Flavin-Mediated Hydroxylation Reactions
Barrie Entsch, School of Science and Technology, University of New England, Armidale, Australia
David P. Ballou, Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan
Flavins react with oxygen and can form stable flavin peroxides in an aprotic solvent or buried in a protein. It is this hydroperoxide or peroxide that is the oxygenating agent in flavoproteins. This property is used in nature to carry out aromatic hydroxylations, halogenations, Baeyer-Villiger oxygenations, hydroxylation of xenobiotics and some metabolites, as well as light emission from luciferase. Several groups of enzymes seem to have evolved hydroxylating properties independently of each other. One group consists of the two-component flavin-dependent hydroxylases that use many of the same principles as the single component hydroxylases, although they also have some special requirements. After a brief introduction to the reactivity of flavins with oxygen, we examine p-hydroxybenzoate hydroxylase as the paradigm for the chemistry and protein functions exhibited by these enzymes. We then discuss unique features of each group of enzymes and the exciting prospects for future research.
In the context of this section, hydroxylation refers to enzymatic catalysis in which one atom of molecular oxygen is incorporated into the structure of a substrate, which causes its oxidation. A large variety of enzymes can carry out this type of reaction, and they are called oxygenases or hydroxylases. They have one common property: They use cofactors in catalysis that are reactive with molecular oxygen. Flavoprotein hydroxylases are a major subgroup of oxygenases, and they are found in all types of aerobic organisms, although particularly in bacteria and fungi. A more detailed overview of flavoprotein hydroxylases can be found in References 1 and 2. These enzymes oxygenate various aromatic compounds and ketones, electron-rich atoms in many compounds, and halogen ions that then halogenate aromatic compounds. However, the enzymes do not hydroxylate less reactive compounds, such as hydrocarbons. We have chosen to emphasize in this section the enzyme p -hydroxybenzoate hydroxylase, because it has been the outstanding model for the chemistry of these enzymes (3, 4) and has been studied extensively. Most other enzymes can be understood as variations on this model.
Reaction of Flavins with Oxygen
The chemistry and biological context of flavins are described in the article, “Chemistry of Flavoenzymes.” Reduced forms of flavins are usually very reactive with O2, which is somewhat surprising because most organic compounds are not very reactive with O2. Although reactions between dioxygen and most organic molecules are extremely favorable in thermodynamic terms (consider what happens in wild fires), the reactions are usually slow. Reactivity is lacking because organic compounds have singlet spin states, whereas O2 has a triplet spin state. However, in the case of reduced flavins and some other molecules, this impediment can be overcome because one-electron reduced states of flavins are readily accessible, and reactions with O2 to form radicals is spin allowed (5). The accidental formation of various reactive oxygen species by electron transfers from reduced flavins to oxygen in cells is generally avoided because free flavins are usually present in very low concentrations. Hy- droxylations can only occur if the formation and reaction of reactive oxygen species are tightly controlled. This control comes from the specific environments of enzyme active sites. One control strategy is to prevent the formation of the reactive reduced cofactor until conditions are suitable for hydroxylation—many flavoproteins use this strategy. Hydroxylases bind to and interact with flavins to decrease the thermodynamic stability of the free-radical form relative to the oxidized and reduced forms. As a result, when the reduced flavin reacts with oxygen inside the protein environment, the first electron is transferred to form a flavin radical and a superoxide radical (O2-) pair. This pair collapses via a much faster electron transfer to form a covalent flavin peroxide or hydroperoxide (the protonated form) at the C4a-atom of the flavin (see Fig. 1) in all flavin-dependent oxygenases studied. In enzymes with known three-dimensional (3-D) structures, there is a cavity suitable for covalent bonding of oxygen to the C4a-position. This C4a-flavin derivative is only stable in an aprotic environment (6) that must be provided by each specific enzyme. It is this flavin peroxide/hydroperoxide that is responsible for hydroxylations. In the absence of the controlled environments of enzymes, reduced flavins react with oxygen autocatalytically to form hydrogen peroxide and superoxide (5). Many flavoproteins that are not hydroxylases can accidentally react in their reduced state with oxygen to form such reactive oxygen species. Aerobic organisms have a battery of defense mechanisms to prevent cellular damage from these reactive oxygen species that frequently form to some extent in a wide range of cellular redox reactions.
Model Hydroxylase-p-Hydroxybenzoate Hydroxylase (PHBH)
PHBH is a typical flavoprotein hydroxylase involved in the breakdown of aromatic compounds that are used in general energy metabolism and as intermediates for biosynthetic processes (2). In general, these aromatic compounds are first oxidized by oxygen insertions, and then broken down into aliphatic compounds that can be incorporated into mainstream metabolism. PHBH is found in many soil bacteria and fungi; the PHBH enzymes that have been studied most extensively come from the bacteria Pseudomonas aeruginosa and Pseudomonas fluorescens. These enzymes differ by only a few amino acids that are not significant for function. The enzymes for study are expressed routinely in Escherichia coli and have the same molecular compositions as the enzymes from natural sources. The many high-resolution structures of PHBH that have been determined have been essential to our extensive knowledge of its function. Structures can be viewed in the Protein Data Bank (important examples include 1pbe—enzyme with p-hydroxybenzoate; 1phh—enzyme with 3,4-dihydroxy benzoate; 1dod—enzyme with 2,4-dihydroxybenzoate; and 1k0l—mutant enzyme R220Q).
PHBH in its native structure is a homodimer that contains one FAD per monomer, and it has a monomer molecular weight of 45,000. The active site of each monomer is constructed from the flavin and amino acid residues in that monomer. There is no clear evidence that a dimer is necessary for activity. PHBH is one member of a large family of similar flavo- proteins that probably have a common evolutionary descent, based on structural and sequence similarities. This family is often referred to as the one-component aromatic hydroxylases. This aspect distinguishes them from another large and diverse group of flavoprotein hydroxylases, which is the two-component flavin-dependent hydroxylases that use two different proteins to carry out the catalysis of hydroxylation. The latter group, which is described in a separate section, has no common ancestry with the one-component enzymes.
