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

Abstract

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 k_cat by less than 3-fold. Furthermore, k_cat/K_m 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 k_cat and k_cat/K_m 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.

Graphical Abstract

INTRODUCTION

Halophenols such as pentachlorophenol, bromoxynil, triclosan, and tetrabromobisphenol A have been widely dispersed in the environment as pesticides, herbicides, antimicrobial agents, and flame retardants. Unfortunately, their beneficial properties are now overshadowed by significant health risks caused by their accumulation in our surroundings. Natural pathways for their degradation are inefficient and waste treatment is problematic.^1-3 A variety of enzyme-based strategies have been proposed for their detoxification but few have yet been applied successfully in the environment.^4-6 A reductive dehalogenation catalyzed by the flavoprotein iodotyrosine deiodinase (IYD) offers a fresh alternative for bioremediation. Homologues of this enzyme are encoded by most metazoa and some bacteria. All variants examined to date prefer halotyrosines over halophenols as substrates.^7-9 Efforts to broaden substrate tolerance by engineering regions of the active site have provided only modest success.¹⁰ High affinity between IYD and its substrates is not sufficient for rapid turnover,⁷ and ligands capable of promoting a disorder-to-order transition of the active site lid do not guarantee effective catalysis.¹⁰ Future manipulation of its substrate specificity remains stymied until the processes controlling catalytic efficiency are identified.

Successful engineering of a multistep enzyme requires a knowledge of the catalytic mechanism, the structural components contributing to efficient turnover, and the kinetic barriers encountered during turnover. Current data on IYD are consistent with a series of one electron transfers from the reduced flavin (FMN_hq) to a dearomatized form of the substrate (Scheme 1).¹¹ These transfers in turn depend on the ability of the substrate to reposition an active site Thr hydroxyl group to form a hydrogen bond with the N(5) position of flavin.^8,12 Otherwise, IYD exhibits a promiscuous ability to promote hydride transfer as evident from a nitroreductase activity that is shared by other enzymes within its nitroreductase structural superfamily.^8,13,14 The desired expansion of substrate specificity for dehalogenation is further complicated by the numerous bond reorganizations suggested in the mechanism of catalysis. For example, substrate aromaticity is disrupted by carbon protonation to create a highly electrophilic intermediate poised for electron transfer and subsequent halide elimination (Scheme 1). Equivalent loss of aromaticity has been proposed in mechanisms such as tyrosine phenol lyase for which an equivalent dienone contributes to the total energy barrier of reaction (Scheme 2).¹⁵ Subsequent steps of IYD such as product release do not likely control the rate of turnover since the steady-state k_cat/K_m for iodotyrosine (I-Tyr) deiodination is similar to the second-order rate constant (k_ox) for I-Tyr-dependent oxidation of the reduced (FMN_hq) IYD.¹⁶ Cleavage of the carbon–halide bond also does not likely control the reaction since the k_cat/K_m values for I-Tyr and bromotyrosine are nearly identical despite the differences in their carbon–halogen bond energy.^17,18 The key steps of substrate protonation and electron transfer are considered most likely to control turnover. These alternatives have now been examined in this report by use of a solvent isotope effect and a series of flavin and substrate analogues. Together, the data suggest that the extent rather than the rate of substrate protonation controls the catalytic rate under neutral conditions.

Scheme 1.

(A) Mechanism for Reductive Dehalogenation by IYD. (B) Isoalloxazine Component of Reduced Flavin Mononucleotide (FMN_hq)

Scheme 2.

Protonation of Tyrosine during Catalysis by Tyrosine Phenol Lyase¹⁵

MATERIALS AND METHODS

General Materials.

Commercial sources for all starting materials and reagents are described in the Supporting Materials. Iodotyrosine deiodinase (Human, HsIYD) lacking its N-terminal transmembrane sequence (residues 2–31) was expressed as a SUMO fusion, purified by Ni²⁺ affinity and size exclusion chromatography and stored as described previously.¹² IYD from Haliscomenobacter hydrossis (HhIYD) was expressed with a C-terminal His₆-tag, purified by Ni²⁺ affinity and stored as described previously.⁷

General Methods.

NMR spectra were recorded on Bruker Avance-300 and Avance-400 spectrometers. UV measurements were performed with an Agilent 8453 spectrophotometer, and fluorescence measurements were performed with a FluoroMax-4 fluorescence spectrophotometer.

FMN and Substrate Analogues.

