Misregulation of bromotyrosine compromises fertility in male Drosophila.

Biological regulation often depends on reversible reactions such as phosphorylation, acylation, methylation, and glycosylation, but rarely halogenation. A notable exception is the iodination and deiodination of thyroid hormones. Here, we report detection of bromotyrosine and its subsequent debromination during Drosophila spermatogenesis. Bromotyrosine is not evident when Drosophila express a native flavin-dependent dehalogenase that is homologous to the enzyme responsible for iodide salvage from iodotyrosine in mammals. Deletion or suppression of the dehalogenase-encoding condet (cdt) gene in Drosophila allows bromotyrosine to accumulate with no detectable chloro- or iodotyrosine. The presence of bromotyrosine in the cdt mutant males disrupts sperm individualization and results in decreased fertility. Transgenic expression of the cdt gene in late-staged germ cells rescues this defect and enhances tolerance of male flies to bromotyrosine. These results are consistent with reversible halogenation affecting Drosophila spermatogenesis in a process that had previously eluded metabolomic, proteomic, and genomic analyses.

The 2'-hydroxy group of flavin mononucleotide influences the catalytic function and promiscuity of the flavoprotein iodotyrosine dehalogenase.

The isoalloxazine ring system of the flavin cofactor is responsible for much of the catalytic power and diversity associated with flavoproteins. While the specificity of these enzymes is greatly influenced by the surrounding protein environment, the ribityl group of the cofactor may also participate in stabilizing transient intermediates formed by substrates and flavin. A conserved interaction between the phenolate oxygen of l-iodotyrosine and the 2'-hydroxy group of flavin mononucleotide (FMN) bound to iodotyrosine deiodianase (IYD) implied such a contribution to catalysis. Reconstitution of this deiodinase with 2'-deoxyflavin mononucleotide (2'-deoxyFMN) decreased the overall catalytic efficiency of l-iodotyrosine dehalogenation (kcat/Km) by more than 5-fold but increased kcat by over 2-fold. These affects are common to human IYD and its homolog from Thermotoga neapolitana and are best explained by an ability of the 2'-hydroxy group of FMN to stabilize association of the substrate in its phenolate form. Loss of this 2'-hydroxy group did not substantially affect the formation of the one electron reduced semiquinone form of FMN but its absence released constraints that otherwise suppresses the ability of IYD to promote hydride transfer as measured by a competing nitroreductase activity. Generation of IYD containing 2'-deoxyFMN also removed steric constraints that had previously limited the use of certain mechanistic probes. For example, l-O-methyl iodotyrosine could be accommodated in the active site lacking the 2'-hydroxy of FMN and shown to be inert to dehalogenation as predicted from a mechanism requiring ketonization of the phenolic oxygen. In the future, ancillary sites within a cofactor should now be considered when engineering new functions within existing protein architectures as demonstrated by the ability of IYD to promote nitroreduction after loss of the 2'-hydroxy group of FMN.

Dynamic accumulation of cyclobutane pyrimidine dimers and its response to changes in DNA conformation.

Photochemical dimerization of adjacent pyrimidines is fundamental to the creation of mutagenic hotspots caused by ultraviolet light. Distribution of the resulting lesions (cyclobutane pyrimidine dimers, CPDs) is already known to be highly variable in cells, and in vitro models have implicated DNA conformation as a major basis for this observation. Past efforts have primarily focused on mechanisms that influence CPD formation and have rarely considered contributions of CPD reversion. However, reversion is competitive under the standard conditions of 254 nm irradiation as illustrated in this report based on the dynamic response of CPDs to changes in DNA conformation. A periodic profile of CPDs was recreated in DNA held in a bent conformation by λ repressor. After linearization of this DNA, the CPD profile relaxed to its characteristic uniform distribution over a similar time of irradiation to that required to generate the initial profile. Similarly, when a T tract was released from a bent conformation, its CPD profile converted under further irradiation to that consistent with a linear T tract. This interconversion of CPDs indicates that both its formation and reversion exert control on CPD populations long before photo-steady-state conditions are achieved and suggests that the dominant sites of CPDs will evolve as DNA conformation changes in response to natural cellular processes.

Substrate Electronics Dominate the Rate of Reductive Dehalogenation Promoted by the Flavin-Dependent Iodotyrosine Deiodinase.

