyl radical whose role was elusive for many years.145 Initially a peroxide reacts with the heme iron, yielding compound I (FeO3+ or FeIV=O PPIX+•) and an alcohol. Compound I oxidizes Tyr-385 to a phenoxy radical, apparently via electron transfer involving the proximal heme ligand His-388. The Tyr-385 radical of intermediate II, a compound II-like species (Figure 30) abstracts the 13-pro-(S) hydrogen of arachidonic acid to initiate the cyclooxygenation process (Figure 30). The first product is prostaglandin G2, a hydroperoxide that can then be reduced to an alcohol (prostaglandin H2) by the peroxidase cycle, regenerating compound I to continue the cycle (Figure 30). Adding an enzyme (e.g., glutathione peroxidase) that scavenges the hydroperoxide will quench the reaction. As might be expected, the initial reaction needs “priming” and there is a lag phase.142,146 One might expect the reaction to continue indefinitely, in that it is a propagative radical reaction (Figure 30). However, the high-valent intermediates are destructive and lead to cross-linking and other destructive processes (Figure 30).142,143 2.5. Lipoxygenase-Coupled Reactions

Lipoxygenases are iron-dependent dioxygenases that catalyze the addition of O2 to allylic or otherwise activated carbons.1 They do not form or cleave C−C bonds by themselves, insofar as we know. However, the hydroperoxide products can be substrates for some unusual P450s and other heme-based enzymes, and a wide variety of natural products result from these processes. 2.5.1. Dicyclobutane Formation. A highly unusual example of C−C bond forming is seen with a cyanobacterial hemoprotein-lipoxygenase fusion protein (Figure 31).147 The reaction begins with a fatty acid hydroperoxide, which can be produced by a dioxygenase reaction (i.e., the lipoxygenase domain of the fusion protein). Two products are observed. One is a leukotriene A-type epoxide. More relevant here is the other product, involving C−C bond formation catalyzed by the hemoprotein. The putative epoxide-carbocation rearranges to create a cyclopropane ring and a new carbocationic center. This

Figure 33. Proposed mechanism of radical and ionic pathways for P450 74 reactions.99

Homolytic scission of the hydroperoxide (produced by a lipoxygenase) initiates the reaction. This process is considered to be very physiological, in that many of the products that emanate from the pathway (Figure 34) have important biological roles in plants and other species (e.g., signaling). Although alkyl hydroperoxides have been utilized with mammalian P450s in oxygen surrogate studies in the laboratory,150 there is no strong evidence to support the view that these play physiological roles. Most studies with these have been done with high concentrations of alkyl hydroperoxides, which are unlikely to be relevant.151−153 Another issue is that homolytic scission of alkyl hydroperoxides generates alkoxy radicals, which can initiate their own nonenzymatic chemistry and may be unrelated to the normal enzyme chemistry.154 Although radical pathways can explain some of the products that have been isolated, ionic intermediates (e.g., carbocations) are preferred to rationalize several products (Figure 33).99 The process of a sequential electron transfer from a carbon radical to compound II (FeOH3+) is generally well-accepted and, as discussed later, a very rational explanation for events related to steroid aromatization (see section 3.1.1.1.3 and Figures 58 and 59, vide inf ra) and hydride and alkyl group transfers in P450 reactions, as well as some other redox enzymes.99,149 (See also Figure 82, vide inf ra.) Several examples of the formation of unusual transformations by these pathways are presented. In green algae, the movement of a carbon outside of the chain to form a formyl group is shown (Figure 35). The formation of a cyclopropyl group and a lactone ring is observed in the synthesis of constanolactones in the red alga Constantinea simplex (Figure 36).149 The red alga Laurencia hybrida synthesizes hybridalactone, a macrocyclic lactone with a

Figure 32. Some general product pathways for P450 74 reactions.

center reacts to generate a highly unusual bicyclobutane ring system. 2.5.2. P450 74 Rearrangements. The rearrangement of oxidized lipids to new biological products was already discussed with P450s 5A1 and 8A1 (section 2.2.4, Figure 14). The P450 74 Family was first discovered in plants, specifically flaxseed.148 Allene oxides are unstable and form a variety of products, many of which include new C−C bonds (Figure 32).61,99 The mechanism for formation of these products is shown (Figure 33).99 6587


