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Published in final edited form as: Arch Biochem Biophys. 2022 Aug 20;729:109378. doi: 10.1016/j.abb.2022.109378

Thermodynamics of iron, tetrahydrobiopterin, and phenylalanine binding to phenylalanine hydroxylase from Chromobacterium violaceum

Mingjie Li †, Bishnu P Subedi ‡, Paul F Fitzpatrick ‡,*, Joseph P Emerson †,*
PMCID: PMC10184773  NIHMSID: NIHMS1894794  PMID: 35995215

Abstract

Phenylalanine hydroxylase (PheH) is a pterin-dependent, mononuclear nonheme iron(II) oxygenase that uses the oxidative power of O2 to hydroxylate phenylalanine to form tyrosine. PheH is a member of a superfamily of O2-activating enzymes that utilizes a common metal binding motif: the 2-His-1-carboxylate facial triad. Like most members of this superfamily, binding of substrates to PheH results in a reorganization of its active site to allow O2 activation. Exploring the energetics of each step before O2 activation can provide mechanistic insight into the initial steps that support the highly specific O2 activation pathway carried out by this metalloenzyme. Here the thermal stability of PheH and its substrate complexes were investigated under an anaerobic environment by using differential scanning calorimetry. In context with known binding constants for PheH, a thermodynamic cycle associated with iron(II), tetrahydrobiopterin (BH4), and phenylalanine binding to the active site was generated, showing a distinctive cooperativity between the binding of BH4 and Phe. The addition of phenylalanine and BH4 to PheH·Fe increased the stability of this enzyme (ΔTm of 8.5 (± 0.7) ºC with an associated δΔH of 43.0 (± 2.9) kcal/mol). The thermodynamic data presented here gives insight into the complicated interactions between metal center, cofactor, and substrate, and how this interplay sets the stage for highly specific, oxidative C—H activation in this enzyme.

Keywords: Energetics, Thermal stability, Nonheme iron(II), Differential scanning calorimetry, cooperativity

1. Introduction

Mononuclear nonheme iron(II) oxygenases catalyze a wide range of chemistries by utilizing dioxygen to oxidize substrates [1,2]. Some of these enzymes require a co-factor or co-substrate to activate O2, where O2 in concert with a non-heme iron centers are converted to a high-valent iron intermediates, which have been shown to catalyze these targeted oxidations [3–7]. For example, the tetrahydrobiopterin (BH4)-dependent nonheme iron(II) dioxygenases use the co-substrate BH4 as a reducing agent to activate O2, leading to C—H activation and eventually hydroxylation of aromatic amino acids [8–10]. The BH4-dependent mononuclear nonheme iron(II) dioxygenases are all aromatic amino acid hydroxylases (AAHs) and include phenylalanine hydroxylase (PheH), tyrosine hydroxylase (TyrH), and tryptophan hydroxylase [10].

PheH hydroxylates the para position of the aromatic side-chain of phenylalanine (Phe) to produce tyrosine, which is essential for neurotransmitter function in higher organisms and serves as a building block in protein synthesis [11,12]. Dysfunctional PheH in humans can lead to the genetic disease phenylketonuria, which is linked to irreversible brain damage in children [13–15]. human PheH (HsPheH) is a homotetramer that exhibits complex cooperativity [16], with each monomer containing a catalytic domain, a regulatory domain, and a C-terminal domain. In comparison, the bacterial PheHs, such as that found in Chromobacterium violaceum (CvPheH), exist as a monomer with only a single catalytic domain. CvPheH has a similar structure to the catalytic domain of eukaryotic PheH [17], including conserved metal-binding and ligand-binding residues (Fig. 1A). This makes CvPheH a simple and well-behaved model of human PheH for targeted biophysical studies.

Figure 1.

Figure 1.