Iodotyrosine deiodinase (IYD) is unusual in its reliance on flavin to promote reductive dehalogenation of halotyrosines under aerobic conditions. Applications of this activity can be envisioned for bioremediation, but expansion of its specificity requires an understanding of the mechanistic steps that limit the rate of turnover. Key processes capable of controlling steady-state turnover have now been evaluated and described in this study. While proton transfer is necessary for converting the electron-rich substrate into an electrophilic intermediate suitable for reduction, kinetic solvent deuterium isotope effects suggest that this process does not contribute to the overall efficiency of catalysis under neutral conditions. Similarly, reconstituting IYD with flavin analogues demonstrates that a change in reduction potential by as much as 132 mV affects kcat by less than 3-fold. Furthermore, kcat/Km does not correlate with reduction potential and indicates that electron transfer is also not rate determining. Catalytic efficiency is most sensitive to the electronic nature of its substrates. Electron-donating substituents on the ortho position of iodotyrosine stimulate catalysis and conversely electron-withdrawing substituents suppress catalysis. Effects on kcat and kcat/Km range from 22- to 100-fold and fit a linear free-energy correlation with a ρ ranging from -2.1 to -2.8 for human and bacterial IYD. These values are consistent with a rate-determining process of stabilizing the electrophilic and nonaromatic intermediate poised for reduction. Future engineering can now focus on efforts to stabilize this electrophilic intermediate over a broad series of phenolic substrates that are targeted for removal from our environment.

Sequence Conservation Does Not Always Signify a Functional Imperative as Observed in the Nitroreductase Superfamily.

Consensus sequences have the potential to help classify the structure and function of proteins and highlight key regions that may contribute to their biological properties. Often, the level of significance will track with the extent of sequence conservation, but this should not be considered universal. Arg and Lys dominate a position adjacent to the N1 and C2 carbonyl of flavin mononucleotide (FMN) bound in the proteins of the nitroreductase superfamily. Although this placement satisfies expectations for stabilizing the reduced form of FMN, the substitution of these residues in three subfamilies promoting distinct reactions demonstrates their importance to catalysis as only modest. Replacing Arg34 with Lys, Gln, or Glu enhances FMN binding to a flavin destructase (BluB) by twofold and diminishes FMN turnover by no more than 25%. Similarly, replacing Lys14 with Arg, Gln, or Glu in a nitroreductase (NfsB) does not perturb the binding of the substrate nitrofurazone. The catalytic efficiency does decrease by 21-fold for the K14Q variant, but no change in the midpoint potential of FMN was observed with any of the variants. Equivalent substitution at Arg38 in iodotyrosine deiodinase (IYD) affects catalysis even more modestly (<10-fold). While the Arg/Lys to Glu substitution inactivates NfsB and IYD, this change also stabilizes one-electron transfer in IYD contrary to predictions based on other classes of flavoproteins. Accordingly, functional correlations developed in certain structural superfamilies may not necessarily translate well to other superfamilies.

The minimal structure for iodotyrosine deiodinase function is defined by an outlier protein from the thermophilic bacterium Thermotoga neapolitana.

The nitroreductase superfamily of enzymes encompasses many flavin mononucleotide (FMN)-dependent catalysts promoting a wide range of reactions. All share a common core consisting of an FMN-binding domain, and individual subgroups additionally contain one to three sequence extensions radiating from defined positions within this core to support their unique catalytic properties. To identify the minimum structure required for activity in the iodotyrosine deiodinase subgroup of this superfamily, attention was directed to a representative from the thermophilic organism Thermotoga neapolitana (TnIYD). This representative was selected based on its status as an outlier of the subgroup arising from its deficiency in certain standard motifs evident in all homologues from mesophiles. We found that TnIYD lacked a typical N-terminal sequence and one of its two characteristic sequence extensions, neither of which was found to be necessary for activity. We also show that TnIYD efficiently promotes dehalogenation of iodo-, bromo-, and chlorotyrosine, analogous to related deiodinases (IYDs) from humans and other mesophiles. In addition, 2-iodophenol is a weak substrate for TnIYD as it was for all other IYDs characterized to date. Consistent with enzymes from thermophilic organisms, we observed that TnIYD adopts a compact fold and low surface area compared with IYDs from mesophilic organisms. The insights gained from our investigations on TnIYD demonstrate the advantages of focusing on sequences that diverge from conventional standards to uncover the minimum essentials for activity. We conclude that TnIYD now represents a superior starting structure for future efforts to engineer a stable dehalogenase targeting halophenols of environmental concern.