Detailed preparations are described in the Supporting Information. Briefly, FMN analogues were synthesized from the appropriately substituted nitroaniline as described previously.¹⁹ These were conjugated with ribose, reduced and condensed with alloxan. Phosphorylation was accomplished with ATP and riboflavin kinase.²⁰ Tyrosine analogues were prepared by a Negishi cross-coupling of the appropriate para-iodophenol with N-Boc-3-iodo-l-alanine methyl ester.²¹ Iodination of the tyrosine analogues typically used iodine in ammonium hydroxide,²² but the cyano derivative (I-CN-Tyr) was prepared with N-iodosuccinimide.²³ The transition-state analogue (N-MePyr) was generated by a similar Negishi cross-coupling using iodomethoxypyridine and N-Boc-3-iodo-l-alanine methyl ester.²¹

Reconstitution of HsIYD with FMN Analogues.

Cofactor exchange was performed as described previously.²⁰ Briefly, a SUMO–HsIYD fusion was expressed and bound to a Ni-affinity column where it was treated with increasing concentrations of guanidinium hydrochloride from 0.6 to 1.5 M in 100 mM NaCl, 50 mM sodium phosphate pH 7.4, 10% glycerol, 20 mM imidazole, and 0.05 mM tris(2-carboxyethyl)-phosphine (TCEP) to release FMN. The gradient of guanidinium hydrochloride was then reversed and the column was equilibrated with 500 mM NaCl, 50 mM sodium phosphate pH 7.9, 10% glycerol, and 0.05 mM TCEP. The column was next washed with the FMN analogue of choice (200 μM) in 100 mM NaCl, 50 mM sodium phosphate pH 7.4, 10% glycerol, and 0.05 mM TCEP for 4 h at 1 mL/min (4 °C). Excess cofactor was removed by an additional wash with the same buffer lacking the FMN analogue. Finally, the reconstituted SUMO–HsIYD fusion was eluted by addition of 250 mM imidazole in 100 mM NaCl, 50 mM sodium phosphate pH 7.4, 10% glycerol, 20 mM imidazole, and 0.05 mM TCEP. The SUMO tag was removed by proteolysis and HsIYD was isolated by size exclusion chromatography.¹²

Catalytic Deiodination.

The product of deiodination was monitored by reverse-phase C18 HPLC after incubating IYD (70–80 nM final) in 111 mM potassium phosphate pH 7.4 (900 μL) with 5% sodium dithionite in 5% sodium bicarbonate (100 μL) at 25 °C as described previously.¹⁷

Binding Affinity to IYD Containing Oxidized FMN (FMN_ox).

Ligand was titrated into a solution of IYD (3 μM) in potassium phosphate (110 mM, pH 7.4) with gentle stirring (25 °C). Fluorescence (λ_ex = 450 nm, λ_em = 516 nm) was measured 2 min after each addition. Dissociation constants were calculated by plotting remaining fluorescence relative to the initial fluorescence ($\Delta F∕F_0$) with respect to total enzyme ($[\mathrm{E}{]}_{\mathrm{t}}$) and total ligand ($[\mathrm{L}]$) using eq 1 (Origin 9.1) as described previously.²⁴

$$\begin{gathered} \frac{F}{F0}=1+ \\ \frac{\Delta F}{F0}\left(\frac{(K_{\mathrm{d}}+[E{]}_{\mathrm{T}}+[L])-\sqrt{(K_{\mathrm{d}}+[E{]}_{\mathrm{T}}+[L]{)}^2-4[E{]}_{\mathrm{T}}[L]}}{2[E{]}_{\mathrm{T}}}\right) \end{gathered}$$ (1)

Reduction of Reconstituted HsIYD Using Xanthine/Xanthine Oxidase.

All assays were performed at 25 °C using xanthine/xanthin e oxidase as described previously.^12,25,26 Briefly, a solution of 1 mM xanthine, 12 μM methyl viologen, 0.5 mM F-Tyr, and 110 mM potassium phosphate pH 7.4 was placed in a sealable quartz cuvette. Molecular oxygen was purged with argon for at least 20 min prior and 2 min subsequent to addition of HsIYD (20 μM). The headspace was then purged with argon for 30 min and reduction was initiated by addition of xanthine oxidase (140 μg/mL). The UV/vis spectral changes were recorded at room temperature every 2 min for over 4 h.

RESULTS AND DISCUSSION

Proton Transfer as a Potential Rate-Determining Step in IYD Catalysis.