PHBH catalyzes the reaction shown in Fig. 1 with a high degree of specificity and a turnover rate of 50-60 s-1 at standard conditions of optimum pH (7.5-8) and 25 °C. A characteristic feature of PHBH and similar enzymes is the formation of some hydrogen peroxide during catalysis when conditions are not ideal for the enzyme (see Fig. 1, k8). This property, which is called uncoupling, comes about when the unstable flavin hydroperoxide (E Fl HOOH-S in Fig. 1) that is essential to catalysis decomposes. All of these flavin-dependent oxygenases function to limit the amount of toxic hydrogen peroxide formed during catalysis.
Figure 1. Catalytic cycle of p-hydroxybenzoate hydroxylase. In the first step, pOHB and NADPH bind (k1) and the FAD becomes reduced (k2). NADP is released (k3) and O2 reacts (k4) to form the C4a-hydroperoxy-FAD (E FlHOOH-S) in complex with substrate. Hydroxylation occurs via k5 to yield the dienone form of product and the C4a-hydroxy-FAD (Int II). Tautomerization yields 3,4-dihydroxybenzoate in complex with the enzyme (E Fl HOH-P). Dissociation of 3,4-DOHB and H2O via k7 leads to free enzyme (E Flox). Uncoupling occurs via the loss of H2O2 from the C4a-hydropeoxy flavin (k8).
Catalysis by PHBH occurs in two distinct half-reactions—the reductive and the oxidative steps. The overall reaction is exergonic and tightly controlled to limit the wasting of energy and regulate the formation of reactive oxygen species. The enzyme has evolved to employ complex molecular dynamic changes that are highly coordinated in time and space to achieve efficient catalysis. Over the last 15 years, extensive studies that involve these structural dynamics have been reported (4, 7). PHBH is one of only a few examples in which protein dynamics is understood. Catalysis begins with the binding of a molecule of p-hydroxybenzoate (pOHB) to the enzyme. The reducing substrate NADPH can also bind to the enzyme, but it does not readily reduce the flavin without bound pOHB. The redox potential difference between NADPH and FAD in the enzyme is suitable for irreversible reduction of FAD, and this potential is nearly the same with and without pOHB. However, the rate of reduction of FAD is 105-fold faster with pOHB bound. When the flavin is reduced, NADP dissociates relatively rapidly from the enzyme, but pOHB remains bound to the active site; the release of pOHB from reduced enzyme is at least 104-fold slower than from oxidized enzyme (8). The combination of the substrate stimulating reduction and remaining bound to the enzyme is vital because reactions with oxygen are only promoted when substrate is present, which avoids formation of hydrogen peroxide by the unintended reaction of reduced flavin with oxygen when the substrate is absent. This general regulatory process is used by many of the flavoprotein oxygenases. PHBH can be fooled into wasteful reduction of FAD by adding a molecule that mimics the structure of pOHB, but cannot be hydroxylated. Examples include 5-hydroxypicolinate and p-fluorobenzoate.
Molecular oxygen reacts with the complex of reduced enzyme and pOHB in a fast second-order reaction with oxygen (described in the previous section) to form a flavin hydroperoxide (Fig. 1). If pOHB is not bound, then this reactive intermediate immediately decomposes to form H2O2 (8). Thus, pOHB is part of the aprotic active site necessary for transient stabilization of the hydroperoxide. Structural modeling of the enzyme with substrate bound indicates that the distal oxygen of the C4a-flavin hydroperoxide is perfectly oriented in the active site for electrophilic attack on the 3-position of pOHB (9). After oxygen transfer, the oxidized nonaromatic product tautomerizes to the aromatic product, 3,4-dihydroxybenzoate (3,4 DOHB, see Fig. 1), which leaves FAD as the flavin-C4a-hydroxide (E Fl HOH-P). Catalysis is completed by dissociation of the product and loss of water from FAD. Thus, one atom of oxygen is incorporated into pOHB, and the other is reduced to water. Depending on the conditions, the primary rate-determining step in catalysis is either the dissociation of NADP from the enzyme after reduction, or the dissociation of 3,4 DOHB after oxidation (or both). The transient chemical intermediates in this oxidative half reaction were initially discovered and studied in the early 1970s by using stopped-flow spectrophotometry and fluorimetry to track the changes in FAD (8). Reduced, oxidized, and C4a-substituted flavins have unique absorption spectra (Fig. 2), and sometimes have unique fluorescence characteristics. For example, the C4a-hydroperoxyflavin and C4a-hydroxyflavin species often have very similar UV-visible spectra. However, the hydroxyflavin is often very fluorescent whereas the hy- droperoxyflavin is not. These properties, which make it possible to study the kinetics directly of the chemical changes in catalysis with considerable specificity and sensitivity, are an enormous help to mechanistic investigations of flavoproteins.
Figure 2. Spectra of reduced (no symbols) m C4a-hydroparoxyflaxin (triangles-dark grey, lower intensity spectrum with max at ~395 nm), C4a-hydroxyflavin (triangles-light grey higher intensity spectrum with max at ~380 nm), C4a=hydroxyflavin plus the product diemone (filled circles) and oxidized flavin (light grey, spectrum with peaks at ~380 and 655 nm). These spectra are intermedials observed by stopped-flow spectrophotometry using 2, 4-dihydroxybeneoate as substrate.
Role of proton exchanges and electrostatics in catalysis
As is the case with most enzymes, catalysis by PHBH is sensitive to pH because proton associations and dissociations are important to the overall process. The structure of PHBH shows that the isoalloxazine in the active site is in a positive electrostatic field (10, 11). When pOHB is bound in the active site, its 4-OH group is linked into a chain of hydrogen bonds to the surface of the protein (Fig. 3 Reference 12). These structural features are highly conserved in PHBH. This enzyme was an excellent model to study the effects of proton exchanges because pH-dependent changes occur to the spectra of both FAD and pOHB in the protein, and it is feasible to make specific stable mutants that involve the hydrogen bond chain and other residues in the enzyme.