Unraveling Reversible DNA Cross-Links with a Biological Machine.

The reversible generation and capture of certain electrophilic quinone methide intermediates support dynamic reactions with DNA that allow for migration and transfer of alkylation and cross-linking. This reversibility also expands the possible consequences that can be envisioned when confronted by DNA repair processes and biological machines. To begin testing the response to such an encounter, quinone methide-based modification of DNA has now been challenged with a helicase (T7 bacteriophage gene protein four, T7gp4) that promotes 5' to 3' translocation and unwinding. This model protein was selected based on its widespread application, well characterized mechanism and detailed structural information. Little over one-half of the cross-linking generated by a bisfunctional quinone methide remained stable to T7gp4 and did not suppress its activity. The helicase likely avoids the topological block generated by this fraction of cross-linking by its ability to shift from single- to double-stranded translocation. The remaining fraction of cross-linking was destroyed during T7gp4 catalysis. Thus, this helicase is chemically competent to promote release of the quinone methide from DNA. The ability of T7gp4 to act as a Brownian ratchet for unwinding DNA may block recapture of the QM intermediate by DNA during its transient release from a donor strand. Most surprisingly, T7gp4 releases the quinone methide from both the translocating strand that passes through its central channel and the excluded strand that was typically unaffected by other lesions. The ability of T7gp4 to reverse the cross-link formed by the quinone methide does not extend to that formed irreversibly by the nitrogen mustard mechlorethamine.

Directing Quinone Methide-Dependent Alkylation and Cross-Linking of Nucleic Acids with Quaternary Amines.

Polyamine and polyammonium ion conjugates are often used to direct reagents to nucleic acids based on their strong electrostatic attraction to the phosphoribose backbone. Such nonspecific interactions do not typically alter the specificity of the attached reagent, but polyammonium ions dramatically redirected the specificity of a series of quinone methide precursors. Replacement of a relatively nonspecific intercalator based on acridine with a series of polyammonium ions resulted in a surprising change of DNA products. Piperidine stable adducts were generated in duplex DNA that lacked the ability to support a dynamic cross-linking observed previously with acridine conjugates. Minor reaction at guanine N7, the site of reversible reaction, was retained by a monofunctional quinone methide-polyammonium ion conjugate, but a bisfunctional analogue designed for tandem quinone methide formation modified guanine N7 in only single-stranded DNA. The resulting intrastrand cross-links were sufficiently dynamic to rearrange to interstrand cross-links. However, no further transfer of adducts was observed in duplex DNA. An alternative design that spatially and temporally decoupled the two quinone methide equivalents neither restored the dynamic reaction nor cross-linked DNA efficiently. While di- and triammonium ion conjugates successfully enhanced the yields of cross-linking by a bisquinone methide relative to a monoammonium equivalent, alternative ligands will be necessary to facilitate the migration of cross-linking and its potential application to disrupt DNA repair.

Migratory ability of quinone methide-generating acridine conjugates in DNA.

The dynamic nature of nucleic acid alkylation by simple ortho quinone methides (QM) and their conjugates has provided numerous opportunities ranging from sequence selective targeting to bipedal walking in duplex DNA. To enhance the diffusion rate of adduct migration, one of two sites for QM generation was deleted from a bisQM conjugate of acridine to remove the covalent anchor to DNA that persists during QM regeneration. This conversion of a bisfunctional cross-linking agent to a monofunctional alkylating agent allowed adduct diffusion to traverse an extrahelical -TT- bulge that previously acted as a barrier for its bisfunctional analog. An electron rich derivative of the monofunctional acridine conjugate was additionally prepared to accelerate the rates of DNA alkylation and QM regeneration. The resulting stabilization of this QM effectively enhanced the rate of its release from adducts attached at guanine N7 in competition with an alternative and detrimental deglycosylation pathway. Intercalation by the acridine component was not sufficient to hold the transient QM intermediates within duplex DNA and consequently these electrophiles diffused into solution and were subject to quenching by solvent and a model nucleophile, ß-mercaptoethanol.

Effect of Nucleosome Assembly on Alkylation by a Dynamic Electrophile.