Protonation of the aromatic ring of I-Tyr is an obvious process that can limit turnover since the loss of aromaticity is highly endergonic even for the electron-rich systems such as a phenol. Since no proton donor has yet been identified in the active sites of various IYDs,^7,9,12,27 site-directed mutagenesis is ineffective for examining such protonation. However, the contribution of proton transfer to the overall rate of turnover can be measured by solvent kinetic isotope effects irrespective of the specific donor. IYD from H. hydrossis (HhIYD) was chosen for this analysis since it is both robust and easily purified.⁷ As usual, excess dithionite was used to reduce IYD since its own native reductase has not yet been identified.¹¹ Reduction by dithionite does not limit turnover based on the similarity of the k_cat/K_m and k_ox values described above.¹⁶

A decrease in turnover is expected after replacing all exchangeable protons with deuterons if proton/deuteron transfer contributes to the overall rate of catalysis. Under neutral conditions used for the standard assays (pL 7.4), the effect of substituting H₂O with D₂O was quite modest on k_cat and generated a solvent kinetic isotope effect (^DODk_cat) of 1.6 (Table 1 and Figure S1). For context, proton transfer was not thought to be rate limiting based on a ^DODk_cat value of 1.7 for tyrosine phenol lyase that requires a carbon protonation most similar to that proposed for IYD (Scheme 2).¹⁵ Alternatively, a corresponding value of 2.3 for ^DODk_ox was considered sufficient to indicate that proton transfer contributes to the rate of flavin oxidation in a monooxygenase SidA.²⁸ An exchange of H₂O with D₂O may also affect catalysis by increasing solvent viscosity and concomitantly decreasing rates of diffusion and conformational dynamics in proteins. The viscosity of D₂O is equivalent to an aqueous solution of 9% glycerol.^29,30 To test if the minimal effect of D₂O on HhIYD was based on viscosity rather than proton transfer, steady-state kinetics were repeated in presence of 9% glycerol (Table 1 and Figure S1). The response to D₂O and 9% glycerol were experimentally equivalent and thus proton transfer appears to contribute little to the overall rate of dehalogenation by HhIYD under neutral conditions. The limited effects of viscosity are not easily interpreted since only k_cat, and not k_cat/K_m, appears sensitive to the glycerol. This result may reflect a viscosity-dependent partitioning of intermediates during catalysis, but its origins will remain ambiguous until extensive studies can sort through the many subtleties of IYD conformation and dynamics that may respond to solvent. A common explanation based on rate-determining product release would not be consistent with the similarity between steady-state rates of catalysis and the second-order rate of FMN_hq oxidation by I-Tyr that is independent of product release.^16,31

Table 1.

Characterization of I-Tyr Turnover by HhIYD in the Alternative Presence of D₂O and Glycerol^a

conditionsb (pL 7.4)	kcat (s−1)	Km μM)	kcat/Km (M−1 s−1) × 102
H2O	0.64 ± 0.08	23 ± 4	280 ± 60
D2O	0.41 ± 0.05	12 ± 4	340 ± 120
H2O, 9% glycerol	0.39 ± 0.05	13 ± 4	300 ± 100

^aData represent an average of two independent measurements and error corresponds to the range of these measurements. For details, see Figure S1.

^bpL maintained by 100 mM potassium phosphate.

A caveat associated with solvent isotope studies arises from the potential for functional group ionization to change after replacing H₂O with D₂O and hence measurements are typically performed under conditions in which catalysis is independent of pH.³² A pH of 7.4 has been the standard for the large majority of IYD assays and was consequently selected for the initial studies above. However, a pH rate profile of diiodotyrosine (I₂-Tyr) deiodination by human IYD (HsIYD) previously indicated a pH sensitivity under these conditions.¹² HhIYD was then subject to an equivalent examination over pH values from 5.5 to 8.5 (Figure 1). The responses of HhIYD and HsIYD are very similar, and both become relatively independent of pH at 6.0 and below. A fit of data from Figure 1 to eq 2 suggests that maximum turnover requires protonation of a group with an apparent pK_a between 7.6 and 7.9 based on the response of V and V/K. The similarity in the pK_a values from these plots is consistent with a common pH-dependent process controlling the two kinetic parameters. This is typically interpreted as a proton-dependent step in the chemical transformation, rather than binding, of substrate. A corresponding pK_a of 7.5–7.6 was previously determined for HsIYD.¹² Together, these values are similar to the pK_a of the α-ammonium groups of I-Tyr (7.9) and I₂-Tyr (7.8).³³ A dependence on these groups is easily rationalized by their coordination to both a fully conserved Glu in the active site lid and the C(4) carbonyl of flavin. While the α-ammonium is not likely responsible for protonating the phenolic ring, it has the potential to influence the chemistry of the flavin very strongly.^34,35

$$\log (\mathrm{y})=\log (\mathrm{y}{)}_{\max }-\log (1+{10}^{\mathrm{p}K\mathrm{a}\text{-pH}})$$ (2)