Figure 3. Reprinted from Biochem. Biophys. Res. Commun. 2005; 338, Ballou, DP, Entsch, B, Cole, LJ., Dynamics involved in catalysis by single-component and two-component flavin-dependent aromatic hydroxylases, Pg. 593, (2005) with permission from Elsevier.
When pOHB binds to the oxidized enzyme at neutral pH in the positive electrostatic field of the active site, the phenolic proton dissociates through the hydrogen bond network, which culminates with the loss of a proton from histidine-72 at the surface of the protein (Fig. 3 and Reference 13). Formation of the substrate dianion (with the carboxylate and phenolate) triggers a conformational change in the structure of the protein that leads to the reduction of FAD by NADPH (see next sub-section). This particular exquisite control mechanism with PHBH probably does not occur in all flavoprotein hydroxylases. Among the known flavoprotein hydroxylases, PHBH has evolved to be exceptional in the level of control that prevents unwanted side reactions with oxygen.
In the positive electrostatic field of the active site, reduced FAD, which is formed by hydride transfer from NADPH, exists primarily as its anion (Fig. 1), which is ideal for its reaction with oxygen. The reaction of O2 with the anionic reduced flavin results in a peroxide that must be protonated to form the hydroperoxide for the subsequent electrophilic attack on pOHB. This protonation is extremely fast and has only been detected at high pH with a mutant form of PHBH (14). It is not known what is the source of the proton, but it is likely to be delivered through a water channel on the re side of the isoalloxazine ring. The hydrogen bond network also has an important function during the oxidative half-reaction. With the formation of the neutral flavin hydroperoxide, the 4-OH proton of pOHB can be removed and transferred to the hydrogen bond network; this transfer promotes electrophilic attack of the distal hydroxyl of the C4a-hydroperoxide on the 3-position of pOHB (14). Thus, the enzyme activates the substrate aromatic ring toward electrophilic attack. This effect was demonstrated by the > 100-fold decrease in the rate of the hydroxylation step in the mutant, Tyr201Phe, which cannot release the 4-OH proton (15). The positive electrostatic field in the active site is also important to the catalysis of hydroxylation. It was shown that making the field more positive increases the rate of hydroxylation, and making the field more negative decreases the rate of hydroxylation (16). These observations are consistent with the fact that the flavin hydroperoxide has a limited ability to carry out electrophilic attacks. For example, PHBH fails to hydroxylate either the less reactive benzoate or p-fluorobenzoate substrate analogs, even though they bind appropriately in the active site. After formation of the 3,4 DOHB product in the active site, the hydrogen bond network can again promote dissociation of the proton from the 4-OH position of 3,4 DOHB as part of a controlled conformational change in the protein that results in release of the 3,4 DOHB product and loss of water from the flavin (see the next section).
Protein dynamics in catalysis
The role of protein dynamics in catalysis has to be inferred from several pieces of mostly indirect evidence, which includes important kinetic observations. For example, why does NADPH reduce FAD in the enzyme 105-fold faster with pOHB bound than without, when the thermodynamics are essentially the same for each reaction? Under specific conditions, the 3-D structure of the enzyme is significantly different (10, 17), and these different structures can be logically ascribed to catalysis. For example, individual mutant forms of PHBH stabilize the structure in one or another particular conformation with specific dramatic effects on catalysis. Recently, single molecule fluorescence studies of PHBH have been used to demonstrate a specific conformational change in PHBH and to suggest a possible role of the protein dimer in promoting binding of pOHB (18). The accumulated evidence has provided the following minimum picture of functional protein dynamics.
The enzyme forms at least three highly interactive conformations during catalysis—named the in, out, and open conformations. Without ligands bound, the enzyme is in rapid equilibrium between the in and open conformations (which were measured in single-molecule studies) (18). It has been suggested that because of this dynamic equilibrium, the native protein fails to form high-resolution crystals without substrates bound. The open conformation has a more open active site and permits solvent access. This conformation has been illustrated by the structure of the Arg220Gln variant of PHBH, which gives a high-resolution crystal structure without any substrate bound (17). To form the open conformation, the highly conserved peptide loop that includes a strained peptide bond between Arg44 and Ala45, and that covers the si side of isoalloxazine ring, rotates to open partially to solvent the active site, which enables pOHB to bind. After binding, the equilibrium shifts toward the in conformation, where the active site is more closed and inaccessible to solvent (Fig. 3). The crystal structure with pOHB bound is the in conformation (19). With NADPH bound, the formation of the phenolate anion of pOHB facilitated by the hydrogen bond network triggers the enzyme into a third conformation, the out form (10). The phenolate anion repels the backbone carbonyl of Pro293, which triggers a structural change that is propagated through the highly conserved protein loop on the re side of the isoalloxazine ring. The critical feature of the out conformation is the position of the isoalloxazine ring, which has been shifted by rotation around the ribityl side chain to a solvent-exposed position at the surface of the protein in just the right orientation and distance from the C4 of the nicotinamide of the bound NADPH to foster rapid hydride transfer and reduction of the flavin. In PHBH, this chemically productive orientation is associated with the formation of a charge-transfer complex between flavin and NADPH that can be monitored spectrally at long wavelengths (20). Similar conformational control of reduction is probably common to many one-component hydroxylases. However, the mechanism for triggering the change is likely to be different with each enzyme.
