Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2012 Mar 22.
Published in final edited form as: Biochemistry. 2011 Feb 16;50(11):1928–1933. doi: 10.1021/bi1019868

Evidence for a High Spin Fe(IV) Species in the Catalytic Cycle of a Bacterial Phenylalanine Hydroxylase†

Aram Joel Panay ‡, Michael Lee ‖, Carsten Krebs ‖,#, J Martin Bollinger Jr ‖,#, Paul F Fitzpatrick §,*
PMCID: PMC3059337  NIHMSID: NIHMS268344  PMID: 21261288

Abstract

Phenylalanine hydroxylase is a mononuclear non-heme iron protein that uses tetrahydropterin as the source of the two electrons needed to activate dioxygen for the hydroxylation of phenylalanine to tyrosine. Rapid-quench methods have been used to analyze the mechanism of a bacterial phenylalanine hydroxylase from Chromobacterium violaceum. Mössbauer spectra of samples prepared by freeze-quenching the reaction of the enzyme/57Fe(II)/phenylalanine/6-methyltetrahydropterin complex with O2 reveal the accumulation of an intermediate at short reaction times (20–100 ms). The Mössbauer parameters of the intermediate, δ = 0.28 mm/s and |ΔEQ| = 1.26 mm/s, suggest that it is a high-spin Fe(IV) complex similar to those that have previously been detected in the reactions of other mononuclear Fe(II) hydroxylases, including a tetrahydropterin-dependent tyrosine hydroxylase. Analysis of the tyrosine content of acid-quenched samples from similar reactions establishes that the Fe(IV) intermediate is kinetically competent to be the hydroxylating intermediate. Similar chemical-quench analysis of a reaction allowed to proceed for several turnovers shows a burst of tyrosine formation, consistent with rate-limiting product release. All three data sets can be modeled with a mechanism in which the enzyme-substrate complex reacts with oxygen to form an Fe(IV)=O intermediate with a rate constant of 19 mM−1s−1, the Fe(IV)=O hydroxylates phenylalanine with a rate constant of 42 s−1, and rate-limiting product release occurs at 6 s−1 at 5°C.

Phenylalanine hydroxylase (PheH)1 is a non-heme-iron-containing monooxygenase that catalyzes the hydroxylation of the aromatic amino acid phenylalanine to yield tyrosine (Scheme 1). The enzyme belongs to the family of pterin-dependent aromatic amino acid hydroxylases, along with tyrosine hydroxylase (TyrH) and tryptophan hydroxylase (1). PheH is found in organisms ranging from eukaryotes to prokaryotes. In mammals PheH is present in the liver, where it catalyzes the first and rate-limiting step in the metabolism of the phenylalanine acquired in the diet; mutations that hamper the function of PheH have been linked to the disease phenylketonuria (2). The structures of the catalytic domains of the three eukaryotic enzymes and of a bacterial PheH from Chromobacterium violaceum (CvPheH) reveal a common fold in which the active site iron is coordinated by two histidines and a glutamate (3–5). A similar arrangement of ligands is found in the α-ketoglutarate-dependent dioxygenases, in which the non-heme iron is typically coordinated by two histidines and an aspartate (6).

Scheme 1.

Scheme 1

Open in a new tab

Reaction catalyzed by phenylalanine hydroxylase showing (i) the generation of the co-product hydroxypterin and the Fe(IV)-oxo intermediate and (ii) hydroxylation of phenylalanine by the Fe(IV)-oxo species as individual steps (7).

The present understanding of the mechanism of the aromatic amino acid hydroxylases is based primarily on studies of the eukaryotic enzymes (7). In the absence of the substrates, the iron cofactor coordinates three water molecules, two histidines, and a monodentate glutamate. Binding of the amino acid substrate and the tetrahydropterin results in loss of two of the waters and conversion of the glutamate to bidentate binding, opening up a site for oxygen (8–10). Reaction of oxygen with the iron and the tetrahydropterin cosubstrate is proposed to yield an Fe(IV)=O (ferryl) intermediate and a 4a–hydroxypterin (Scheme 1) (7, 11, 12). Reaction of the amino acid with the ferryl intermediate by an electrophilic aromatic substitution mechanism would yield the hydroxylated amino acid (13–17), returning the iron to the Fe(II) form. In the case of the bacterial PheH, the structure of the ferric enzyme shows a bidentate glutamate (3), in contrast to the eukaryotic enzymes. Still, the reactivities of the hydroxylating intermediates in the eukaryotic and prokaryotic enzymes appear to be essentially identical on the basis of their similar abilities to catalyze aromatic, benzylic, and aliphatic hydroxylation (17–19).