Quinone methides are reactive electrophiles that are generated during metabolism of various drugs, natural products, and food additives. Their chemical properties and cellular effects have been described previously, and now their response to packaging DNA in a nucleosome core is described. A model bisquinone methide precursor (bisQMP) was selected based on its ability to form reversible adducts with guanine N7 that allow for their redistribution and transfer after quinone methide regeneration. Assembly of Widom's 601 DNA with the histone octamer of H2A, H2B, H3, and H4 from Xenopus laevis significantly suppressed alkylation of the DNA. This result is a function of DNA packaging since addition of the octamer without nucleosome reconstitution only mildly protected DNA from alkylation. The lack of competition between nucleophiles of DNA and the histones was consistent with the limited number of adducts formed by the histones as detected by tryptic digestion and ultraperformance liquid chromatography-mass spectrometry. Only three peptide adducts were observed after reaction with a monofunctional analogue of bisQMP, and only two peptide adducts were observed after reaction with bisQMP. Histone reaction was also suppressed when reconstituted into the nucleosome core particle. However, bisQMP was capable of cross-linking the DNA and histones in moderate yields (∼20%) that exceeded expectations derived from reaction of cisplatin, nitrogen mustards, and diepoxybutane. The core histones also demonstrated a protective function against dynamic alkylation by trapping the reactive quinone methide after its spontaneous regeneration from DNA adducts.

Redox control of iodotyrosine deiodinase.

The redox chemistry of flavoproteins is often gated by substrate and iodotyrosine deiodinase (IYD) has the additional ability to switch between reaction modes based on the substrate. Association of fluorotyrosine (F-Tyr), an inert substrate analog, stabilizes single electron transfer reactions of IYD that are not observed in the absence of this ligand. The co-crystal of F-Tyr and a T239A variant of human IYD have now been characterized to provide a structural basis for control of its flavin reactivity. Coordination of F-Tyr in the active site of this IYD closely mimics that of iodotyrosine and only minor perturbations are observed after replacement of an active site Thr with Ala. However, loss of the side chain hydroxyl group removes a key hydrogen bond from flavin and suppresses the formation of its semiquinone intermediate. Even substitution of Thr with Ser decreases the midpoint potential of human IYD between its oxidized and semiquinone forms of flavin by almost 80 mV. This decrease does not adversely affect the kinetics of reductive dehalogenation although an analogous Ala variant exhibits a 6.7-fold decrease in its kcat /Km . Active site ligands lacking the zwitterion of halotyrosine are not able to induce closure of the active site lid that is necessary for promoting single electron transfer and dehalogenation. Under these conditions, a basal two-electron process dominates catalysis as indicated by preferential reduction of nitrophenol rather than deiodination of iodophenol.

The importance of a halotyrosine dehalogenase for Drosophila fertility.

The ability of iodotyrosine deiodinase to salvage iodide from iodotyrosine has long been recognized as critical for iodide homeostasis and proper thyroid function in vertebrates. The significance of its additional ability to dehalogenate bromo- and chlorotyrosine is less apparent, and none of these functions could have been anticipated in invertebrates until recently. Drosophila, as most arthropods, contains a deiodinase homolog encoded by CG6279, now named condet (cdt), with a similar catalytic specificity. However, its physiological role cannot be equivalent because Drosophila lacks a thyroid and its associated hormones, and no requirement for iodide or halotyrosines has been reported for this species. We have now applied CRISPR/Cas9 technology to generate Drosophila strains in which the cdt gene has been either deleted or mutated to identify its biological function. As previously shown in larvae, expression of cdt is primarily limited to the fat body, and we now report that loss of cdt function does not enhance sensitivity of the larvae to the toxic effects of iodotyrosine. In adult flies by contrast, expression is known to occur in testes and is detected at very high levels in this tissue. The importance of cdt is most evident in the decrease in fertility observed when either males or females carry a deletion or mutation of cdt Therefore, dehalogenation of a halotyrosine appears essential for efficient reproduction in Drosophila and likely contributes to a new pathway for controlling viability in arthropods.

The distribution and mechanism of iodotyrosine deiodinase defied expectations.