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Structural overlays of Chromobacterium violaceum PheH with the catalytic domain of human PheH. Protein backbone structures are shown as a solid tube for HsPheH and a red line ribbon for CvPheH. Residue numbers are show for both the Homo sapiens (or human) and the C. violaceum PheH systems in black and blue, respectively. Illustrated side-chains and other cofactors/substrates are shown with C atoms in gray for HsPheH and light gray for CvPheH. All overlays are generated by aligning the αC associated with the 2H-1E/D facial triad found in these proteins. (A) Overlay of catalytic domain of HsPheH (PDB: 1J8T) and CvPheH (PDB: 1LTV). The active site of PheH is formulated with an iron(II) center and highly conserved 2H-1E/D facial triad. (B) Overlay of catalytic domain of the PheH·Fe·BH4 complex from both the human protein (PDB: 1J8U) and C. violaceum system (PDB: 1LTZ), showcasing similar BH4 binding domains. (C) The active site pocket of HsPheH·Fe·BH4·substrate complex (PDB: 1KW0), where a non-activated substrate analogue (thienylalanine) was bound to the protein.

The metal site of CvPheH is made up of two histidine residues (His138 and His143) and a glutamate residue (Glu184) (Fig. 1A) and shows high structural homology to the active center of HsPheH. A similar binding mode is commonly found in nonheme iron(II) oxygenases, and has been termed the 2-His-1-carboxylate facial triad [18]. A range of structural and spectroscopic approaches has been demonstrated that binding of both amino acid and a tetrahydropterin (BH4) to an AAH results in changes in the active site that allow productive O2 binding and activation [19–25]. Structures are available of the catalytic domain of HsPheH that illustrate these changes (Fig. 1B and 1C). Although there is no structure of the phenylalanine complex, Fig 1C shows the structure of HsPheH in complex with iron(II), BH4, and a substrate analogue. For CvPheH, structures are available of the apo-enzyme and of the iron(III)-bound enzyme in the absence and presence of dihydrobiopterin. Fig. 1B shows the CvPheH·Fe bound to BH4 [17]. The individual rate constants for hydroxylation of Phe by CvPheH reaction have been determined [26]. A summary of the pre-O2 activation mechanism is shown in Figure 2, where these rate data can be used to calculate thermodynamic driving forces (i.e. K and ΔG values) associated with this system. Further analysis of the thermodynamics of iron(II) and substrate binding to PheH would provide further insight into how this system is switched on for O2 activation, one of the last major gaps in understanding this class of oxygenases.

Figure 2.

Figure 2.

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Precatalytic kinetic values and proposed mechanism associated with substrate and cofactor binding to PheH·Fe. Two different binding modes of PheH·Fe are described here, where the PheH’·Fe·BH4 is assigned as a non-productive binding mode. Data adapted from reference 26.

Previous studies have probed the thermodynamics of Phe and BH4 binding to PheH by using isothermal titration calorimetry (ITC) [27,28]. However, ITC is not well-suited for measuring thermodynamic parameters associated with low affinity binding equilibria, such as the binding of Phe to PheH·Fe [27]. Here we report a complementary thermodynamic study using differential scanning calorimetry (DSC) to explore the thermostability of a number of PheH complexes, where we have used the enthalpy terms measured from these data to determine the thermodynamic properties of iron(II), BH4, and Phe binding to the active site of PheH. A generalized thermodynamic cycle was generated for iron(II), BH4, and Phe binding to PheH that shows distinctive cooperativity between these binding events.

2. Materials and methods

2.1. Reagents and general procedures

All buffers, and metal salts were purchased at the highest grade available and used as received. Phe was obtained from Sigma-Aldrich and used as received. BH4 was purchased from Schircks Laboratories (Jona, Switzerland). Ultrapure water (18 MΩ) was generated using a Millipore Ultrapurification system and used to make all solutions described here.