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Figure 34. Proposed role of epoxy allylic carbocation in the formation of marine oxylipins.149

2.6. Rieske Oxygenases RedG and PR4B68 and Prodiginine Synthesis

Prodiginines are red tripyrrolic alkaloids with a variety of biological activities,155 formed by oxidation reactions (Figure 39). In the complex bacterium S. coelicolor, the process involves a family of Rieske nonheme iron monooxygenases, exemplified by RedG. (Rieske iron sulfur clusters were first discovered in 1964 in mitochondrial electron transfer chain proteins156 and have also been implicated in both monooxgenases and dioxygenases, vide inf ra.) In the marine bacterium Pseudoalteromonas rubin, the reaction is catalyzed by PR4B680, an integral membrane diiron oxygenase that is unrelated to RedG but a member of the fatty acid hydroxylase family and closely related to metazoan alkyl glycerol monooxygenase.155 2.7. Radical SAM and Cobalamin Reactions

We have already seen the utility of hypervalent iron−oxygen complexes in creating carbon radicals that lead to C−C bond cleavage and rearrangements to yield new C−C bonds (e.g., Figures 4, 8, 9, 12, and 13). Complex rearrangements have been observed with both radical SAM and cobalamin (vitamin B12) enzymes, with redox reactions driving the initiation of radical chemistry.1,157 The methyltransferases in the radical SAM enzyme superfamily have been divided into four classes, which differ in their requirements for prosthetic groups and cofactors.158 Class A includes RNA base methylases (vide inf ra). Class B consists of cobalamin-dependent methyltransferases [e.g., TsrM, Fom3 (vide inf ra), GenK, CysS, ThuK, Sven0516, and PoyC].158 Class C enzymes are cobalamin-independent. Class D enzymes use N5,N10-methylene tetrahydrofolate. Formation of the hypermodified tRNA base wyosine is considered as an example.134,159 Electron transfer from a flavoprotein (Figure 24) is involved in activation of the iron−

Figure 35. Formation of a branched-chain (Acrosiphonia coalita) trienal.149

cyclopropyl group (Figure 37).149 Finally, the marine diatom Nitzschia pungens forms an unusual bicyclic lactone (Figure 38).149 6588


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Figure 36. Formation of constanolactone (C. simplex).149

Figure 37. Formation of hybridalactone (L. hybrida).149

the synthesis of OXT-A (Figure 43).168 A proposed mechanism is shown in Figure 44.168 Radical SAM reactions are relatively common, and >113000 proteins have been annotated as such.157,161 Many are found in bacteria. One important mammalian example of a radical SAM enzyme participating in C−C bond formation is in molybdopterin biosynthesis, involving the protein MOCS1A with its two [4Fe-4S] clusters (Figure 45).169 Molybdopterin deficiency is a pleiotropic genetic disorder. The human enzymes sulfite oxidase, xanthine oxidoreductase, and aldehyde oxidase require this prosthetic group, and administration of Moco precursor Z (Figure 45) rescues Z-deficient knockout mice displaying the human deficiency phenotype.170,171 Another example of Class B is TbtI, which involves the methylation of a thiazole ring during posttranslational modification in the biosynthesis of the peptide antibiotic thiomuracin (Figure 46).158 A proposed mechanism is shown in Figure 47.158