Figure 1.

pH dependence of HhIYD catalysis. The pH profile of (A) log V and (B) log(V/K) were fit by nonlinear least-square regression using eq 2. Reductive deiodination with 0.5% dithionite was measured using standard protocols in the presence of 2-(N-morpholino)-ethanesulfonic acid (100 mM, MES) (■), potassium phosphate (100 mM) (○), and Tris-HCl (100 mM) (▲) at the indicated pH values (Figure S2). Error bars represent the range of values of two independent measurements.

Solvent deuterium isotope effects were repeated at a pL of 6.0 to check turnover under conditions that are independent of pL. In this case, a solvent kinetic isotope effect (^DODk_cat) of 2.3 was observed and represents a perturbation well beyond the 14% suppression of k_cat caused by an increase in viscosity by 9% glycerol at pL of 6.0 (Figure S3). However, k_cat/K_m values measured at this pL in D₂O, H₂O, and 9% aqueous glycerol differed by no more than 12% from an average value of 250 × 10² M⁻¹s⁻¹. Thus, the overall energetics for one complete cycle of IYD catalysis remains relatively unperturbed by the change of solvent. In contrast, the sensitivity of k_cat may suggest the presence of an internal partitioning that is altered by D₂O. The different responses at pL of 6.0 and 7.4 could result from a change in the balance of processes contributing to the multistep catalysis of reductive dehalogenation promoted by HhIYD. Ultimately, the relative significance of the solvent effects depends on the complementary effects of FMN redox potential and substrate electronics as described below. These further investigations returned to the standard pH of 7.4 to evaluate each parameter when solvent isotope effects are minimal.

Electron Transfer as a Potential Rate-Determining Step in IYD Catalysis Using FMN Analogues.

Substitution of the methyl groups on the flavin C7 and C8 influences its redox potential as well as the basicity and nucleophilicity of its N5 group as predicted by inductive effects (Scheme 1).^19,36-38 Linear free-energy correlations between such perturbations and enzyme turnover typically reveal key mechanistic constraints limiting catalysis.^39,40 If electron transfer from reduced flavin to the protonated substrate was rate determining, then catalytic efficiency should increase as the reducing power increases.³⁶ Accordingly, the 7-chloro- and 8-methoxy-desmethylFMN (7-Cl-FMN, 8-MeO-FMN) were prepared by literature procedures^19,41 and used to reconstitute HhIYD with an on-column method described previously (Supporting Information).²⁰ 7-Cl-FMN and 8-MeO-FMN were selected as representatives with higher and lower potentials relative to the native FMN (Table 2). Unfortunately, HhIYD was not amenable to the reconstitution protocol optimized for HsIYD. Various concentrations of guanidinium HCl and KBr were later examined but none reconstituted HhIYD in reasonable yields. A switch to HsIYD seemed reasonable since HhIYD and HsIYD share similar substrate specificities, pH profiles, and active site structures.^7,8,12 Additionally, HsIYD has been reconstituted with cofactor occupancies of 100% and enzyme recoveries of 97%.²⁰ Steady-state characterization of these enzymes by I-Tyr dehalogenation indicated that catalytic efficiency (k_cat/K_m) remained greatest for IYD reconstituted with the native FMN (Table 2). In contrast, the k_cat value did trend with reducing power and an enhancement of 2.8-fold was observed between analogues that differ by 132 mV. Electron transfer may then control the dehalogenation chemistry in part but the response is mild compared to the precedence of bacterial luciferase that induced a 6.6-fold acceleration of rate with a change in reduction potential of 108 mV⁴⁴ and adrenodoxin reductase that induced a 35-fold acceleration with a change in potential of 136 mV.³⁶

Table 2.