Iodotyrosine deiodinase (IYD) is unusual for its reliance on flavin to promote reductive dehalogenation under aerobic conditions. As implied by the name, this enzyme was first discovered to catalyze iodide elimination from iodotyrosine for recycling iodide during synthesis of tetra- and triiodothyronine collectively known as thyroid hormone. However, IYD likely supports many more functions and has been shown to debrominate and dechlorinate bromo- and chlorotyrosines. A specificity for halotyrosines versus halophenols is well preserved from humans to bacteria. In all examples to date, the substrate zwitterion establishes polar contacts with both the protein and the isoalloxazine ring of flavin. Mechanistic data suggest dehalogenation is catalyzed by sequential one electron transfer steps from reduced flavin to substrate despite the initial expectations for a single two electron transfer mechanism. A purported flavin semiquinone intermediate is stabilized by hydrogen bonding between its N5 position and the side chain of a Thr. Mutation of this residue to Ala suppresses dehalogenation and enhances a nitroreductase activity that is reminiscent of other enzymes within the same structural superfamily.

Conversion of a Dehalogenase into a Nitroreductase by Swapping its Flavin Cofactor with a 5-Deazaflavin Analogue.

Natural and engineered nitroreductases have rarely supported full reduction of nitroaromatics to their amine products, and more typically, transformations are limited to formation of the hydroxylamine intermediates. Efficient use of these enzymes also requires a regenerating system for NAD(P)H to avoid the costs associated with this natural reductant. Iodotyrosine deiodinase is a member of the same structural superfamily as many nitroreductases but does not directly consume reducing equivalents from NAD(P)H, nor demonstrate nitroreductase activity. However, exchange of its flavin cofactor with a 5-deazaflavin analogue dramatically suppresses its native deiodinase activity and leads to significant nitroreductase activity that supports full reduction to an amine product in the presence of the convenient and inexpensive NaBH4 .

Active Site Binding Is Not Sufficient for Reductive Deiodination by Iodotyrosine Deiodinase.

The minimal requirements for substrate recognition and turnover by iodotyrosine deiodinase were examined to learn the basis for its catalytic specificity. This enzyme is crucial for iodide homeostasis and the generation of thyroid hormone in chordates. 2-Iodophenol binds only very weakly to the human enzyme and is dehalogenated with a kcat/Km that is more than 4 orders of magnitude lower than that for iodotyrosine. This discrimination likely protects against a futile cycle of iodinating and deiodinating precursors of thyroid hormone biosynthesis. Surprisingly, a very similar catalytic selectivity was expressed by a bacterial homologue from Haliscomenobacter hydrossis. In this example, discrimination was not based on affinity since 4-cyano-2-iodophenol bound to the bacterial deiodinase with a Kd lower than that of iodotyrosine and yet was not detectably deiodinated. Other phenols including 2-iodophenol were deiodinated but only very inefficiently. Crystal structures of the bacterial enzyme with and without bound iodotyrosine are nearly superimposable and quite similar to the corresponding structures of the human enzyme. Likewise, the bacterial enzyme is activated for single electron transfer after binding to the substrate analogue fluorotyrosine as previously observed with the human enzyme. A cocrystal structure of bacterial deiodinase and 2-iodophenol indicates that this ligand stacks on the active site flavin mononucleotide (FMN) in a orientation analogous to that of bound iodotyrosine. However, 2-iodophenol association is not sufficient to activate the FMN chemistry required for catalysis, and thus the bacterial enzyme appears to share a similar specificity for halotyrosines even though their physiological roles are likely very different from those in humans.

Functional analysis of iodotyrosine deiodinase from drosophila melanogaster.

The flavoprotein iodotyrosine deiodinase (IYD) was first discovered in mammals through its ability to salvage iodide from mono- and diiodotyrosine, the by-products of thyroid hormone synthesis. Genomic information indicates that invertebrates contain homologous enzymes although their iodide requirements are unknown. The catalytic domain of IYD from Drosophila melanogaster has now been cloned, expressed and characterized to determine the scope of its potential catalytic function as a model for organisms that are not associated with thyroid hormone production. Little discrimination between iodo-, bromo-, and chlorotyrosine was detected. Their affinity for IYD ranges from 0.46 to 0.62 µM (Kd ) and their efficiency of dehalogenation ranges from 2.4 - 9 x 103 M-1 s-1 (kcat /Km ). These values fall within the variations described for IYDs from other organisms for which a physiological function has been confirmed. The relative contribution of three active site residues that coordinate to the amino acid substrates was subsequently determined by mutagenesis of IYD from Drosophila to refine future annotations of genomic and meta-genomic data for dehalogenation of halotyrosines. Substitution of the active site glutamate to glutamine was most detrimental to catalysis. Alternative substitution of an active site lysine to glutamine affected substrate affinity to the greatest extent but only moderately affected catalytic turnover. Substitution of phenylalanine for an active site tyrosine was least perturbing for binding and catalysis.

