Review

Nature Chemical Biology 4, 186 - 193 (2008)
Published online: 15 February 2008 | doi:10.1038/nchembio.71

Versatility of biological non-heme Fe(II) centers in oxygen activation reactions

Elena G Kovaleva1 & John D Lipscomb1

Oxidase and oxygenase enzymes allow the use of relatively unreactive O2 in biochemical reactions. Many of the mechanistic strategies used in nature for this key reaction are represented within the 2-histidine-1-carboxylate facial triad family of non-heme Fe(II)-containing enzymes. The open face of the metal coordination sphere opposite the three endogenous ligands participates directly in the reaction chemistry. Here, data from several studies are presented showing that reductive O2 activation within this family is initiated by substrate (and in some cases cosubstrate or cofactor) binding, which then allows coordination of O2 to the metal. From this starting point, the O2 activation process and the reactions with substrates diverge broadly. The reactive species formed in these reactions have been proposed to encompass four oxidation states of iron and all forms of reduced O2 as well as several of the reactive oxygen species that derive from O-O bond cleavage.

Dioxygen serves at least three quite different roles that profoundly impact aerobic life. The most commonly appreciated function is its role as the terminal electron acceptor in processes such as oxidative phosphorylation that yield the central energy-rich molecules used throughout metabolism. The second, no less important role is to serve as the source for many of the oxygen atoms found in the essential molecules of biological systems, such as steroid hormones, aromatic amino acids, neurotransmitters, signaling molecules and regulatory factors1. Also, processes operating on a global scale, such as the recovery of the enormous quantities of carbon sequestered as lignin in plant life or the oxidation of the billions of tons of methane generated by anaerobes before it can enter the atmosphere, also involve oxygen incorporation from O2 (refs. 2,3). On a more local scale, biodegradation of both aliphatic and aromatic toxic compounds often begins with the incorporation of oxygen4. The third and least appreciated role played by dioxygen in aerobic organisms involves neither energy conversion nor oxygen incorporation. Rather, some enzymes can convert dioxygen to alternative forms that are, in effect, highly specialized reagents that are used to catalyze the synthesis of important biomolecules. An excellent example of the latter role for O2 is the biosynthesis of penicillin-type antibiotics5, 6, which is discussed in more detail later in this review.

Dioxygen is an attractive reagent for use in biological systems because its high potential reactivity is held in check by its molecular structure. The triplet ground state of O2 that results from the presence of two unpaired electrons in degenerate molecular orbitals makes the direct reaction with singlet molecules, the spin-paired state of most potential reaction partners, a forbidden process7. The central question that has faced chemists and biochemists for the half century since the oxygen incorporation into a biological molecule from O2 was first demonstrated8 is how O2 can be made to react at ambient temperature and with high specificity. Of course, the answers to this question are of fundamental importance in understanding the myriad of interacting, O2-dependent metabolic processes that make life possible while living in a sea of oxygen. However, an understanding of nature's oxygen activation strategies also has many direct applications, including the design of (i) new pharmaceuticals relevant to a wide variety of diseases, (ii) targeted biodegradation systems for the environment and (iii) catalysts for the conversion of abundant molecules for fuels and industrial synthons. On the horizon, such an understanding could allow genetic alteration of oxygen activating enzymes to mitigate the effects of aberrant catalysis.

Dioxygen is activated from its abundant triplet ground state to reactive singlet or doublet (radical) species by oxidase and oxygenase enzymes. In many cases, these same enzymes generate species of even greater reactivity by cleaving the O-O bond. All of these reactions are tightly regulated both spatially and temporally so that specific chemistry occurs to yield biomolecules that are compatible with downstream meta-bolic pathways or function. When loss of regulation occurs, the well-characterized deleterious effects of reactive oxygen species ensue. The problem of specifically activating O2 has been addressed and solved in a remarkably varied manner by nature. Generally, the solution involves either a transition metal (such as iron, copper or manganese), an organic cofactor (such as flavin or pterin) or both, as is the case for heme. In all biological systems, the O2 is reductively activated because simple inversion of an oxygen electron to yield the singlet state directly is highly endothermic. The source of the activating electrons is again quite varied; the list includes the metal, the organic substrate itself, a cosubstrate, a second redox-active metal or cofactor, reduced pyridine nucleotide and for some oxidases, ascorbate.