sulfur cluster needed to generate a 5′-deoxyadenosyl radical, which is utilized to abstract a hydrogen atom from a substrate (Figure 40), akin to the process in cobalamin chemistry.1 At least two mechanistic proposals have been made for the formation of wyosine by TYW1. The first (Figure 40A) is based on covalent catalysis involving a conserved lysine residue in TYW1, which holds the substrate pyruvate via a Schiff base linkage. Hydrogen atom abstraction from the guanine N1-methyl group leaves a methylene radical, which reacts with the C-2 carbon of the pyruvate that in turn is liganded to the iron−sulfur cluster. HCO2H is released, and nucleophilic attack on the Schiff base by the guanyl N2 atom, followed by a series of rearrangements, yields wyosine. An alternate mechanism (Figure 40B)160 invokes a role for the iron−sulfur cluster in the later steps. Another example of a C−C bond forming reaction involving radical SAM chemistry is the biosynthesis of the antibiotic fosfomycin (Figure 41).161−164 This [FeS] cluster-containing enzyme requires SAM and a methylcorrinoid as substrates. Cobalamin reactions (vitamin B12) are also known and use similar mechanisms involving adenosine CH2• radicals. A classic case is glutamate mutase (Figure 42A), which uses the general radical mechanism shown for 1,2-alkyl migrations (Figure 42B).1,165−167 An interesting example of a cobalamin-dependent reaction is that of Bacillus megaterium OxsB, involved in a ring contraction in

2.8. Vitamin K-Mediated Carboxylation

Vitamin K has long been known to be important in blood coagulation, but its mechanism remained enigmatic for many years. The reaction of interest is the formation of γcarboxyglutamate moieties in protein side chains and a redox cycle is involved in the C−C bond formation. The reaction is driven by generation of a strong anion that can deprotonate the γ6589


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Figure 38. Formation of bacillariolides I and II (N. pungens).149 HPETE: hydroperoxypentatetraenoic acid. Figure 39. Biosynthesis of prodiginines by Rieske/diiron monooxygenases.155 (A) Two coupling reactions. (B) Postulated mechanism of the reaction with prodigiosin, as proposed by de Rond et al.155

carbon of a glutamate residue and cause it to react with CO2 (Figure 48). The carboxylase is a nonheme single-iron dioxygenase, with some sequence identity with lipoxygenases.172,173 18O2 incorporation studies showed one 18O in the epoxide oxygen and 5−20% in one of the quinone oxygens.174,175 The epoxidation mechanism proposed by Dowd et al.174 (Figure 49) is realistic when one considers the kinetics of carbonyl oxygen exchange with H2O.176−178 The vitamin K epoxide gem-diol is considered to be a strong base and abstracts a proton from a γ-glutamate residue,179 allowing the formal carbanion to attack CO2 and form the γcarboxyglutamate products. To complete the catalytic cycle, the epoxide must be reduced to the hydroquinone (Figure 50). The oxidative and reductive steps of the cycle are accomplished by the enzyme vitamin K oxidoreductase (VKOR) (Figure 49). Along with P450 2C9 (the major enzyme involved in warfarin metabolism),180,181 genetic differences in VKORC1 contribute to dose−response patterns and safety with the anticoagulant drug warfarin. (There are two forms of VKOR in different tissues, VKORC1 and VKORL1. 182) Warfarin (also used as a rodenticide) acts by inhibiting VKOR. The electrons used to reduce VKOR do not come directly from pyridine nucleotides but can be supplied from the simple reagent dithiothreitol in vitro. Two (membrane-imbedded) thiols in VKOR (Cys-132 and Cys-135) are apparently involved in a disulfide linkage, which is reduced by Cys-43 and Cys-51.183 Rishavy et al.183,184 have shown that a thioredoxin/thioredoxin reductase system is capable of supporting the reaction in vitro. A plausible mechanism is presented in Figure 50. VKOR is capable of reducing vitamin K quinone to hydroquinone but other enzymes are postulated to also contribute.185