Steady-State Dehalogenation of I-Tyr by HsIYD Reconstituted with FMN and Its Analogues^a

cofactor	redox potential (mV)b	kcat (s−1 × 10−2)	Km (μM)	kcat/Km (M−1 × s−1) × 102	pKae (FMNhq N5)
8-MeO-FMN	−260c	9.0 ± 0.3	37 ± 3	25 ± 2	5.32
FMN	−205d	5.2 ± 0.2	10 ± 1	50 ± 7	5.17
7-Cl-FMN	−128d	3.3 ± 0.2	23 ± 2	15 ± 0.2	3.42

^aData represent an average of two independent measurements under standard assay conditions (pH 7.4) and the error represents their range (see Figure S4 for details).

^bRedox potentials are reported for these derivatives when free in neutral aqueous solution at 25 °C.

^cFrom reference 42.

^dFrom reference 43.

^eFrom reference 19.

Insights Provided by FMN Analogues.

Additional FMN analogues were not pursued based on the weak correlation between catalytic efficiency and redox potential, but the properties of HsIYD alternatively containing 7-Cl-FMN and 8-MeO-FMN were examined for their ability to form their one-electron-reduced flavin semiquinone (FMN_sq). This species does not form in detectable quantities in the native HsIYD containing FMN in the absence of a halotyrosine derivative bound in its active site.^8,12 In presence of the inert substrate analogue fluorotyrosine, however, FMN_sq accumulates as an intermediate during reductive titration of HsIYD and this feature correlates with the dehalogenase activity of the flavoprotein.^8,11,45 No information was found in the literature by these authors on 7-Cl-FMN_sq and 8-MeO-FMN_sq and thus their ability to form the one-electron-reduced species was confirmed by redox titration of the reconstituted HsIYDs using xanthine and xanthine oxidase in the presence of 3-fluorotyrosine (F-Tyr, Figure 2). HsIYD containing 7-Cl-FMN produced a long-wavelength absorbance between 550 and 650 nm that is reminiscent of the absorbance of the neutral FMN_sq of native HsIYD.¹² Equivalent reduction of HsIYD containing 8-MeO-FMN_sq generated a related absorbance at a shorter wavelength (500–600 nm) with respect to those of 7-Cl-FMN_sq and FMN_sq. Stabilization of these semiquinone states is consistent with the ability of the reconstituted HsIYD to support the dehalogenation described above. Otherwise, significant destabilization of the semiquinone intermediate would have severely limited dehalogenation as observed previously with 5-deazaFMN.²⁰

Figure 2.

Formation of FMN_sq during reduction of IYD. HsIYD reconstituted alternatively with (A) 7-Cl-FMN and (B) 8-MeO-FMN was then reduced under anaerobic conditions by xanthine and xanthine oxidase in the presence of 0.5 mM F-Tyr over 2 h.^12,25,26 Titrations began with the oxidized cofactors indicated by the FMN_ox designation.

Further analysis of the reconstituted IYDs provided additional mechanistic information beyond the redox steps of catalysis. Previously, the acid/base chemistry of FMN had been shown to control the efficiency of type II isopentenyl diphosphate:dimethylallyl diphosphate (IPP:DMAPP) isomerase through a correlation between the estimated pK_a of N5 and catalytic efficiency.¹⁹ The acidic form of the N5 position within a reduced flavin has the potential to donate a proton to the halogenated carbon of I-Tyr since these atoms are separated by only ~4 Å in the crystal of I-Tyr and HsIYD containing oxidized FMN (FMN_ox) (Figure 3).¹² No other residues within a 5 Å radius are better positioned for proton transfer. However, the rate of dehalogenation is not enhanced by increasing the estimated acidity of the N5 by substituting FMN with 7-Cl-FMN (Table 2). Hence, the source of a proton for I-Tyr activation remains to be determined and does not likely involve FMN. Flavoproteins within the same structural superfamily appear to rely on solvent as a proton source during reduction of nitroaromatic substrates.¹⁴ Currently, this is the most reasonable source for IYD as well.

Figure 3.

Amino acid residues within 5 Å of the site of I-Tyr protonation in HsIYD based on PDB 4TTC.¹² Carbon atoms of I-Tyr, FMN_ox, and amino acid side chains are rendered in light magenta, yellow, and light cyan, respectively.