The understanding of biological oxygen activation mechanisms has been advanced in recent years through the coordinated use of chemical, structural, spectroscopic and computational approaches9, 10, 11, 12. A surprisingly comprehensive overview of the lessons learned from these studies can be acquired by focusing on specific representative members of the large family of non-heme iron-containing oxygenases and oxidases that bind the metal using weakly charge-donating ligands. Most of the members of this class use two histidyl and one glutamyl or aspartyl endogenous ligands arranged on one face of the iron coordination sphere (2-His+Asp/Glu facial triad motif)13. The opposite face is usually occupied by solvents that can be directly or indirectly displaced as substrates or cosubstrates and then O2 bind to the enzyme14, 15. This allows the iron to participate in the oxygen activation and substrate oxidation process in a variety of ways. Here, five representative types of non-heme oxygenases and related oxidases that have adopted different solutions to the oxygen activation process will be considered.

Extradiol catecholic dioxygenases

Roughly 30% of the mass of woody plant material on earth is comprised of aromatic compounds2, 16. The inherent stability of these compounds would make them a natural sink for a significant fraction of the bioavailable carbon were it not for the action of dioxygenase enzymes that cleave the aromatic ring and initiate pathways leading to central metabolites17, 18, 19, 20. Invariably, the aromatic compound is first functionalized, generally by the introduction of two or more hydroxyl or amino substituents on the aromatic ring to increase its reactivity. The aromatic ring is then cleaved by one of several types of dioxygenase enzymes. The extradiol dioxygenases cleave the aromatic ring of catechol analogs adjacent to the vicinal hydroxyl substituents by inserting both atoms of oxygen from O2 to yield muconic semialdehyde adducts. Structural studies show that these enzymes bind Fe(II) in the 2-His+Asp/Glu facial triad coordination environment21, 22. So far, there is no evidence that the iron changes redox state during the catalytic cycle. Moreover, no external reductant is needed for catalysis, which shows that the electrons required to activate O2 derive from the substrate. The structure and utility of the 2-His+Asp/Glu facial triad were first recognized in the extradiol dioxygenases13, 14, 15, and elements of their catalytic mechanism are carried through most other members of the broad family. Thus, it is instructive to first examine the extradiol dioxygenases in some detail.

The extradiol dioxygenases form an active site Fe(II)-chelate complex with the catecholic substrate and then bind O2 in an adjacent metal ligand site14, 20, 23, 24, 25. This allows a concerted activation of substrate and O2 by transfer of an electron from the former to the latter via the iron (Fig. 1). Recombination of the resulting radicals in a position enforced by the geometry of the metal complex gives a specific Fe(II)-bound alkylperoxo intermediate. This intermediate was originally proposed to break down by a concerted Criegee rearrangement to yield a lactone intermediate14, 23, 26. The heterolytic O-O bond cleavage during this reaction would be facilitated by protonation of the oxygen atom bound to the iron by an active site acid catalyst. Finally, the open ring product would result from hydrolysis of the lactone by the second oxygen atom from O2, which remains bound to the iron at the level of water. Recent computational studies are in accord with this mechanism, except that they predict formation of the lactone by a multistep process involving an intermediate unstable epoxide27, 28.

Figure 1: Oxygen activation by extradiol dioxygenases.

Figure 1 : Oxygen activation by extradiol dioxygenases.

This type of activation involves simultaneous activation of O2 and substrate, once both are bound to the active site Fe(II). It proceeds through formation of an alkylperoxo intermediate in which activated dioxygen attacks the substrate before O-O bond cleavage. The R groups of the substrates considered here are CH2COO (homoprotocatechnic acid, HPCA, 1) and NO2 (4-nitrocatechol, 4NC, 2). The structures shown are the Brevibacterium fuscum HPCD 4-nitrocatechol-semiquinone-Fe(II)-[O2]dot– (top) and Fe(II)- alkylperoxo (bottom) intermediates (Protein Data Bank (PDB) code 2IGA)29. Atom color code: gray, carbon (enzyme residues); yellow, carbon (substrate); blue, nitrogen; red, oxygen; cyan, iron. Red and gray dashed lines show hydrogen bonds and potential bonds to iron, respectively.