2.2. PheH and PheH complexes solution preparation

Expression of CvPheH in Escherichia coli, purification of the enzyme, and preparation of the apo-enzyme were performed as previously described [29]. The enzyme concentration was determined by using UV-vis spectroscopy with a Ɛ280 = 56,505 M−1 cm−1 calculated with ProtParam (https://web.expasy.org/protparam/). Samples for DSC analysis were prepared in the following manner: PheH apoprotein was dialyzed for 18 h against 1 L 50 mM ammonium acetate, 100 mM NaCl, and 200 µM EDTA (pH 7.2) to remove residual metal ions. The PheH apoprotein stock was then dialyzed against 1 L of buffer containing 50 mM ammonium acetate overnight, with buffer changes after 3 and 12 h. Stock solutions of Phe, BH4, and Fe(NH4)2(SO4)2 were made in the final dialysis buffer. All solutions were purged with Ar to remove O2. To reconstitute the iron-containing enzyme 1 equivalent of Fe(NH4)2(SO4)2 was added anaerobically to 16 μM apoprotein. The PheH·Fe·BH4, PheH·Fe·Phe, and PheH·Fe·BH4·Phe complexes were generated by adding Phe and/or BH4 to PheH·Fe solutions to a final concentration of 1 mM of Phe and/or BH4. The excess Phe and BH4 pressure the associated equilibria to shift toward substate and cofactor bound forms of PheH.

2.3. Differential scanning calorimetry

DSC was used to follow the thermal unfolding of CvPheH complexes using a Calorimetry Sciences Nano-DSC constructed with gold capillary cells for reference and sample solutions. A glove bag was attached to the top of DSC, providing an anaerobic working environment to load both reference and sample cells. Argon gas was purged through the glove bag for at least 15 min before loading samples into cells, and was kept at positive pressure during sample loading. The pressure cap of the DSC instrument was then sealed and maintained at 3 atm before removing the Ar gas flow. A scan rate of 2 °C per min was used to monitor the thermal denaturation of 16-20 μM CvPheH complexes from 15 to 95 °C in both the forward and reverse directions. Raw data were analyzed as previously described [30], and specific examples of fits are shown in Appendix A. Supplementary data. The CPcalc software from the Calorimetry Sciences Corp. data analysis software package was used to fit the data. A final temperature scan of the forward direction was set as the baseline in each experiment. All data were fit to one or more two-state models, where the best fitting modes were decided by the goodness of fit values. The final data for the PheH complexes were averaged from 2-3 replicated runs, where error bars were obtained from the standard deviation of the mean.

3. Results

The change of enthalpy (ΔH) for a reaction is a fundamental thermodynamic quantity that can be determined using calorimetric techniques like DSC and isothermal titration calorimetry (ITC). Here, DSC was used to investigate the changes in enthalpy associated with iron(II) and substrate binding to PheH. Frequently, with nonheme iron(II) proteins, unfolding is rapidly followed by protein aggregation, which can impact the accuracy of data collected by DSC [31]. In this study, a scan rate of 2 °C per min was used to effectively quantify the thermal unfolding before aggregation events occurs. The heat capacity (Cp) curves of PheH and its complexes are shown in Figure 3 and the resulting thermodynamic data are found in Table 1.

Figure 3.

Figure 3.

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Heat capacity curves for the thermal denaturation of CvPheH species: PheH apoprotein (black), PheH·Fe (purple short dashes), PheH·Phe (orange), PheH·Fe·Phe (blue), PheH·Fe·BH4 (green), and the quaternary complex (red long dashes) consisting of PheH with bound iron(II), Phe and BH4. All samples were prepared in 50 mM ammonium acetate buffer, pH 7.2. The concentration of PheH complexes varies from 16 to 20 μM and concentrations of Phe and BH4 are 1 mM.

Table 1.