An Activator of an Adenylation Domain Revealed by Activity but Not Sequence Homology.

Nonribosomal peptide synthetases (NRPSs), which are responsible for synthesizing many medicinally important natural products, frequently use adenylation domain activators (ADAs) to promote substrate loading. Although ADAs are usually MbtH-like proteins (MLPs), a new type of ADA appears to promote an NRPS-dependent incorporation of a dihydropyrrole unit into sibiromycin. The adenylation and thiolation didomain of the NRPS SibD catalyzes the adenylation of a limited number of amino acids including l-Tyr, the precursor in dihydropyrrole biosynthesis, as determined by a standard radioactivity exchange assay. LC-MS/MS analysis confirmed loading of l-Tyr onto the thiolation domain. SibB, a small protein with no prior functional assignment or sequence homology to MLPs, was found to promote the exchange activity. MLPs from bacteria expressing homologous biosynthetic pathways were unable to replace this function of SibB. The discovery of this new type of ADA demonstrates the importance of searching beyond the conventional MLP standard for proteins affecting NRPS activity.

Targeting duplex DNA with the reversible reactivity of quinone methides.

DNA alkylation and crosslinking remains a common and effective strategy for anticancer chemotherapy despite its infamous lack of specificity. Coupling a reactive group to a sequence-directing component has the potential to enhance target selectivity but may suffer from premature degradation or the need for an external signal for activation. Alternatively, quinone methide conjugates may be employed if they form covalent but reversible adducts with their sequence directing component. The resulting self-adducts transfer their quinone methide to a chosen target without an external signal and avoid off-target reactions by alternative intramolecular self-trapping. Efficient transfer is shown to depend on the nature of the quinone methide and the sequence-directing ligand in applications involving alkylation of duplex DNA through a triplex recognition motif. Success required an electron-rich derivative that enhanced the stability of the transient quinone methide intermediate and a polypyrimidine strand of DNA to associate with its cognate polypurine/polypyrimidine target. Related quinone methide conjugates with peptide nucleic acids were capable of quinone methide transfer from their initial precursor but not from their corresponding self-adduct. The active peptide nucleic acid derivatives were highly selective for their complementary target.

Single Amino Acid Switch between a Flavin-Dependent Dehalogenase and Nitroreductase.

A single mutation within a flavoprotein is capable of switching the catalytic activity of a dehalogenase into a nitroreductase. This change in function correlates with a destabilization of the one-electron-reduced flavin semiquinone that is differentially expressed in the nitro-FMN reductase superfamily during redox cycling. The diversity of function within such a superfamily therefore has the potential to arise from rapid evolution, and its members should provide a convenient basis for developing new catalysts with an altered specificity of choice.

Rapid kinetics of dehalogenation promoted by iodotyrosine deiodinase from human thyroid.

Reductive dehalogenation such as that catalyzed by iodotyrosine deiodinase (IYD) is highly unusual in aerobic organisms but necessary for iodide salvage from iodotyrosine generated during thyroxine biosynthesis. Equally unusual is the dependence of this process on flavin. Rapid kinetics have now been used to define the basic processes involved in IYD catalysis. Time-dependent quenching of flavin fluorescence was used to monitor halotyrosine association to IYD. The substrates chloro-, bromo-, and iodotyrosine bound with similar rate constants (kon) ranging from 1.3 × 10(6) to 1.9 × 10(6) M(-1) s(-1). Only the inert substrate analogue fluorotyrosine exhibited a significantly (5-fold) slower kon (0.3 × 10(6) M(-1) s(-1)). All data fit a standard two-state model and indicated that no intermediate complex accumulated during closure of the active site lid induced by substrate. Subsequent halide elimination does not appear to limit reactions of bromo- and iodotyrosine since both fully oxidized the reduced enzyme with nearly equivalent second-order rate constants (7.3 × 10(3) and 8.6 × 10(3) M(-1) s(-1), respectively) despite the differing strength of their carbon-halogen bonds. In contrast to these substrates, chlorotyrosine reacted with the reduced enzyme approximately 20-fold more slowly and revealed a spectral intermediate that formed at approximately the same rate as the bromo- and iodotyrosine reactions.