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Direct evidence for several aspects of this mechanistic proposal has come from our recent study in which reaction intermediates of the extradiol dioxygenase homoprotocatechuate 2,3-dioxygenase (HPCD) were trapped in a single crystal29. In this study, crystals of HPCD were allowed to react with the slow alternative substrate 4-nitrocatechol (2) in the presence of a low concentration of O2 before cryofreezing and structure solution. Three intermediates of the reaction cycle were found in different subunits of the homotetrameric enzyme, of which two are shown in Figure 1. One subunit contained the chelated substrate–Fe(II)–O2 complex. The oxygen in this complex is bound in the side-on configuration, thereby aligning it for reaction with the correct ring carbon of 4-nitrocatechol. Also, the ring of the 4-nitrocatechol is not planar, which suggests that it gives up an electron to become a semiquinone radical. The long Fe-O (2.4 Å) bond lengths are consistent with the iron remaining in the Fe(II) state, which suggests that the electron from the 4-nitrocatechol is transferred to the oxygen to form a bound superoxide.

Two of the other subunits of HPCD in the crystal contained the Fe(II)-alkylperoxo intermediate with continuous electron density from the iron through both oxygen atoms to the C2 of 4-nitrocatechol, which becomes fully tetrahedral. Finally, the fourth subunit of HPCD contained the Fe(II)–product complex, thereby showing that the enzyme is capable of turnover in the crystal.

The structural studies indicate that two residues in the second sphere of the metal coordination sphere play important roles in the catalytic process. The first is Tyr257, which is within hydrogen bonding distance of the deprotonated 4-nitrocatechol hydroxyl substituent at C2. This distance becomes shorter (2.4 Å) in the observed intermediates, which suggests that formation of a strong hydrogen bond by Tyr257 draws the reaction toward the intermediates. The second residue is His200, which is positioned to donate a proton to the alkylperoxo intermediate to promote heterolytic O-O bond fission. Alteration of the reaction kinetics and specificity caused by mutation of His200 to residues that cannot act as acid catalysts supports its essential function30, 31. Without a histidine at position 200, an activated oxygen species is stabilized in solution, greatly slowing the reaction cycle30. Thus, O2 activation and subsequent reaction occur by a collaboration of electron donation from the substrate and acid catalysis by an active site histidine.

Rieske cis-diol–forming dioxygenases

One way in which the catecholic substrates for the extradiol dioxygenases are formed in nature is through the direct addition of both atoms of oxygen from O2 to adjacent carbons of an unactivated aromatic ring in the reaction catalyzed by cis-dihydrodiol–forming dioxygenases32, 33. Re-aromatization of the cis-dihydrodiol product by the next enzyme in the pathway yields the catechol, which in turn becomes a substrate for downstream ring-cleaving dioxygenases. In the cis-diol–forming reaction, one more bond is formed than is broken in the substrate and O2, so two external electrons must be supplied. This also serves a mechanistic purpose because it allows activation of O2 to a more potent form than occurs in the extradiol dioxygenase mechanism—a necessity when the goal is to attack an unactivated aromatic ring.

The Rieske cis-diol dioxygenases fall into four subfamilies with differing numbers and types of protein components and subunits, but a similar active site region is retained4. Crystal structures show that each enzyme has a substrate-binding site located in the oxygenase component that contains a mononuclear iron bound by the 2-His+Asp/Glu motif in which the carboxylate ligand is often bidentate34, 35, 36, 37, 38. Spectroscopic studies show that, in solution, the iron is six-coordinate in the absence of substrate, which indicates that additional solvent molecules or weak protein-derived ligands are bound39, 40. Approximately 12 Å away from the mononuclear iron, and across a subunit boundary, is a Rieske-type [2Fe-2S] cluster that acts to store and supply an additional electron for the reaction. A separate reductase component, sometimes acting through electron transfer proteins, couples NADH oxidation to Rieske cluster reduction.