After FAD is reduced, the enzyme reverts to the in conformation, probably because of the electrostatic attraction between the newly formed reduced flavin anion and the positive electrostatic field of the active site after protonation of pOHB by the hydrogen bond network (14). The conformational change is somehow linked to the dissociation of NADP, which is the rate-determining step in catalysis under optimal conditions for catalysis. The reaction with oxygen and the subsequent hydroxylation occurs with the protein in the in conformation. As with all of these hydroxylases, the enzyme reacts rapidly with oxygen in the enclosed hydrophobic active site. For PHBH, a hydroxylase with average reactivity with oxygen, the rate constant is 2 x 105 M-1 s-1 at 4 °C, which is at least 103-fold faster than the reaction of oxygen with free reduced flavin. A structural explanation probably existed for this reaction. PHBH has a hydrophobic pocket on the re face of the flavin with just the right space for oxygen to contact the isoalloxazine ring for reaction. Because the redox potential for the first electron transfer to oxygen (forming O2-) is not favorable, the electron transfer occurs over a short distance to oxygen, which is in the pocket; thus, the O2- that is initially formed as a caged pair with the flavin radical rapidly collapses to form the peroxide. This property of short-range electron transfer may help to avoid the formation of free superoxide that might otherwise escape to solution. In the in conformation, the flavin hydroperoxide has sufficient stability to execute hydroxylation of pOHB with 100% efficiency under the right conditions (pH between 6 and 7). The ring of the product, 3,4 DOHB, is rotated in the active site by comparison to pOHB, which helps trigger a conformational shift to the open form, permitting product dissociation and loss of water from the flavin to complete the catalytic cycle. PHBH, like most of these hydroxylases, is subject to substrate (pOHB) inhibition. Kinetic analysis has shown that mM concentrations of pOHB rapidly bind to PHBH after dissociation of the 3,4 DOHB product and thus stabilize the in conformation with the flavin trapped as the flavin-C4a-hydroxide (Fig. 1). This dead-end complex inhibits catalysis.
For PHBH to function as an efficient catalyst, the series of four conformational changes in a catalytic cycle have to be fast and coordinated compared with the chemical reactions of catalysis. For example, the observation that the reduction of flavin under optimal conditions for catalysis exhibits a full primary deuterium isotope effect (13) implies that the rate of reduction of flavin is limited by hydride transfer and not by conformational rearrangements. However, when the enzyme is stabilized in the in conformation (as with the mutant form, Ala45Gly), then a large fraction of flavin reduction becomes much slower under the same conditions and shows only a small deuterium isotope effect (21).
Two-Component Flavin-Dependent Hydroxylases
The one-component hydroxylases like PHBH have been recognized since the process of biological oxygenation was first demonstrated in the 1960s. Many similar enzymes have been discovered over the years. For example, although phenol hydroxylase from yeast has very little sequence similarity to PHBH, it has a 3-D structure with the same folding pattern as PHBH (22), and almost certainly undergoes similar protein dynamics in catalysis. In the 1990s, another completely different group of flavin-dependent hydroxylases was found in bacteria—the two-component enzymes. These two-component hydroxylases consist of one protein that catalyzes the reduction of flavin and an oxygenase that binds the reduced flavin product and carries out the hydroxylation step. These enzyme systems are often called two component-flavin diffusible monooxygenases (TC-FDM). In this section, we examine briefly p-hydroxyphenylacetate hydroxylase as a model for a large subset of this group of enzymes that, like PHBH, hydroxylate aromatic compounds. Another group of two-component enzymes has evolved with a different function—to halogenate substrates. We use tryptophan halogenase as an example of this expanding group. Finally, we refer to bacterial luciferase. This unique enzyme was the first two-component flavin-dependent hydroxylase to be recognized about 40 years ago. In the past 15 years, hydroxylations that involve catalysis by two-component enzymes of a huge diversity of substrates have been found. A summary of these can be found in Reference 2. The special case of epoxidation reactions should be noted. The best example is styrene monooxygenase (23, 24). As a two-component enzyme, it is similar to other enzymes of this type and uses a C4a-hydroperoxyflavin as a hydroxylating intermediate (24). However, the oxygenase component has no sequence similarity to other equivalent oxygenase components (23). We look forward to detailed mechanistic studies on hydroxylation by this enzyme because it may provide new insights into the process of oxygen insertion into double bonds by flavinperoxides as well as aromatic hydroxylations.
Para-hydroxyphenylacetate hydroxylase (HPAH)
This enzyme system, which has been isolated from several different organisms, subdivides into two distinct groups. The enzyme system from Acinetobacter baumannii is a model for one group, and the enzyme system from Pseudomonas aeruginosa is a model for the other. The P. aeruginosa hydroxylase is very similar to HPAH from E. coli (25), but it is more amenable for mechanistic studies.
HPAH from A. baumannii is isolated as a reductase that contains flavin mononucleotide (FMN) and an oxygenase without flavin. The reductase is a homodimer with monomer molecular weight of 35,000, whereas the oxygenase is a homotetramer with monomer mass of 47,000 Da. The two proteins together carry out a catalytic reaction analogous to that of PHBH; curiously, no detectable complex formation exists between the reductase and oxygenase proteins (26). The reductase reaction is almost identical to the reductive half-reaction of PHBH. NADH reduces the bound FMN slowly unless p-hydroxyphenylacetate (HPA) is present. However, when HPA is bound, the reductase binds NADH tightly and rapid reduction of the FMN ensues (27). The reduced FMN (FMNH-) is bound much less tightly to the reductase than is the oxidized FMN. In contrast, the oxygenase binds FMNH- tightly and then catalyzes a reaction similar to the oxidative half-reaction of PHBH. Nevertheless, notable differences from PHBH are observed. Without HPA, the oxygenase forms a stable flavin hydroperoxide, then binds HPA to form the product, 3,4-dihydroxyphenylacetate, and finally liberates FMN into solution (28). To complete catalysis, the separate proteins must coordinate their functions. It has been shown that when FMN is reduced by the reductase in the presence of HPA, FMNH- dissociates into solution with a rate constant of 80 s-1 at 4 °C (26). FMNH- binds very rapidly to the oxygenase and reacts with oxygen with a rate constant of 1.1 x 106 M-1s-1 at 4 °C. Because these events are so fast, the reaction of oxygen with free FMNH- in transit between the two proteins is negligible, providing that the oxygenase is in excess over the available FMN, which is generally the case (25). This two-component system is most effective with no external flavin added and with a slight excess of oxygenase active sites over reductase sites. Under these conditions, coupling of reducing equivalents from NADH to hydroxylation is maximal. How does the performance of this system compare with PHBH? Under the same conditions, turnover of HPAH is ~2 s-1 compared with ~50 s-1 for PHBH. Moreover, HPAH always generates a small amount of H2O2, but PHBH generates almost none.