The aromatic ring of halotyrosine stacks intimately with the isoalloxazine ring of FMN_ox in all crystal structures characterized to date (Figure 3). Similar stacking is expected in the active complex with FMN_hq and this is inferred by the tight and competitive binding of an N-methylpyridone derivative (N-MePyr) that mimics the proposed protonated substrate intermediate (Scheme 1 and Table 3).⁴⁶ The FMN analogues provided an opportunity to correlate this stacking between the electron-rich FMN_hq species and the electron-poor intermediate analogue. Competitive inhibition of I-Tyr dehalogenation was compared for HsIYD reconstituted with 7-Cl-FMN, 8-MeO-FMN, and native FMN (Table 3 and Figure S6). The pyridone derivative strongly inhibited all of the reconstituted HsIYDs as expected from a prior examination of IYD that had been extracted from porcine thyroids.⁴⁶ Additionally, the binding affinity as determined by K_i was enhanced by ~60% when the electron-poor 7-Cl-FMN was replaced by the electron-rich 8-MeO-FMN in the HsIYD active site. Thus, FMN likely participates in substrate binding and orientation as well as serving as a donor of electrons. The significance of the π stacking is also evident from the ability of 2-iodophenol to adopt a similar orientation as the corresponding atoms of I-Tyr above FMN in a crystal structure of HhIYD.⁷ Still, the overall contribution of this noncovalent association is not easily deciphered since the trend of K_i does not match the trend of the K_m values for I-Tyr dehalogenation (Tables 2 and 3). Because neither proton nor electron transfer strongly controls turnover of IYD, attention was directed back to the most obvious high-energy species proposed in catalysis, the dearomatized substrate intermediate (Scheme 1).

Table 3.

Competitive Binding of N-MePyr to Reconstituted HsIYDs^a

	Cofactor	KI (nM)
	8-MeO-FMN	121 ± 17
	FMN	163 ±4
	7-Cl-FMN	198 ± 2

^aK_I values represent an average of two independent trials and the error represents their range (see Figure S6 for details).

Correlation between Dehalogenation Efficiency and Substrate Electronics.

As a complement to the use of flavin analogues above, a series of substrate analogues were designed to perturb the electronic properties of I-Tyr and consequently those of its intermediates formed during catalysis. Electron-donating substituents ortho to the phenol of I-Tyr have the potential to stabilize the dearomatized, electron-deficient intermediate while concurrently diminishing its propensity for accepting an electron from FMN_hq (Scheme 1). Electron-withdrawing groups induce the reciprocal response to dearomatization and electron transfer. Accordingly, substrates alternatively containing −OMe, −Me, −F, and −CN were prepared by iodination of their tyrosine precursors that were generated from a Negishi cross-coupling between an iodoalanine derivative and the appropriate para-iodophenol (see the Supporting Information). The consequences of these substituents on binding to IYD containing FMN_ox were conveniently surveyed by the ability of bound substrate to quench flavin fluorescence.^24,47 The native substrate I-Tyr bound most tightly to HsIYD, but all of the analogues bound with K_d values in the low μM range (Table 4). No trend based on electronics was apparent and the greatest influence could be the size of the substituent because substrates containing the −OMe and −CN bound somewhat weaker than those containing −Me, −F, and −H. In vivo, HsIYD is thought to process both I-Tyr and I₂-Tyr and hence tolerance to the 3,5-substituted tyrosines had been anticipated.¹¹ Previously, the K_d of I₂-Tyr with HsIYD was determined to be similar to that of Me-I-Tyr and one order of magnitude larger than that for I-Tyr.¹²

Table 4.

Substituent Effects on Binding and Deiodination with HsIYD^a

HsIYD	Kdb (μM)	kcat c (s−1)	Km c (μM)	kcat/Km (M−1s−1) × 102
I-CN-Tyr	7.0 ± 0.03	0.02 ± 0.003	30 ± 10	7 ± 2
I-F-Tyr	0.9 ± 0.1	0.06 ± 0.008	36 ± 8	17 ± 4
I-Tyr	0.20 ± 0.04	0.12 ± 0.008	19 ± 3	60 ± 10
I-MeO-Tyr	6.3 ± 0.3	0.72 ± 0.12	77 ± 18	90 ± 30
I-Me-Tyr	2.6 ± 0.03	0.76 ± 0.05	50 ± 5	150 ± 20

^aData represent averages of two independent trials under standard conditions and the error represents their range. 3-Iodo-5-methyltyrosine (I-Me-Tyr), 3-iodo-5-methoxytyrosine (I-MeO-Tyr), 3-fluoro-5-iodotyrosine (I-F-Tyr), and 3-cyano-5-iodotyrosine (I-CN-Tyr).