As has been found to be the case for each of the O2 activating enzymes discussed here, studying the enzyme reaction under single turnover conditions has provided important clues to the mechanism of the Rieske cis-diol–forming enzymes41, 42, 43, 44. Exposure of the fully reduced oxygenase component (in which each metal center is one-electron-reduced) to substrate and O2 resulted in one turnover to form a nearly stoichiometric yield of cis-dihydrodiol product in the case of naphthalene dioxygenase (NDO). At the end of the single cycle, both metal centers were found to be oxidized, which suggests that the O2 is activated by a two-electron reduction process. This led to the hypothesis that the reactive form of oxygen is a peroxo or hydroperoxo species bound to Fe(III) (Fig. 2). In heme-containing oxygenases and methane monooxygenase (MMO), the equivalent species undergoes O-O bond cleavage upon protonation to yield a species at the oxidation level of Fe(V)=O, which is the true hydroxylating reagent25, 45, 46. In each of the latter species, there is more than one oxidized center in close proximity, so the highest localized valence for iron is Fe(IV). A similar formal Fe(V)=O species can be proposed for the Rieske dioxygenases, although in this case there is no obvious candidate for the second nearby oxidized center, so the oxidizing potential may not be delocalized. Also, a mechanism to retain both atoms of oxygen for incorporation into the substrate must be proposed. One solution to both of these issues is to retain both atoms of oxygen in the metal coordination sphere to form an Fe(V)-oxo-hydroxo moiety in which the hydroxo substituent would partially stabilize the high-valent metal-oxo41.

Figure 2: Oxygen activation by Rieske cis-diol dioxygenases.

Figure 2 : Oxygen activation by Rieske cis-diol dioxygenases.

This type of reaction requires formation of a more reactive species than is found in the extradiol dioxygenase pathway of Figure 1. One electron from the Rieske cluster and one from the Fe(II) are used to form Fe(III)-OOH, which may then form Fe(V)=O-OH as the reactive species. The structure shown is of the hydroperoxo intermediate of Pseudomonas sp. strain NCIB 9816-4 NDO with the substrate analog indole bound nearby (PDB code 1O7N)52. Atom color code: gray, carbon (enzyme residues); yellow, carbon (substrate); blue, nitrogen; red, oxygen; cyan, iron.

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An alternative mechanistic hypothesis has received support from studies of phthalate dioxygenase, which gives only approx50% yield during single turnover47. It was suggested that after achieving the formal Fe(V)=O state, the mononuclear iron accepts another electron from a Rieske cluster located in a remote subunit of the multimeric enzyme. Thus, the true reactive species would be either an Fe(II)-(hydro)peroxo or an Fe(IV)-oxo-hydroxo, which would presumably be more readily stabilized in a biological system.

Both mechanistic scenarios have received support from computational studies48, 49. One direct approach to resolving this controversy has been to show that hydrogen peroxide added to the substrate complex of naphthalene or benzoate dioxygenase (BZDO) in the resting state with the Rieske cluster oxidized and the mononuclear iron reduced yields nearly stoichiometric amounts of product (a peroxide shunt)50, 51. In this case, 18O labeling studies showed that the peroxide is the source of the oxygen in the product, and because it can supply only two electrons, the two-electron-activated oxygen proposal is favored. In the case of BZDO, a form with both the Rieske and mononuclear iron sites fully oxidized was prepared, and this state was also active in the peroxide shunt despite the lack of any source for a third electron51.

It has thus far not proven possible to distinguish between an Fe(III)-(hydro)peroxo and an Fe(V)-oxo-hydroxo reactive species. Computational studies favor the former for enzyme reactions, but they show that the latter is a possible reactive species in small-molecule model compounds where the low spin state of the metal can be accessed48, 49. The crystal structure of the side-on–bound hydroperoxo complex of NDO has been solved after exposing the substrate-bound crystal of NDO to O2 (Fig. 2)52. Also, an intermediate with electron paramagnetic resonance and Mössbauer parameters similar to those of a side-on–bound peroxo adduct of model complexes has been found after adding H2O2 to fully oxidized BZDO in solution51. Results favoring the (perhaps subsequent) formation of an intermediate Fe(V)-oxo-hydroxo species have derived from the use of probe molecules that are monooxygenase substrates for NDO (ref. 53). Both norcarane and bicyclohexane form high percentage yields of products that reveal the intermediate formation of long-lived substrate radicals with no evidence for cation intermediates. Studies of a variety of oxygenases in recent years have shown that Fe(III)-hydroperoxo intermediates are unlikely to yield radical intermediates, whereas this is the expected route for formal Fe(V)=O species46.