The HPAH from P. aeruginosa consists of a reductase that is a homodimer with monomer molecular weight of 19,000 and is isolated with only some protein that contains FAD. The oxygenase is isolated as a colorless homotetramer with monomer mass of 58,500. In this system, in contrast to the A. baumannii HPAH, the reductase does not bind HPA, and it catalyzes reduction of FAD by NADH and release of FADH- into solution with no regulation of catalysis by HPA. Thus, for this HPAH to be effective, regulation of the reaction with oxygen must reside with the oxygenase. The reductase that is present reduces any free FAD for immediate uptake by the oxygenase, which, as established in E. coli (25), is present in cells at higher concentrations than the reductase. It has been found that the oxygenase forms a very stable C4a-hydroperoxyflavin, and this species is probably the predominant form of the enzyme in cells (25, 29). When the C4a-hydroperoxyflavin form of oxygenase binds HPA, it rapidly converts the HPA to product and releases it into solution. The release of FAD from the oxygenase follows and is the rate-determining step in catalysis (29). This HPAH has a turnover rate similar to the enzyme from A. baumannii. The structures of the oxygenases from the two types of HPAHs are different in detail (particularly the active sites) but have similar core scaffolds (30). A great deal of research must be performed to understand the unique dynamics of the P. aeruginosa-type oxygenase that probably do not occur in the A. baumannii -type oxygenase.
Many thousands of halogenated metabolites in nature become halogenated by reactions catalyzed by flavoproteins. To date, only a couple of enzymes have been studied in any detail. TH has now become the model for these enzymes. TH catalyzes the chlorination of the 7-position of tryptophan for the synthesis of pyrrolnitrin, which is an antifungal agent from P. fluorescens, and for the synthesis of rebeccamycin, which is an anticancer agent from Saccharothrix aerocolonigenes. TH consists of a flavin reductase and a halogenase. The reductase is similar to the simple reductase of HPAH from P. aeruginosa, whereas the halogenase has structural similarity to PHBH (31). Thus, in general terms, TH is a two-component system with FAD that diffuses between two proteins, but TH has evolutionary links to the one-component hydroxylases.
Recent transient-state kinetic analysis of the reaction of the halogenase component of TH has been very revealing (32). The halogenase binds FADH- and reacts with oxygen to form a stabilized C4a-hydroperoxyflavin. With Cl- present, this intermediate converts to a fluorescent C4a-hydroxyflavin, which finally decomposes to FAD and water. Tryptophan has no influence on the kinetics of the flavin reactions. The implication of these observations is that Cl- is hydroxylated by the hydroperoxide to form HOCl, which is subsequently used in the chlorination of tryptophan. The details of the regiospecific chlorination remain an outstanding question, although it is known from the crystal structures of TH from both P. fluoresens (PrnA) and from S. aerocolonigenes (RebF) that tryptophan is separated from FAD by ~10A, and they are connected by a channel in the active site. Chlorination is a slow reaction (first-order with a rate constant of 0.05 s-1 at 25 °C) and inefficient—only ~38% of starting reduced FAD equivalents result in chlorinated product (32). A static crystal structure is probably inadequate for a complete understanding of the details of the catalytic process. Unlike other hydroxylases, the halogenase undergoes some dynamic changes in the oxygen reactions that are not understood. For example, if the halogenase binds FADH- anaerobically, then the subsequent reaction with oxygen fails to form the flavin hydroperoxide, and only FAD and H2O2 are produced. In contrast, if reduced flavin is added to RebH and O2, full formation of the flavin C4a-hydroperoxide is observed. Recently, it was shown that when RebH, FADH-, Cl-, and O2 react in the absence of tryptophan, a long-lived intermediate (t1/2 = 63 h at 4 °C) formed that could halogenate tryptophan (33). This result suggested that the initially formed HOCl reacted with a group on the enzyme to produce an intermediate, such as a chloramine, that was responsible for halogenating tryptophan. Lysine 97, which is required for catalysis, is a likely candidate, and it is in a position that could halogenate tryptophan in the 7-position.
A luciferase is an enzyme that catalyzes chemiluminescence. This property is widespread in marine environments (particularly deep-sea), in which many higher organisms cultivate specific bacteria for the production of light. All known bacterial luciferases are homologous. Although this enzyme has been studied for decades, its mechanism is still partly a mystery. Like the enzymes just discussed, luciferase is composed of a reductase and an oxygenase. These proteins are usually expressed from a single operon similar to other two-component enzymes (34). The reductase catalyses an unregulated reduction of FMN by NADH (34). The oxygenase, which is usually referred to as luciferase, is structurally and functionally unique. It catalyses the hydroxylation of a fatty aldehyde to form a fatty acid in a manner similar to the Baeyer-Villiger enzymes discussed in the next section. However, mysteriously, the free energy liberated in the reaction is captured in an excited-state flavin. The usual intermediates in an oxygenase reaction are observed (35, 36)—formation of a flavin-C4a-peroxide, followed by a flavin hydroxide, return to oxidized flavin, and release of flavin from the enzyme. It is the excited-state C4a-hydroxyflavin that is the light emitter. Hypotheses put forward for the capture of an excited state can be found in Reference 37. Currently, we do not even know how FMN is bound in luciferase. When we have a clear picture of the active site of this enzyme (showing the interactions between protein and FMN in different oxidation states) and the complex dynamics that seem to be part of the process, we may finally understand this chemistry.