^bSee Figure S7.

^cSee Figure 4A.

Steady-state kinetic characterization of these tyrosine derivatives reveals that the native substrate I-Tyr exhibits the lowest K_m value, but otherwise there is no obvious trend between ortho substitution and K_m (Table 4 and Figure 4A). In contrast, both k_cat and k_cat/K_m reveal a clear dependence on substrate properties. Electron-deficient substrates were processed much less efficiently than the electron-rich substrates by a range of 38-fold as measured by k_cat and 29-fold by k_cat/K_m. This trend suggests the importance of stabilizing the C-protonated intermediate proposed in the mechanism over a dependence on electron transfer (Scheme 1). Additionally, the substituent effects fit well to a linear free-energy correlation using σ_m (Figure 4B).⁴⁸ This parameter was selected based on the placement of the substituents meta to the halogen-bearing carbon. The sensitivity factor (ρ) derived from the k_cat/K_m values is −2.2 ± 0.3 with an R² value of 0.934. Similar treatment of the k_cat values provides a ρ of −2.4 ± 0.5 with an R² of 0.851 (Figure S11). A negative value is consistent with an electron-deficient species in a rate-controlling process. The magnitude of this ρ is similar to that used to confirm the generation of an electron-deficient species during key steps in turnover of two flavin-dependent enzymes, UDP-galactose mutase and IPP:DMAPP isomerase.^19,38

Figure 4.

Substituent effects on catalytic deiodination by HsIYD. (A) Concentration dependence of deiodination for the indicated substrates in 100 mM potassium phosphate pH 7.4 and 0.5% sodium dithionite. Each data point represents an average of two individual observations and error bars represent their range. Kinetic parameters were determined from the best fit of data (solid lines) to Michaelis–Menten kinetics using Origin 9. (B) Linear free-energy correlation between turnover and substituent constant σ_m.⁴⁸ The sensitivity to substituents ρ is defined as the slope of the linear best fit to the data.

The 29- to 38-fold effect on catalysis created by perturbing the substrate electronics is significantly more pronounced than the effects of the FMN reducing potential and solvent properties (viscosity and deuteration). To test the generality of this major determinant, binding and turnover of the same set of substrates were repeated with the bacterial HhIYD used in the initial studies. This enzyme exhibited even less variation than HsIYD in its affinity (K_d) for the substrates, and binding of I-Tyr to HhIYD was among the weakest variant examined (Table 5). These values should be considered most useful for comparing relative affinities since they reflect binding to the oxidized (FMN_ox) state of IYD while catalysis involves the reduced state containing FMN_hq. Association of I-Tyr and other substrates to the reduced enzyme is not easily detected due to the immediate reoxidation of FMN_hq as part of the standard turnover. However, the affinity of an inert analogue, fluorotyrosine, to the reduced enzyme was estimated from its competitive inhibition of I-Tyr dehalogenation by HhIYD. Its K_i (10.7 ± 0.1 μM, Figure S8) is within the same order of magnitude as its K_d (46 ± 2 μM) measured previously with HhIYD containing FMN_ox.⁷

Table 5.

Substituent Effects on Binding and Deiodination of I-Tyr with HhIYD^a

HhIYD	Kdb (μM)	kcatc (s−1)	Kmc (μM)	kcat/Km (M−1s−1 × 102
I-CN-Tyr	7.3 ± 0.4	0.04 ± 0.008	74 ± 25	5 ± 2
I-F-Tyr	6.9 ± 0.3	0.27 ± 0.05	38 ± 13	70 ± 30
I-Tyr	7.1 ± 0.4	0.58 ± 0.005	49 ± 10	120 ± 20
I-MeO-Tyr	4.3 ± 0.4	1.4 ± 0.15	30 ± 5	500 ± 100
I-Me-Tyr	1.7 ± 0.1	0.72 ± 0.10	16 ± 4	500 ± 100

^aData represent averages of two independent under standard conditions and the error represents their range. All measurements used enzyme from a single preparation and differed from preparation used for solvent effects (Tables 1 and 2).

^bSee Figure S8.

^cSee Figure S9.