Dioxygenases using a 2-oxo acid as a cosubstrate

Many mammalian and bacterial hydroxylating dioxygenases reductively activate O2 by using a mechanism that is substantially different from those considered above for extradiol and Rieske cis-diol dioxygenases54. X-ray crystallographic studies have shown that the 2-oxo acid–linked dioxygenase family uses a mononuclear Fe(II) in a 2-His+Asp/Glu ligand motif that chelates a 2-oxo acid (generally alpha-ketoglutarate, 3) cosubstrate in ligand sites adjacent to the probable O2 binding site on the iron (Fig. 3)55, 56, 57, 58. This is analogous to substrate binding to extradiol dioxygenases, and similarly, the bound O2 attacks the oxo acid. However, the reactions diverge in several respects at this point (Fig. 3). First, the 2-oxo acid is much less electron donating than the catecholate substrate of the extradiol dioxygenases, and thus the reactive oxygen species is likely to be an Fe(III)-[O2]dot– rather than the Fe(II)-[O2]dot–. Second, the role of these dioxygenases is to hydroxylate a substrate, which is similar to the roles of Rieske dioxygenases, cytochrome P450 and MMO, and thus a high-valent iron-oxo species of some sort is likely to be required. This mandates the input of electrons to break the O-O bond. In the case of the alpha-ketoglutarate dioxygenases, these electrons are derived not from reduced pyridine nucleotide, but rather from bond cleavage of the cosubstrate to yield the intermediate products CO2 and succinate, the latter containing one atom of oxygen from O2. Finally, the Fe(IV)=O species also generated in this reaction is used to incorporate one oxygen atom into the substrate to form a hydroxylated product. This means that the dioxygenase stoichiometry is satisfied by addition of the two atoms from O2 to different molecules.

Figure 3: Oxygen activation by 2-oxo acid dioxygenases.

Figure 3 : Oxygen activation by 2-oxo acid dioxygenases.

An initial Fe(III)-[O2]dot– species attacks the iron-bound alpha-ketoglutarate (alphaKG) cosubstrate to yield an Fe(IV)=O reactive species. This species in turn attacks the substrate by hydrogen atom abstraction. The structure shown is of the initial reactive complex of Escherichia coli TauD with alpha-ketoglutarate bound to the iron and taurine bound nearby (PDB code 1GQW)55. Atom color code: gray, carbon (enzyme residues); yellow, carbon (substrate); bronze, carbon (cosubstrate); blue, nitrogen; red, oxygen; green, sulfur; cyan, iron.

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The use of transient enzyme kinetic techniques in combination with spectroscopy has allowed very significant progress in understanding the mechanism of 2-oxo acid dioxygenases in recent years59, 60. Oxygen binding in this class is triggered by the binding of both substrate and 2-oxo acid cosubstrate. In studies of the enzyme taurine–alpha-ketoglutarate dioxy-genase (TauD), an enzyme that hydroxylates 2-amino-1-ethane sulfonic acid (taurine, 4) as part of a pathway that allows bacteria to release sulfite as a sulfur source for growth, it was shown that rapid mixing of the TauD–alpha-ketoglutarate–taurine complex with O2-containing buffer results in the formation of at least two intermediates. One intermediate, termed 'J', was trapped by rapid freezing and shown by Mössbauer spectroscopy to have parameters similar to those of known Fe(IV)-containing intermediates, in particular intermediate X of ribonucleotide reductase and Q of MMO (refs. 60–62). Formation of J required alpha-ketoglutarate, and the rate of its decay exhibited a very large kinetic isotope effect (KIE) upon deuteration of the C1 of taurine63, 64. Resonance Raman spectra of the intermediate showed a stretching mode characteristic of Fe(IV)=O (ref. 65), and EXAFS spectra showed the appropriate Fe-O distance66. Together these approaches revealed the first mononuclear non-heme Fe(IV)=O intermediate and offered strong support for the proposed mechanism that invokes a high-valent iron-oxo species to attack the substrate (Fig. 3). The large KIE suggests that the mode of this attack is hydrogen atom abstraction from the substrate followed by rebound of hydroxyl radical to yield the hydroxylated product.

It is useful to note that the 2-oxo acid dioxygenases, in effect, activate oxygen twice using different strategies that favor the specific chemistry that follows activation. In the first activation step, an Fe(III)-[O2]dot– species is generated to carry out the relatively facile attack on the 2-oxo acid. This leads to the second activation step, in which the much more potent Fe(IV)=O species is generated by O-O bond cleavage. Because the iron begins and ends its cycle in the Fe(II) state, the even more reactive formal Fe(V)=O state characteristic of the reactive species of Rieske cis-diol dioxygenases, cytochrome P450 and MMO is not generated and is apparently not required.