Other One-Component Hydroxylases
Although the one-component aromatic hydroxylases (represented by PHBH above) are a major group of flavoproteins, they are not the only one-component flavin-dependent oxygenases. Many flavoproteins oxygenate aliphatic compounds that are susceptible to nucleophilic attack by flavinperoxides. Oxygenation of aldehydes and ketones is a common reaction catalyzed. The mechanism is considered to involve a Baeyer-Villager rearrangement. We briefly examine the model enzyme cyclohexanone monooxygenase, which oxygenates the carbonyl of cyclohexanone and forms a lactone (cyclic ester) via a Baeyer-Villiger rearrangement. Another group of flavoproteins is important in the defensive armory of animals against electron-rich foreign compounds, and it is also involved in some metabolic pathways in animals and plants. An appropriate model for this group of enzymes is mammalian flavin monooxygenase. We finish with an example of a recently discovered enzyme BluB that although structurally unrelated to other flavoprotein hydroxylases, it catalyzes the oxygenation and conversion of FMN to dimethylbenzimidazole using flavin C4a-intermediates. Other novel flavoprotein hydroxylases/oxygenases are yet to be discovered.
Cyclohexanone monooxygenase (CHMO)
The CHMO that has been studied most extensively comes from a strain of Acinetobacter calcoaceticus. The monomeric form of the enzyme contains one FAD and has a molecular weight of 62,000. The structure of a homolog of this enzyme (phenylacetone monooxygenase) has been published (38). The structure and kinetic analysis of CHMO show that the overall catalytic cycle is similar to that of PHBH, although there are important differences (39). In contrast to PHBH, the reductive half-reaction is not regulated by substrate. Thus, the formation of cellular reactive oxygen species is prevented by a different mechanism. After NADPH binds and reduces the FAD, independently of cyclohexanone, the NADP product remains bound to the enzyme, and the resultant reduced enzyme reacts rapidly (> 106 M-1s1) to form a stable NADP-flavin C4a-peroxide complex. In fact, it is this species that is the predominant form of the enzyme in the cell. The bound NADP has a critical role in stabilizing the flavin peroxide. When cyclohexanone binds to the enzyme, it is converted rapidly to the 7-membered ring, ε-caprolactone, with the ring-oxygen coming from the peroxide, which leaves the flavin in the form of a C4a-hydroxy-FAD. After it loses water to form oxidized FAD, the rate-determining step in catalysis is the dissociation of NADP. Kinetic and structural evidence suggests that the mechanism involves a nucleophilic attack of the flavin peroxide on the carbonyl carbon to form a Criegee intermediate that collapses to form product and flavin hydroxide (Fig. 4). These enzymes avoid producing reactive oxygen species because the C4a-peroxy-FAD is very stable until substrate binds and reacts rapidly with it. There is great potential for research into the relationship between the protein structure and function of these enzymes that use C4a-flavin peroxides.
Figure 4. Chemistry of the oxygenation of cyclohexanone by cyclohexanone monooxygenase—a Baeyer-Villager rearrangement. In the first stage, NADPH binds and reduces the FAD to form E Flred-NADP. The NADP remains bound to the enzyme during the ensuing reactions. Oxygen reacts to form a C4a-peroxy-FAD (E Fl HOO-). In the next phases, cyclohexanone binds and reacts with the peroxyflavin to form a Criegee adduct, which breaks down to form the lactone product and the C4a-hydroxy-FAD (E Fl HOH-P). When the NADP dissociates, the hydroxy-flavin loses H2O to reform the oxidized FAD (E Flox).
Flavin monooxygenase (FMO)
The first known enzyme of this class was isolated from pig liver and was described as liver microsomal FAD-containing monooxygenase (40). It has several properties similar to CHMO, but it mainly oxygenates a wide range of heteroatom-containing soft nucleophiles, which are generally electron-rich compounds. Mammalian FMOs participate with cytochrome P450s in the oxygenation of hydrophobic xenobiotic compounds, which includes many drugs, making them more water soluble and ready for coupling with glutathione and other compounds so they can be excreted. Extensive rapid and steady-state kinetics studies have been carried out on pig liver FMO (40-42). FMO is first reduced by NADPH and, like CHMO, the enzyme retains the NADP product. The reduced flavin reacts with O2to form a stable NADP-C4a-hydroperoxyflavin. Its return to oxidized FAD with release of H2O2 occurs over a period of many minutes, which depends on the conditions. However, if a suitable heteroatom-containing substrate is present, FMO reacts quickly to oxygenate that substrate, which forms hydroxy amines, sulfenic acids, and so on. The resulting C4a-hydroxyflavin releases water to reform the oxidized FAD in what is often the rate-determining step of catalysis. Therefore, the turnover numbers for most substrates are very similar. In the absence of NADP, the C4a-hydroperoxide is so unstable that it is almost undetectable. Five documented functional genes and some pseudogenes for FMO are observed in the human genome (43). FMOs also exist in plants (44) where they participate in numerous activities, which include the biosynthesis of the plant hormone, auxin.
The biosynthesis of vitamin B12 has intrigued some of the most famous chemists, and research on this topic has resulted in four Nobel prizes. Yet until recently, the synthesis of dimethylbenzimidazole (DMB), which is the alpha-axial ligand to B12, has remained a mystery, except that it was known to derive from FMN. It has now been demonstrated that the BluB proteins from Sinorhizobium meliloti (45) and Rhodospirillum rubrum (46) catalyze the reaction of FMNH- with O2 to form DMB and erythrose-4-phosphate. The C1’ from the ribityl moiety of FMN becomes the C2 of DMB, and the dimethyl-diamino benzene portion comes from the isoalloxazine ring of the flavin. This amazing reaction is unprecedented, and clearly, it must involve some unusual chemistry. Recent unpublished studies (D. Ballou and M. Taga) have shown that BluB binds FMNH-, and this complex reacts with O2 with a rate constant ≥ 106 M-1s1 at 4 °C to form a C4a-hydroperoxy-FMN. This species oxygenates itself and decays in several steps to form ~50% DMB with the remaining product being FMN. Thus, BluB is a special case of a flavin oxygenase. Future identification of intermediates along the pathway to DMB will surely provide some novel chemistry.