For HhIYD, I-Tyr may not be a native substrate since the physiological role of IYD has not yet been well documented in organisms other than Chordata.^11,49 However, at least one of the halotyrosines is likely to be physiologically important because tyrosine derivatives in general are bound and processed by HhIYD much more efficiently than their simple halophenol counterparts.⁷ Dehalogenation of the substrates by HhIYD established the same trend as that observed for HsIYD (Figure S9). The electron-rich I-Me-Tyr and I-MeO-Tyr were processed most efficiently, and the electron-poor I-F-Tyr and I-CN-Tyr were processed least efficiently. Sensitivity to these substituents (ρ = −2.8 ± 0.9) was experimentally equivalent to that of HsIYD, although the linear correlation was weaker with an R² of 0.777 (Figure S10). The k_cat values for HhIYD also responded similarly with a ρ of −2.1 ± 0.2 and an R² of 0.867 (Figure S11). Thus, substrate electronics dominate the efficiency of turnover for both the human and bacterial enzymes.

The substituents on I-Tyr also control the acidity of the phenolic proton and thus affect the balance of the protonated and deprotonated substrate under the assay conditions of pH 7.4. The phenolate form is thought to bind with significantly more affinity to HsIYD than the phenol form and this preference has been invoked to explain K_d values as low as 0.2 μM for I-Tyr but greater than 1000 μM for Tyr.¹² Association of the phenolate anion to the active site of IYD is likely stabilized by hydrogen bonding offered by the protein backbone and the 2′-hydroxy group of FMN.^11,12 If the phenolate form is then the true substrate (Scheme 1), K_d values calculated for the total substrate may not reflect the true affinity of the phenolate forms of the ligands. More importantly for this study, the steady-state rate constants and their response to the substituents may need a re-evaluation based on the approximate concentrations of phenolate rather than the total substrate. At the two extremes, 98% of I-CN-Tyr should exist in its phenolate form based on an estimated pK_a of 5.7, whereas only 3.4% of I-Me-Tyr should exist in its phenolate form based on an estimated pK_a of 8.9 (Table S1). Under this scenario, the differences between their k_cat/K_m values would increase to almost 1000-fold. The actual state of protonation within the active site is not yet known, so this analysis may exaggerate the power of the substituents but at least the trend remains consistent whether the total substrate or only its phenolate form is considered when calculating the steady-state constants.

CONCLUSIONS

The dominant effect of substituents on I-Tyr dehalogenation by human and bacterial IYD most likely arises from the varying stability and steady-state concentrations of the dearomatized intermediate that subsequently accepts an electron from FMN_hq before halide elimination. This sensitivity to substrate electronics would not have been observed if enzyme reduction by dithionite or halide release had controlled turnover. Contrary to expectations, neither electron nor proton transfer strongly control the efficiency of dehalogenation under neutral conditions. The weak dependence on reduction potential indicates electron transfer only moderately contributes to the overall rate of catalysis and the subsequent lysis of the carbon–halogen bond was already shown to be relatively fast based on the nearly equivalent rates of deiodination and debromination.^16,17

Proton transfer associated with dearomatization of the substrate is similarly not rate limiting since solvent deuterium isotope effects are minimal under neutral conditions. However, this transfer within an internal partitioning of intermediates may begin to limit catalysis under acidic conditions (pL ≤ 6.0) that supports turnover efficiency greater than that at neutral conditions. The proton source remains to be determined and does not likely involve the reduced FMN_hq since an inverse correlation was observed between the acidity of its N5 proton and the k_cat of I-Tyr dehalogenation. This contrasts the behavior of another flavin-dependent enzyme, IPP:DMAPP isomerase, that does utilize the N5 proton.¹⁹ An inverse correlation was evident for the acidity of the substrate phenol and turnover of HsIYD and HhIYD. Thus, the phenol is also not a likely candidate for proton transfer particularly since the substrate may bind in the phenolate form without its proton. In addition, the pH rate profile does not reflect the pK_a of the phenol and the pH response of HhIYD with I-Tyr is equivalent to that of HsIYD with I₂-Tyr despite the difference in the phenol acidities of 2 pK_a units.¹²

The largest variable in catalysis is the steady-state concentration of the electrophilic intermediate as evident from the strong correlation between rate and substrate substituents. A very similar conclusion was reached after extensive analysis of tyrosine phenol lyase, a PLP-dependent enzyme that also requires proton transfer to dearomatize its tyrosine substrate before bond lysis (Scheme 2).^15,50 Proton transfer did not control this later enzyme as much as stabilization of the dearomatized intermediate. Future efforts to expand the scope of substrates for IYD may now focus on electron-rich halophenols and active site mutations that stabilize the electron-deficient intermediate of substrates lacking the zwitterion of halotyrosine.