Enzymes similar to the 2-oxo acid dioxygenases

The number of recognized 2-oxo acid dioxygenase enzymes is large and growing rapidly as modern proteomic techniques are used. However, two other types of enzymes seem to use analogous O2 activation mechanisms. The first includes enzymes such as 4-hydroxyphenylpyruvate dioxygenase (HPPD), which is part of the tyrosine metabolic pathway67. In this case, the 2-oxo acid is the pyruvate substituent of the substrate, which binds directly to the iron to allow O2 binding and activation. Following Fe(III)-[O2]dot– attack and release of CO2, the presumed Fe(IV)=O intermediate carries out aromatic ring hydroxylation. The reaction differs from the mechanism shown in Figure 3 in that it is proposed to occur by direct attack of the Fe(IV)=O species on the aromatic ring to yield an intermediate Fe(II)-O-substrate arenium cation. The presence of the cation is thought to allow ketonization and substituent migration before formation of homogentisate product.

The second related enzyme class catalyzes halogenation of amino acids and related molecules bound to carrier proteins in preparation for nonribosomal peptide formation in a variety of natural products68. These enzyme use Fe(II), O2 and alpha-ketoglutarate like the traditional 2-oxo acid dioxygenases, but they also require a halide, and the normal carboxylic acid from the 2-His+Asp/Glu Fe(II) binding motif is absent. A crystal structure of syringomycin halogenase shows that the place in the ligand sphere normally occupied by the carboxylic acid residue is occupied by the halogen that will be transferred69. It is proposed that the normal oxygen activation scheme for a 2-oxo acid dioxygenase (Fig. 3) proceeds through the substrate hydrogen atom abstraction step. Then, instead of OHdot rebound, the halogen reacts with the substrate radical to complete the reaction. In accord with this mechanistic proposal, a transient Fe(IV)=O species has now been trapped and spectroscopically characterized70, 71.

Tetrahydropterin-containing oxygenases

The neurotransmitters 3,4-dihydroxyphenylalanine (4) and serotonin (5) and the amino acid tyrosine are synthesized by mammals in reactions catalyzed by hydroxylases containing an Fe(II) bound in a 2-His+Asp/Glu motif72. In addition, crystal structures show that a tetrahydropterin cofactor binds near the Fe(II) (Fig. 4)73. The reactions of these enzymes require an activated oxygen such as the high-valent Fe(IV)=O species found in the 2-oxo acid dioxygenases. The binding of substrate near, but not to, the Fe(II) facilitates O2 binding (Fig. 4). Two electrons are required to break the O-O bond to generate the high-valent iron-oxo species. It is postulated that the intermediate in this case contains a peroxo bridge between the Fe(II) and the tetrahydropterin74. Thus, an intermediate iron-superoxo or peroxo species is not formally required, although one is likely to transiently form. The pterin is believed to supply the two electrons required to cleave the O-O bond in much the same way that a flavin stabilizes the bound O2 in the peroxy state in flavomonooxygenases. Heterolytic cleavage of the O-O bond would yield a 4a-hydroxy pterin and an Fe(IV)=O species prepared for hydroxylation chemistry. Recently, the Fe(IV)=O species has been directly detected and trapped75. The mechanism of hydroxylation remains under investigation. It is possible that electrophilic attack of the Fe(IV)=O species on the aromatic ring of the substrate results in a two-electron oxidation to yield an Fe(II)-O-substrate arenium cation in one step72. Alternatively, the Fe(IV)=O species may abstract one electron to yield an intermediate ring radical and an Fe(III)-oxo species. Subsequent reaction of the oxo species with the substrate radical would yield the same arenium cation as the first mechanism76. The second mechanism is more in line with reactions of high-valent iron-oxo in other systems, but the final intermediate in either case would collapse to yield the hydroxylated product and leave the Fe(II) resting state of the enzyme.

Figure 4: Oxygen activation by tetrahydropterin-dependent hydroxylases.

Figure 4 : Oxygen activation by tetrahydropterin-dependent hydroxylases.

A peroxo bridge is formed between Fe(II) and an electron-donating tetrahydropterin cofactor in this O2 activation mechanism. The stability of the 4a-peroxypterin adduct allows the heterolytic O-O bond cleavage to yield an Fe(IV)=O species that reacts in turn with the aromatic substrate by electrophilic aromatic substitution. The structure shown is of human phenylalanine hydroxylase complexed with a substrate analog and tetrahydropterin (PDB code 1MMK)73. Atom color code: gray, carbon (enzyme residues); yellow, carbon (substrate); bronze, carbon (cosubstrate); blue, nitrogen; red, oxygen; green, sulfur; cyan, iron.