2.9. Lignin Synthesis

Our discussion of C−C bond-forming reactions would not be complete without mention of what may be the most plentiful natural substance on the earth aside from cellulose, namely lignin. Lignin is of interest in that it is needed for plants to stand erect, but degradation of lignin is also an environmental necessity (see section 3.13). 2.9.1. Structural Lignins. Lignin is formed by the polymerization of phenolic substances derived from phenylalanine (Figure 51A). The P450 enzymes cinnamate 4hydroxylase (P450 73A), p-coumarate 3-hydroxylase (P450 98A), and ferulate 5-hydroxylase (P450 84A) are involved in generating mixtures of the phenols, which yield different types of lignins and mixtures, resulting in polymers with different textures. Lignin polymerization is the result of oxidative coupling of phenolic radicals (i.e., the diradical process discussed earlier for P450s and some other redox enzymes). Many different peroxidases (and some laccases)187 are involved, in that most plants have large gene families involved (and gene-knockouts are often ineffective due to the extensive gene redundancy).186 An example of the polymerization process is shown for the dimerization of coniferyl alcohol in Figure 51B. Peroxidases are able to abstract electrons from aryl rings, especially when electron-donating groups (e.g., methoxy) are attached (compound I abstracts an electron, forming compound II). For instance, rabbit P450 1A2 was demonstrated to be able to abstract an electron from 1,2,4,5-tetramethoxybenzene (E1/2 6590


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Figure 40. Mechanistic proposals for synthesis of wyosine by TYW1. See also Figure 24. (A) Mechanism involving covalent catalysis.159 (B) Alternative mechanism.160

Figure 41. Radical SAM enzyme-catalyzed methylation in the formation of fosfomycin.161,163

0.81 V vs SCE, or 1.05 V vs NHE),188 yielding a stable radical.189 The resonance forms are shown (Figure 51B), and diradical

couplings yield various products that impart different properties to lignins in various plants (and individual tissues). 6591


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3.1. P450 Reactions

A previous section (section 2.2) dealt with P450-catalyzed C−C bond-forming reactions. The list of P450 C−C bond-breaking reactions is probably just as long or longer. However, the cleavage of C−C bonds by P450s is not as facile as the cleavage of C−N or C−O bonds, which is readily done by C-hydroxylation to form unstable carbinolamines or hemiacetals.61 Cleavage of C−C bonds generally requires oxidation reactions at two adjacent carbon atoms (i.e., a multistep reaction). For instance, all of the C−C bond-breaking steps observed in mammalian steroid metabolism involve successive reactions (Figures 55 and 56). P450 C−C cleavage reactions involve steroids, drugs, model, and industrial chemicals, and many natural products. These fall into a general mechanistic pattern in the formation of unstable products that break C−C bonds during decomposition, either within or outside of the enzyme. 3.1.1. Steroids. The biosynthesis of steroids in mammals involves four P450 C−C cleavage reactions: lanosterol 14αdemethylation (P450 51A1), cholesterol side chain cleavage (P450 11A1), the 17α-hydroxylation/lyase reactions with pregnenolone and progesterone (P450 17A1), and the aromatization of androgens to form estrogens (P450 19A1) (Figure 56).111,192 (4-Demethylation of sterols is catalyzed by a nonheme diiron protein, to be discussed later, section 3.9.) Genetic deficiencies in any of these four enzymes are clinically problematic.192,193 On the other hand, the 17α-hydroxylation/ lyase (P450 17A1) and aromatization (P450 19A1) reactions are targets for inhibition by drugs in cancer therapy because of the roles of androgens and estrogens in growth of certain tumors.194−198 14α-Demethylation is critical in formation of ergosterol for membrane synthesis in yeast and fungi, and the enzymes catalyzing this reaction are the most common targets in the development of antifungal therapies.199,200 Some P450 enzymes that catalyze sterol oxidative cleavages are also targets for drugs to treat bacterial infections (e.g., tuberculosis).201,202 This section will focus on the mechanism of the C−C bond cleavage process by P450 enzymes. The C−C bond cleavage substrates of P450s 19A1 and 51A1 are both aldehydes, which are cleaved to form the alkene and HCO2H products, while P450s 17A1 and 11A1 require an α-hydroxy ketone (e.g., 17αhydroxypregnenolone) and a vicinal diol (20R,22R-dihydroxycholesterol), respectively, as their substrates to form ketone products [e.g., dehydroepiandrosterone (DHEA) and pregnenolone]. All of these P450s (i.e., P450s 11A1, 17A1, 19A1, and 51A1) cleave C−C bonds in a multistep process from the initial substrate, with varying degrees of processivity.203−205 3.1.1.1. P450 19A1. P450 19A1 (also known as steroid aromatase) is the enzyme responsible for the formation of 18carbon estrogens and formic acid from 19-carbon androgens.206 Inhibition of this enzyme is a mechanism for treating estrogendependent cancers, and several third-generation selective

Figure 42. Glutamate mutase and cobalamin chemistry.165−167 (A) Overall reaction; (B) general cobalamin 1,2-migration mechanism. In Part A, the carbon atoms are labeled (*, #) to indicate the rearrangement.