Thermodynamic data for the thermal denature of CvPheH and its enzyme·substrate complexes.a

Sample Tm1 ΔH1 Tm2 ΔH2 Tm3 ΔH3
° C kcal/mol ° C kcal/mol ° C kcal/mol
PheH 49.7 (± 0.1) 135.2 (± 1.6)
PheH·Phe 53.6 (± 0.1) 130.6 (± 0.2)
PheH·Fe 54.6 (± 0.1) 149.5 (± 1.5)
PheH·Fe·Phe 55.1 (± 0.2) 147.5 (± 0.5)
PheH·Fe·BH4 54.3 (± 0.5) 78.0 (± 1.5) 60.3 (± 0.3) 159.3 (± 0.5)
PheH·Fe
BH4·Phe		 54.9 (± 0.1) 149.0 (± 0.1) 60.5 (± 0.5) 159.5 (± 0.5) 63.1 (± 0.7) 192.5 (± 2.5)
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a

Data presented here represents averages of 3 separate DSC experiments. The error associated with these data are one standard deviation of the mean. This error is significantly higher than the error associated with any individual fit.

We first analyzed the binding of iron(II) to the apo-enzyme. The unfolding of both PheH apoprotein (Fig. 3 black trace) and the iron(II)-reconstituted enzyme (Fig. 3 purple dashed trace) were fit well to a single two-state model. The difference in the unfolding enthalpies of the PheH·Fe and PheH yielded −14.3 (± 2.2) kcal/mol for the enthalpic stabilization energy associated with iron(II) binding to the apo-enzyme (δΔHbinding = ΔHPheH - ΔHPheH·Fe).

The thermodynamics of phenylalanine binding were analyzed next. A large excess of Phe was mixed with the apo-PheH to produce the complex. The unfolding of PheH·Phe could be fit to a single two-state model (Fig. 3 orange and Table 1). The difference in the enthalpy changes between PheH·Phe and PheH is +4.6 (± 1.6) kcal/mol, and the substrate-enzyme complex unfolds at a higher Tm than the apo-enzyme. The DSC melting curve of the PheH·Fe·Phe complex was also fit to a single two-state model (Fig. 3, blue trace). The difference in the enthalpy changes between PheH·Fe·Phe and PheH·Fe is +2.0 (± 1.6) kcal/mol, and the PheH·Fe·Phe complex unfolds at a Tm about 2 °C higher than PheH·Fe. The enthalpic difference in the unfolding of the PheH·Fe·Phe and PheH·Phe complexes yields a δΔHbinding of −16.9 (± 0.6) kcal/mol for binding of iron(II) to PheH·Phe, very close to the value in the absence of Phe.

The thermodynamics of binding of the co-substrate BH4 to PheH·Fe are more complex. Fitting the data required a combination of two separate unfolding events, each described by a two-state model (Fig. 3 green trace and Table 1). The first event contained approximately 40% of the enzyme and occurred at a Tm of 54.3 (± 0.5) °C, while the second occurred at a Tm of 60.3 (± 0.3) °C. The presence of two species in solution is indicative that BH4 binding to PheH·Fe occurs in two different binding modes, with unfolding enthalpies of 78.0 (± 1.5) and 159.3 (± 0.5) kcal/mol for these processes, respectively. Previous analysis of the kinetic mechanism of this enzyme showed the formation of two PheH·Fe·BH4 complexes, where only one is a catalytically active pathway [26]. The higher Tm unfolding event was assigned as unfolding of the productive state PheH·Fe·BH4, while the lower Tm unfolding process is the unproductive PheH’·Fe·BH4 species.

Binding of both pterin and amino acid substrate to PheH·Fe and the closely related AAH TyrH·Fe from mammalian sources results in significant rearrangements of the active site, with the iron switching from hexacoordinate to pentacoordinate, the relative orientation of the substrates to the iron changing, and active site loops closing over the active site [20–25,32]. The quaternary complex was generated by mixing iron(II), Phe, and BH4 with CvPheH. The unfolding of this complex fit well to a combination of three separate two-state events (Fig. 3 long-dashed red trace and Table 1), indicating that there are a number of species in solution. The Tm and ΔH values for the first unfolding event match well with those for unfolding of PheH·Fe·Phe (Table 1); although PheH·Fe also has very similar Tm and ΔH values, this species was not observed in the unfolding of PheH·Fe·Phe. The first unfolding event contained approximately 9% of the enzyme sample. The values for the second event match well with those for unfolding of PheH·Fe·BH4, where the unfolding event occupied about 43% of the enzyme. The values for the third unfolding are significantly different from those of the other complexes, so that it can be assigned to unfolding of the quaternary species (approximately 50% of the enzyme). Thus, binding of Phe to PheH·Fe·BH4 to form the oxygen-reactive complex is associated with an enthalpy change of 33.2 (± 2.6) kcal/mol. Similarly, binding of BH4 to PheH·Fe·Phe is associated with an enthalpy change of 45.0 (± 2.6) kcal/mol.