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In the tetrahydropterin-containing oxygenases, elements of the O2 activation mechanism described above are combined with the novel application of a reduced cofactor that participates directly in the reaction to yield the reactive oxygen species. The initial peroxo complex is similar to that formed by the extradiol dioxygenases in that iron remains formally Fe(II), and an O-O bridge is formed with a molecule that can supply reducing equivalents. However, the stability of the 4a-hydroxy pterin causes the heterolytic O-O bond cleavage to occur in the opposite direction such that the high-valent Fe(IV)=O species is formed rather than the Fe(II)-hydroxy intermediate of the extradiol dioxygenases. Similar to the Rieske cis-diol dioxygenases, a second electron-donating cofactor is required to activate oxygen. However, the reduced pterin can supply both electrons whereas the Rieske cluster of the Rieske cis-diol dioxygenases can supply only one, thereby requiring the Fe(II) to supply the second electron for bond cleavage, thus becoming Fe(III). The formation of these distinct intermediate states dictates that the product of heterolytic cleavage is at the level of a formal Fe(V)=O in the case of the Rieske cis-diol dioxygenases and a Fe(IV)=O for the pterin-linked hydroxylases.

Oxidase enzymes in the 2-His+Asp/Glu family

Although the most familiar oxidase enzymes serve as terminal electron acceptors in energy storage pathways, there are many oxidases that make use of the oxidizing potential of O2 to promote biosynthetic reactions. Through the application of the analytical techniques developed in the study of the oxygenase enzymes described here, and with the advent of X-ray crystal structures of several members of the biosynthetic oxidase family, it is now known that many of these enzymes belong to the 2-His+Asp/Glu family77, 78. The mechanisms of O2 activation proposed for this subgroup are very diverse, but they contain elements of the mechanisms discussed here. The general strategy appears to be to bind both the substrate and O2 to the iron, as in the extradiol and 2-oxo acid dioxygenases, to promote electron transfer away from the substrate and (in most cases) the iron. The reduced oxygen then undergoes O-O bond cleavage to yield a molecule of water and leave an Fe(IV)=O species, as in the 2-oxo acid dioxygenases and tetrahydropterin-linked hydroxylases. Finally, the high-valent iron species is used as a reagent to complete a second part of the reaction, and in doing so accepts two more electrons to form the second mole-cule of water.

Some well-studied examples of 2-His+Asp/Glu oxidases are isopenicillin N-synthase (IPNS)79, 80, 81, fosfomycin synthase (FOS)82, 83 and 1-aminocyclopropane-1-carboxylate oxidase (ACCO), which catalyzes the formation of ethylene, a hormone in plants84, 85, 86. These enzymes activate O2 and ultimately form water using either four electrons from the substrate (IPNS), two from the substrate and two from NADH (FOS), or two from the substrate and two from ascorbate (ACCO). The proposed mechanism of IPNS, of which two key steps are shown (Fig. 5), will serve as an example of the mechanistic strategy.

Figure 5: Oxygen activation in the mechanism of formation of isopenicillin N by IPNS.

Figure 5 : Oxygen activation in the mechanism of formation of isopenicillin N by IPNS.

Binding of O2 to the active site Fe(II) yields an ACV-bound Fe(III)-[O2]dot– reactive intermediate. Hydrogens (orange) and electrons derived from the closure of the beta-lactam ring are then used to break the O-O bond and generate an Fe(IV)=O intermediate that is proposed to serve as a reagent to effect the thiazolidine ring closure reaction. The structure shown is of Aspergillus nidulans IPNS complexed with ACV (PDB code 1BK0)80. Atom color code: gray, carbon (enzyme residues); yellow, carbon (substrate); blue, nitrogen; red, oxygen; green, sulfur; cyan, iron.