C−C bond-breaking of lignin is done by similar enzymes, presented in section 3.13. 2.9.2. Dietary Lignins. Not all lignans are structural in plants. Some are metabolites that do not have known functions in plants but have biological properties, or at least flavors, when consumed by mammals. An example is sesame. In the biosynthesis of sesame, two molecules of coniferyl alcohol are coupled by a dirigent protein, in a radical process, to form (+)-pinoresinol (Figure 52). Two oxidations by P450 81Q1 yield the dimethylenedioxyphenyl structure (+)-sesamin (Figure 52).190 Further oxidation (of (+)-sesamin) by P450 92B14 yields both C−O and C−C coupled products [i.e. (+)-sesamolin and (+)-sesaminol (Figure 53)].190 2.10. MauG and Tryptophan Cross-Linking

We now return to diradical cross-linking by heme proteins. Bacteria have a number of unsual modifiations of amino acids to generate new prosthetic groups. One is tryptophan tryptophylquinone (TTQ), which functions in some redox reactions. The biosynthesis involves the diheme enzyme MauG (Figure 54), which utilizes long-range electron transfer to form two tryptophan radicals that link, as shown earlier for some of the isoquinoline alkaloids (Figure 4) and indolocarbazole alkaloids (Figure 10).191

3. C−C BOND BREAKING REACTIONS C−C bond cleavage does not occur with unfunctionalized methylene carbons, olefins, and aromatic molecules and requires functionalization before cleavage. In some cases this functionalization is an inherent part of the cleavage enzyme.

Figure 43. Conversion of dAMP to OXT-A.168 6592


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Figure 44. Proposed mechanism for oxsB.168 The circled P denotes a phosphate group.

Arguments against mechanisms A, B, and C (Figure 57) have been advanced, and these three have generally been dismissed. One controversial aspect has been the proposal of the active iron species directly involved in cleaving the C10−C19 bond in its androgen substrate to form the estrogen product. Because the direct C−C bond cleavage substrate in the aldehyde form (e.g., 19-oxoandrostenedione) is inherently electrophilic at C19, it has been proposed that a nucleophilic ferric peroxide can attack the aldehyde substrate to form a tetrahedral peroxohemiacetal intermediate, which can break down to form the estrogen product (e.g., estrone) and HCO2H (Figure 57E). In contrast, compound I has been proposed to abstract the C1β-H bond of the hydrated form of 19oxoandrostenedione, a 19,19-gem-diol intermediate, to form a C1-radical androgen intermediate. This intermediate can transfer its electron to the iron center of compound II, forming a carbocation intermediate at C1 (Figure 57D).177,226 The resulting carbocation can break down to form HCO2H and estrone.

Figure 45. Biosynthesis of Moco precursor Z by MoaA.161

inhibitors are in wide clinical use today (e.g., exemestane, anastrazole, letrozole).207,208 The C−C bond cleavage process of P450 19A1 occurs in three steps (Figure 56). The first two steps involve hydroxylation of the C19-methyl of the androgen substrate (i.e., testosterone or androstenedione), and the third step is the C−C bond cleavage reaction to afford HCO2H and the estrogen product. The mechanism of the C−C bond cleavage step has been controversial since the 1970’s, and various mechanisms had been proposed by experimentalists and theoreticians (Figure 57)177,209−227.

Figure 46. Methyl transfer in the synthesis of thiomuracin by TbtI.158 The course of hydrogens is shown in color (red). 6593


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Figure 47. Proposed mechanism for TbtI.158 The course of hydrogens is shown in color (red).