1. Palfey BA, Massey V. Flavin-dependent enzymes. In: Comprehensive Biological Catalysis. Sinnott M, ed. 1998. Academic Press, New York. pp. 83-154.
2. Van Berkel WJ, Kamerbeek NM, Fraaije MW. Flavoprotein monooxygenases, a diverse class of oxidative biocatalysts: review. J. Biotechnol. 2006; 124:670-689.
3. Entsch B, van Berkel WJH. Structure and mechanism of parahydroxybenzoate hydroxylase: review. FASEB J. 1995; 9:476-483.
4. Entsch B, Cole LJ, Ballou DP. Protein dynamics and electrostatics in the function of p-hydroxybenzoate hydroxylase: review. Arch. Biochem. Biophys. 2005; 433:297-311.
5. Massey V. Activation of molecular oxygen by flavins and flavoproteins: review. J. Biol. Chem. 1994; 269:22459-22462.
6. Bruice TC. Flavin oxygen chemistry brought to date. Flavins and Flavoproteins. Bray RC, Engel PC, Mayhew SG, eds. 1984. Walter de Gruyter, Berlin. pp. 45-54.
7. Ballou DP, Entsch B, Cole LJ. Dynamics involved in catalysis by single-component and two-component flavin-dependent aromatic hydroxylases: review. Biochem. Biophys. Res. Commun. 2005; 338:590-598.
8. Entsch B, Ballou DP, Massey V. Flavin-oxygen derivatives involved in hydroxylation by p-hydroxybenzoate hydroxylase. J. Biol. Chem. 1976; 251:2550-2563.
9. Schreuder HA, Hol WGJ, Drenth J. Analysis of the active site of the flavoprotein p-hydroxybenzoate hydroxylase and some ideas with respect to its reaction mechanism. Biochemistry 1990; 29:3101-3108.
10. Gatti DL, Palfey BA, Lah MS, Entsch B, Massey V, Ballou DP, Ludwig ML. The mobile flavin of 4-OH benzoate hydroxylase. Science 1994; 266:110-114.
11. Moran GR, Entsch B, Palfey BA, Ballou DP. Electrostatic effects on substrate activation in para-hydroxybenzoate hydroxylase: studies of the mutant Lysine 297 Methionine. Biochemistry 1997; 36:7548-7556.
12. Gatti DL, Entsch B, Ballou DP, Ludwig ML. pH-Dependent structural changes in the active site of p-hydroxybenzoate hydroxylase point to the importance of proton and water movements during catalysis. Biochemistry 1996; 35:567-578.
13. Palfey BA, Moran GR, Entsch B, Ballou DP, Massey V. Substrate recognition by “password” in p-hydroxybenzoate hydroxylase. Biochemistry 1999; 38:1153-1158.
14. Ortiz-Maldonado M, Entsch B, Ballou DP. Oxygen reactions in p-hydroxybenzoate hydroxylase utilize the H-bond network during catalysis. Biochemistry 2004; 43:15246-15257.
15. Entsch B, Palfey BA, Ballou DP, Massey V. Catalytic function of tyrosine residues in para-hydroxybenzoate hydroxylase as determined by the study of site-directed mutants. J. Biol. Chem. 1991; 266:17341-17349.
16. Ortiz-Maldonado M, Cole LJ, Dumas SM, Entsch B, Ballou DP. Increased positive electrostatic potential in p-hydroxybenzoate hydroxylase accelerates hydroxylation but slows turnover. Biochemistry 2004; 43:1569-1579.
17. Wang J, Ortiz-Maldonado M, Entsch B, Massey V, Ballou DP, Gatti DL. Protein and ligand dynamics in 4-hydroxybenzoate hydroxylase. Proc. Natl. Acad. Sci. U.S.A. 2002; 99:608-613.
18. Brender JR, Dertouzos J, Ballou DP, Massey V, Palfey BA, Entsch B, Steel DG, Gafni A. Conformational dynamics of the isoalloxazine in substrate-free p-hydroxybenzoate hydroxylase: single-molecule studies. J. Am. Chem. Soc. 2005; 127:18171-18178.
19. Schreuder HA, Prick PA, Wierenga RK, Vriend G, Wilson KS, Hol WG, Drenth J. Crystal structure of the p-hydroxybenzoate hydroxylase-substrate complex refined at 1.9 A resolution. Analysis of the enzyme-substrate and enzyme-product complexes. J. Mol. Biol. 1989; 208:679-696.
20. Ortiz-Maldonado M, Entsch B, Ballou DP. Conformational changes combined with charge-transfer interactions are essential for reduction in catalysis by p-hydroxybenzoate hydroxylase. Biochemistry 2003; 42:11234-11242.
21. Cole LJ, Gatti DL, Entsch B, Ballou DP. Removal of a methyl group causes global changes in p-hydroxybenzoate hydroxylase. Biochemistry 2005; 44:8047-8058.
22. Enroth C, Neujahr H, Schneider G, Lindqvist Y. The crystal structure of phenol hydroxylase in complex with FAD and phenol provides evidence for a concerted conformational change in the enzyme and its cofactor during catalysis. Structure 1998; 6:605-617.
23. Otto K, Hofstetter K, Rothlisberger M, Witholt B, Schmid A. Biochemical characterization of StyAB from Pseudomonas sp. strain VLB120 as a two-component flavin-diffusible monooxygenase. J. Bacteriol. 2004; 186:5292-5302.
24. Kantz A, Chin F, Nallamothu N, Nguyen T, Gassner GT. Mechanism of flavin transfer and oxygen activation by the two-component flavoenzyme styrene monooxygenase. Arch. Bio- chem. Biophys. 2005; 442:102-116.