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Spectroscopic and crystallographic studies show that the substrate, delta-(l-alpha-aminoadipoyl)-l-cysteinyl-d-valine (ACV, 6), binds to the iron via its cysteinyl sulfur80, 87. The O2 analog NO can be bound in an adjacent site, which suggests that oxygen and substrate bind at the same time to the iron88. Recent coordinated spectroscopic studies of the substrate–IPNS–nitrosyl complex have been extended computationally to the oxy complex81. These show that electron donation from the S-Fe bond would stabilize a Fe(III)-[O2]dot– in an orientation that would favor abstraction of a hydrogen atom from the beta-carbon of the ACV cysteinyl moiety. This would prevent formation of a peroxo bridge, which would lead to oxygenase chemistry as in the cases of extradiol and the 2-oxo acid dioxygenases. Instead, a second hydrogen atom is transferred to the activated O2 to give O-O bond cleavage coincident with formation of the beta-lactam ring of penicillin N (7). The Fe(IV)=O species resulting from the O-O bond cleavage then becomes a reagent for the second ring-closing reaction. It is postulated that it abstracts a hydrogen atom from the beta-carbon of the valinyl moiety of ACV (refs. 79,87). This radical intermediate rebounds to the original cysteinyl sulfur ligand rather than the hydroxyl ligand to form the thiazolidine ring of penicillin N and provide the final electron required to form the second water from the oxidase reaction. The rebound reaction to an atom other than oxygen is reminiscent of the halogenase reaction described above and prevents oxygenase chemistry from occurring during the second half of the reaction.

Conclusion

The broad application of the 2-His+Asp/Glu Fe(II) binding motif (Fig. 6) demonstrates the ability of nature to adapt a functional structural building block to catalyze a remarkable variety of oxygen activation chemistries. The key to this flexibility appears to be the open face of the iron coordination sphere, which can be used to bind O2 and in some cases substrate or cosubstrate to carry out a determining first step in the catalytic process. The organization of the second sphere environment of this open face can also be tuned by the strategic placement of acid/base catalysts, hydrogen bonding partners, nonbonding substrates, electron-supplying metal clusters and cofactors. We have illustrated here that the combination of these elements allows the enzymes of the 2-His+Asp/Glu class to activate O2 as: (i) Fe(II)-[O2]dot–, (ii) Fe(II)-alkylperoxo, (iii) Fe(III)-[O2]dot–, (iv) Fe(III)-(hydro)peroxo, (v) Fe(IV)=O and possibly (vi) Fe(V)=O-hydroxo. All of these activation mechanisms share the common theme that the O2 is reduced as part of the activation process and the O-O bond is broken during the reaction. However, by controlling the structural elements of the 2-His+Asp/Glu Fe(II) center and its local environment, the point at which the O-O bond breaks during the reaction cycle can be regulated. Moreover, it is possible to direct the distribution of electrons in the immediate product of the O-O bond cleavage reaction. The result is exquisite control over the O2 activation process and the specific oxygenated and oxidized products that result.

Figure 6: Oxygen activation by the flexible 2-His+Asp/Glu facial triad motif.

Figure 6 : Oxygen activation by the flexible 2-His+Asp/Glu facial triad motif.

All reactions begin by binding a substrate or cofactor to or near the iron, resulting in release of solvents to open an O2 binding site on the iron. A myriad of downstream chemistries are possible, as determined by the nature of the exogenous molecules and active site residues near the catalytic surface of the iron. The three ligand sites shown to be occupied by H2O in the central structure can be vacant, occupied by OH- or occupied by a weak protein ligand in different enzymes from the 2-His+Asp/Glu family.

Full size image (53 KB)

The past decade has witnessed a great expansion in our appreciation of the detailed mechanisms used by non-heme Fe(II)-containing oxidases and oxygenases. This is attributable to the application of new kinetic techniques that allow intermediates in the reaction cycles to be recognized in combination with structural and spectroscopic techniques that allow them to be characterized. As a result, several of the key oxo-, peroxo- and high-valent metal complexes described here have quickly moved from the hypothetical to the anchors in the mechanistic proposals. The search for additional intermediates in the reaction cycles in the future can now be much more focused, but at the same time it is likely that these intermediates will be less stable and more difficult to detect and trap. Though the route to success is unknown in this endeavor, it is likely to build on our ability to rationally control the progress of reactions through changes in the local metal environments, and our ability to modulate the dynamics of the global enzyme structures by carrying out the reactions in atypical media such as crystals. It is also likely that computational studies will play a significant role by forecasting the properties of intermediates so that experiments can be designed to optimize their stability and hence our ability to trap them for characterization.



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Acknowledgments

The authors acknowledge support from US National Institutes of Health grant GM24689.

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