4. Discussion

DSC was used here to measure the changes in enthalpy associated with the binding of iron(II), Phe, and BH4 to CvPheH by comparing the difference in globular stability of these enzyme and enzyme-substrate complexes. This has allowed us to generate a thermodynamic cycle for iron(II), BH4, and Phe binding (Fig. 4). Combining the previously reported K values [26] with these DSC-obtained enthalpies allows us to generate a full thermodynamic profile for the binding of BH4 and Phe to PheH·Fe (Table 2).

Figure 4.

Figure 4.

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Summary of thermodynamic data collected as part of the DSC study. δΔH terms associated with each equilibrium are shown in parentheses and have units of kcal/mol.

Table 2.

Thermodynamic profile of substrate/co-substrate binding to CvPheH species.

ΔGa (kcal/mol) ΔHb (kcal/mol) −TΔS (kcal/mol)
Phe binding to PheH·Fe −3.4 2.0 (±1.6) −5.4
BH4 binding to PheH·Fe −5.9 −9.8 (±1.6) 3.9
BH4 binding to PheH·Fe·Phe −6.2 −45.0 (±2.6) 38.8
Phe binding to PheH·Fe·BH4 −4.3 −33.2 (±2.6) 28.9
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a

Free energy terms calculated from values reported in reference 26.

b

Enthalpy terms from DSC data collected in this study.

Iron(II) binding to the 2-His-1-carboxylate facial triad in PheH is an enthalpically favorable reaction (Fig. 4). This is also the case for two other 2-His-1-carboxylate binding motif metalloenzymes: taurine/2-oxoglutarate dioxygenase (TauD, −12.7 kcal/mol) [30] and ethylene forming enzyme (EFE, −10.1 kcal/mol).[33] The approximately 4 °C increase in stability associated with iron(II) binding to PheH likely has to do with the partial bidentate binding mode of Glu184 to the iron(II) center in the PheH·Fe (Fig. 1A) compared to the monodentate binding mode of the carboxylic acid in other 2-His-1-carboxlate-facial triad containing enzymes.

The DSC analyses presented here show only evidence for a single Phe binding mode. This observation is consistent with the kinetics of CvPheH, which showed no evidence for a second binding mode of Phe [26]. These data are not consistent with the multiple binding modes for Phe described by Ronau et al. [28], which could be an artifact of the conditions where this system crystallized. Binding of Phe to PheH·Fe to form the PheH·Fe·Phe complex is entropically driven, with an unfavorable ΔH that is just outside of the error associated with this experiment. Binding of Phe and iron(II) are not enthalpically coupled, since the ΔH values for each are, within error, unaffected by the presence of the other. The energetics of Phe binding are consistent with the order of binding established by kinetic analyses, which showed that the preferred order of binding is BH4 before Phe, with binding of BH4 increasing the affinity for Phe by an order of magnitude [26].