25. Louie TM, Xie XS, Xun L. Coordinated production and utilization of FADH2 by NAD(P)H-flavin oxidoreductase and 4-hydroxy-phenylacetate 3-monooxygenase. Biochemistry 2003; 42:7509 -7517.
26. Sucharitakul J, Phongsak T, Entsch B, Svasti J, Chaiyen P, Ballou DP. Kinetics of a two-component p-hydroxyphenylacetate hydroxylase explain how reduced flavin is transferred from the reductase to the oxygenase. Biochemistry 2007; 46:8611-8623.
27. Sucharitakul J, Chaiyen P, Entsch B, Ballou DP. The reductase of p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii requires p-hydroxyphenylacetate for effective catalysis. Biochemistry 2005; 44:10434-10442.
28. Sucharitakul J, Chaiyen P, Entsch B, Ballou DP. Kinetic mechanisms of the oxygenase from a two-component enzyme, p-hydroxy phenylacetate 3-hydroxylase from Acinetobacter baumannii. J. Biol. Chem. 2006; 281:17044-17053.
29. Entsch B, Cole LJ, Ballou DP. Protein dynamics in catalysis by flavoprotein hydroxylases. In: Flavins and Flavoproteins. Nishino T, Miura R, Tanokura M, Fukui K, eds. 2005. ARchiTech, Tokyo, Japan. pp. 143-154.
30. Kim S-H, Hisano T, Takeda K, Iwasaki W, Ebihara A, Miki K. Crystal structure of the oxygenase component (HpaB) of the 4-hydroxyphenylacetate 3-monooxygenase from Thermus thermophilus HB8. J. Biol. Chem. 2007; 282:33107-33117.
31. Dong C, Flecks S, Unversucht S, Haupt C, van Pee K-H, Naismith JH. Tryptophan-7-halogenase (PrnA) structure suggests a mechanism for regioselective chlorination. Science 2007; 309:2216-2219.
32. Yeh E, Cole LJ, Barr EW, Bollinger JM, Ballou DP, Walsh CT. Flavin redox chemistry precedes substrate chlorination during the reaction of the flavin-dependent halogenase RebH. Biochemistry 2006; 45:7904-7912.
33. Yeh E, Blasiak LC, Koglin A, Drennan CL, Walsh CT. Chlorination by a long-lived intermediate in the mechanism of flavin- dependent halogenases. Biochemistry 2007; 46:1284-1292.
34. Nijvipakul S, Wongratana J, Suadee C, Entsch B, Ballou DP, Chaiyen P. LuxG is a functioning flavin reductase for bacterial luminescence. J. Bacteriol. 2008; 190:1531-1538.
35. Suadee C, Chaiyen P, Svasti J, Entsch B, Ballou DP. Luciferase from Vibrio cambellii- a few changes in amino acids shine new light on the problem. In: Flavins and Flavoproteins. Nishino T, Miura R, Tanokura M, Fukui K, eds. 2005. ARchiTect, Tokyo, Japan. pp. 611-616.
36. Macheroux P, Ghisla S, Hastings JW. Spectral detection of an intermediate preceding the excited-state in the bacterial luciferase reaction. Biochemistry 1993; 32:4183-4186.
37. McCapra F. Chemical generation of excited states: the basis of chemiluminescence and bioluminescence, volume 305. In: Methods in Enzymology. Baldwin TO, Ziegler MM, eds. 2000. Academic Press, New York. pp. 3-47.
38. Malito E, Alfieri A, Fraaije MW, Mattevi A. Crystal structure of a Baeyer-Villiger monooxygenase. Proc. Natl. Acad. Sci. U.S.A. 2004; 101:13157-13162.
39. Sheng D, Ballou DP, Massey V. Mechanistic studies of cyclohexanone monooxygenase: chemical properties of intermediates in catalysis. Biochemistry 2001; 40:11156-11167.
40. Poulsen LL, Ziegler DM. The liver microsomal FAD-containing monooxygenase. Spectral characterization and kinetic studies. J. Biol. Chem. 1979; 254:6449-6455.
41. Beaty NB, Ballou DP. The oxidative half-reaction of liver microsomal FAD-containing monooxygenase. J. Biol. Chem. 1981; 256:4619-4625.
42. Jones KC, Ballou DP. Reactions of the 4a-hydroperoxide of liver microsomal flavin-containing monooxygenase with nucleophilic and electrophilic substrates. J. Biol. Chem. 1986; 261:2553-2559.
43. Cashman JR, Zhang J. Human flavin-containing monooxygenases: review. Ann. Rev. Pharm. Toxic. 2006; 46:65-100.
44. Schlaich NL. Flavin-containing monooxygenases in plants: looking beyond detox. Trends Plant Sci. 2007; 12:412-418.
45. Taga ME, Larsen NA, Howard-Jones AR, Walsh CT, Walker GC. BluB cannibalizes flavin to form the lower ligand of vitamin B12. Nature 2007; 446:449-453.
46. Gray MJ, Escalante-Semerena JC. Single-enzyme conversion of FMNH2 to 5,6-dimethylbenzimidazole, the ligand of B12. Proc. Natl. Acad. Sci. U.S.A. 2007; 104:2921-2926.
The further reading list is incorporated into the References list above (especially Refs. 3-5) in the form of review articles and book chapters. Breaking news about the field of flavoproteins can be found in Flavins and Flavoproteins 2008, which will be published in 2008. This book will contain proceedings from the 16th International Symposium on Flavins and Flavoproteins to be held in Spain in June 2008.
Cytochrome P450 Monooxygenases, Chemistry of
Enzyme Catalysis, Roles of Structural Dynamics in
Enzyme Catalysis, Chemical Strategies for
NAD+ Dependent Enzymes, Chemistry of
Oxygen-Activating Enzymes, Chemistry of
Transient State Enzyme Kinetics
Flavoenzymes, Chemistry of