Figure 50. Proposed mechanism of reduction of vitamin K epoxide.183,184

The incorporation of an 18O atom from molecular oxygen ( O2) into the HCO2H product from the incubation of P450 19A1 with its 19-oxo androgen substrate would support a ferric peroxide (compound 0) mechanism, while the lack of 18O in HCO2H derived from the lyase substrate would support a role for compound I (Figure 58).177 Analysis of the formic acid product from the incubation of P450 19A1 with the 19-oxo androgen substrate is not trivial. The main challenges with detecting derivatized formate compounds by mass spectrometry are related to the abilities (i) to distinguish between the formic acid from the enzyme incubation and endogenous formic acid and (ii) to detect small amounts of compound (∼100 ng to 1 μg scale). Formic acid is ubiquitous and is present in solvents, air, and enzyme preparations. In the analysis of formic acid produced by P450 19A1, differentiating between the background source of formic acid from the 18

Figure 48. Vitamin K and γ-glutamate carboxylation.

3.1.1.1.1. 18O-Isotopic Labeling Studies. The strongest evidence supporting the active oxoiron species responsible for cleaving the C−C bond has come from oxygen isotope-labeling experiments (Figures 58 and 59).220,222,223 The difference between the compound I mechanism and the ferric peroxide mechanism to form estrogens from 19-oxo androgen precursors is the source of one of the oxygens in the side-product HCO2H. One of the oxygen atoms in HCO2H comes from molecular oxygen in the ferric peroxide mechanism, while oxygen comes from water in the compound I mechanism (Figures 58 and 59).

Figure 49. Mechanism of vitamin K epoxidation.174 6594


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100000) to distinguish between the formic acid produced by P450 19A1 and the endogenous formic acid (i.e., M/ΔM = 58000) (Figure 52B). With the newer technology (i.e., sensitive derivatization method, high-resolution mass spectrometry, and purified enzyme source), it was concluded that P450 19A1 did not incorporate an oxygen atom from molecular oxygen (18O2) into the formic acid product, and therefore only a compound I active iron species could be responsible for cleaving the C10− C19 bond of androgens to form estrogens. Spectral studies using resonance Raman measurements have also been interpreted as being consistent with a compound I intermediate.225 No evidence was seen for the accumulation of a compound 0 intermediate in EPR work.235 However, the formation of multiple products (e.g., 19-oic acid, vide inf ra) makes assignments of spectral intermediates ambiguous, in that one intermediate may give rise to only some products. Nevertheless, in this case, the spectral conclusions are consistent with our own isotope labeling work. 3.1.1.1.2. Hydration of the 19-Aldehyde to Form the gemDiol and Catalytic Competency. The 19-carbon androgen intermediate that is directly converted to the estrone product is 19-oxoandrostenedione. This 19-aldehyde intermediate can potentially exist in equilibrium with its 19,19-gem-diol form in water. In the ferric peroxide mechanism of C10−C19 bond cleavage of P450 19A1 (Figure 57E), the only substrate that can react with the nucleophilic ferric peroxide intermediate is the 19aldehyde form, which is electrophilic, and not the 19,19-gem-diol. Therefore, determination of the ratio of the 19-aldehyde and the 19,19-gem-diol in water (pH 7.4) is mechanistically informative. If the 19-oxo androgen intermediate exists only as the 19,19-gemdiol in water, then the enzyme will have to catalyze a dehydration of the 19,19-gem-diol to the 19-aldehyde intermediate before ferric peroxide can attack the substrate. On the other hand, if only the 19-aldehyde exists, the enzyme may readily react with the 19aldehyde in the ferric peroxide mechanism. In addition, the second step of P450 19A1 is the second hydroxylation of its androgen substrate (i.e., 19-hydroxyandrostenedione) to form 19,19-dihydroxyandrostenedione (i.e., 19,19-gem-diol), which must dehydrate to 19-oxoandrostenedione (i.e., 19-aldehyde) at a faster rate than the conversion of 19-hydroxyandrostenedione to estrone by P450 19A1 (kcat 0.13 s−1) in the ferric peroxidebased mechanism. If this nonenzymatic dehydration rate is

Formation and Cleavage of CâC Bonds by Enzymatic Oxidation

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