The unfolding of PheH·Fe·BH4 reflects the presence of two BH4 complexes. This is consistent with the kinetic studies that show the existence of an unproductive BH4 complex [26]. The structure of the iron(III)-containing CvPheH with dihydrobiopterin shows that it binds to the active site through interactions with water molecules and the carboxylate of Asp139 [17]. A similar arrangement is seen in the BH4 complex of the iron(II)-containing human enzyme (Fig. 1C) [34], so this is likely a valid model for a BH4 complex of CvPheH. In the ternary complex of PheH with Fe2+, BH4, and an amino acid, there are fewer crystallographically defined water molecules present and the glutamate that corresponds to CvPheH Asp139 interacts directly with BH4 (Fig. 1) [20]. It is possible that the first structure is the unproductive complex, while the arrangement in the complex with both amino acid and BH4 reflects the productive complex. We have assigned as the productive species the one leading to the greater Tm and enthalpy values. The enthalpy of BH4 binding to form this species of −9.8 (± 1.6) kcal/mol is fairly consistent with a previously reported ITC-obtained ΔH value for BH4 binding (−6.4 kcal/mol), although that analysis did not distinguish between the two binding modes [28]. The increase in enthalpy could be associated with the ionic interactions between the BH4 and Asp139, as well as the tighter interactions with backbone residues. It also may result from the change in the metal ligand Glu184 to a true bidentate mode from partial bidentate binding [17]. The Gibbs free energy for this process is mainly driven by the enthalpic contribution with a small entropic penalty (−TΔS = 3.9 kcal/mol).

Formation of the complex of PheH·Fe with both Phe and BH4 from the single-substrate complexes results in large changes in enthalpy and unfavorable entropy changes (Table 2). BH4 binding is highly enthalpically favorable, where the enthalpy value is balanced by a large entropy term (−TΔS = 38.8 kcal/mol). This complex is the O2-activating form of the enzyme, which is eventually responsible forming the Fe4+=O hydroxylating intermediate responsible for C—H activation [3,4]. Structural and spectroscopic studies of the eukaryotic AAHs have shown that formation of this complex results in a change in the iron(II) from hexacoordinate to pentacoordinate, movement of the substrates relative to the iron, and closing of active site loops [19–25]. The large increase in entropy upon formation of this complex can be attributed to a general tightening up of the active site and the local protein structure. It is not obvious from the available structures where the sizable change in the enthalpy comes from that drives this process. The thermodynamic data in Table 2 are consistent with a model in which the formation of the complex with both substrates triggers the structural changes leading to a productive reaction with oxygen.

5. Conclusions

In summary, the present results provide a thermodynamic model associated with iron(II), BH4, and Phe binding to the active site of PheH. The thermodynamic data collected on CvPheH also provides some insight into the chemical steps before O2 activation by the pterin-dependent AAHs. Interestingly, there appears to be compensation between enthalpy and entropy terms reported within this study. This observation is highlighted by the enthalpically driven BH4 binding to PheH·Fe·Phe and Phe binding to PheH·Fe·BH4 equilibria, which both overcome significant entropic penalties to generate the O2 activating complex, PheH·Fe·BH4·Phe. The relationship between thermodynamic parameters measured here and dynamic structural changes in the active site of PheH are not well-characterized and out-side the scope of this study. However, one could envision the formation of the O2 activating species would be highly entropically unfavorable, where highly organized active sites could limit aberrant reactivity. A strategy that seems to be favored in a range of non-heme iron(II) oxygenases.

Supplementary Material

Supporting Information

Highlights.

  • The thermodynamics of iron(II), BH4, and substrate binding to CvPheH are reported.

  • The energetic profile for BH4 binding is dramatically influenced by binding of the amino acid substrate.

  • Two BH4 binding modes are observed for PheH·Fe.

Acknowledgements

We thank Kyle R. Carter for his time in reading and reviewing a penultimate draft of this article. This work was partially supported by the National Institutes of Health’s Center of Biomedical Research Excellence Program funded through the Center for Research Capacity Building (CRCB) in the National Institute for General Medical Sciences to J.P.E. (grant number P20GM103646). We also thank the National Science Foundation (CHE-1404129) and The Welch Foundation (AQ-1245) to P.F.F for partially supporting this effort.

Footnotes

Declaration of competing interest

The authors declare that they have no conflicts of interest with the contents of this article.

Data availability

Data will be made available on request.

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Supplementary Materials

Supporting Information
NIHMS1894794-supplement-Supporting_Information.docx (582.8KB, docx)

Data Availability Statement

Data will be made available on request.

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