Although it has been proposed that ferryl complexes are the substrate-hydroxylating intermediates in the reactions of all the aromatic amino acid hydroxylases, only in the case of TyrH has direct spectroscopic evidence for such a complex been provided (12). The Mössbauer spectrum of that enzyme trapped at short reaction times by the freeze-quench method showed that it contained iron in the +IV oxidation state; this was tentatively assigned as an Fe(IV)=O species on the basis of the similarity of its spectroscopic parameters to those of the extensively characterized high-spin Fe(IV)=O intermediate in taurine:α-ketoglutarate dioxygenase (TauD) (20–24). Here we describe the use of rapid-quench approaches to monitor the catalytic cycle of CvPheH. The results provide evidence that an Fe(IV) species, most likely a ferryl complex, is also the hydroxylating intermediate in this bacterial aromatic amino acid hydroxylase.

EXPERIMENTAL PROCEDURES

Materials

The E. coli strain BL21(DE3), used for protein expression, was from Novagen (Madison, WI). 6-Methyltetrahydropterin (6-MePH4) was from Schircks Laboratories (Jona, Switzerland). L-Phenylalanine, HEPES, and ferrous ammonium sulfate were from Sigma-Aldrich Chemical Co. (Milwaukee, WI). Dithiothreitol was from Inalco (Milan, Italy).

Enzyme expression and purification

CvPheH was expressed in E. coli and purified as previously described (17). The ferric iron present in the purified enzyme was removed by incubation with 5 mM EDTA and 5 mM nitrilotriacetate on ice for 2 hours followed by dialysis against 50 mM Hepes (pH 7.2) containing 50 mM NaCl.

Chemical-quench analyses

A solution of 100 mM HEPES buffer (pH 7.2) containing apo-CvPheH, 50 mM NaCl, and 5 mM phenylalanine was made anaerobic in a tonometer through 20 argon-vacuum cycles over a period of 1 hour. A stoichiometric amount of ferrous ammonium sulfate from a 1.5 mM stock was then added to the tonometer under argon. An aliquot of 6-MePH4 sufficient for a final concentration of 5 mM was placed in one arm of the tonometer. Additional argon-vacuum cycles were performed before mixing the 6-MePH4 with the CvPheH:Fe(II):Phe complex in the main body of the tonometer. A solution of 1.9 mM O2 was generated by bubbling oxygen into an ice-cold solution of 100 mM HEPES (pH 7.2) and 50 mM NaCl. Both oxygenated buffer and anaerobic enzyme solutions were loaded into a SFM-400/Q rapid-mixing instrument from Bio-Logic (Claix, France) calibrated using the reaction of 2,4-dinitrophenylacetate and hydroxide. The reactions were performed at 5 °C and quenched with an equal volume of 5 M HCl. The collected samples were chilled on ice until further analysis.

The precipitated protein was removed by centrifugation for 5 min at 13,700g. An aliquot of the sample was diluted in water and injected onto a Waters HPLC equipped with a Model 2475 fluorescence detector. Phenylalanine and tyrosine were separated using a Phenomenex C18 column using 10 mM sodium phosphate with 1% THF (pH 7.0) as the mobile phase (25). The intrinsic fluorescence of the amino acids was used for their detection with an excitation wavelength of 270 nm and an emission wavelength of 310 nm. The amount of tyrosine in each reaction was determined by comparison to a tyrosine standard curve.

Freeze-quench Mössbauer spectroscopy

A solution of 100 mM HEPES buffer (pH 7.2) containing 1.6 mM apo-CvPheH, 50 mM NaCl, and 5 mM phenylalanine was made anaerobic in a round bottom flask through several argon-vacuum cycles over a period of 1 h. This solution was placed in an MBraun anoxic chamber with a nitrogen atmosphere (Peabody, MA) where it was made 5 mM in 6-MePH4 by the addition of an aliquot from a stock solution prepared by dissolving the solid compound in anaerobic 100 mM HEPES buffer (pH 7.2) and 50 mM NaCl. A 50 mM stock solution of 57Fe(II) was made by dissolving 57Fe(0) in a volume of anaerobic 1 M H2SO4 sufficient to deliver 4 moles H+ per mole 57Fe(0). In order to avoid precipitating the enzyme, the stock solution of 57Fe(II) was mixed with 0.5 volume equivalents of anaerobic 1 M Tris·HCl (pH 7.6) before its addition to the CvPheH:Phe:6-MePH4 complex at a final concentration of 1.5 mM. An anaerobic control sample was prepared by taking an aliquot of the CvPheH:57Fe(II):Phe:6-MePH4 complex and transferring it to a Mössbauer cell. The cell was sealed in a plastic vial, removed from the glove box, frozen in liquid nitrogen, removed from the vial, and stored in liquid nitrogen.

The procedure for the preparation of freeze-quench Mössbauer samples has been described before (21). The CvPheH:57Fe(II):Phe:6-MePH4 complex was transferred to a rapid-quench syringe in the glove box. The syringe was removed from the glove box and positioned within the rapid quench instrument. A second syringe filled with oxygen-saturated (at 0 °C) buffer (1.9 mM O2) was also positioned within the instrument. These two solutions were mixed in a 1:1 ratio at 5 °C and allowed to flow through an aging line before being quenched by direct injection into 2-methylbutane at −150 °C. The total reaction time was calculated by adding 10 ms (the "quench time" that we have determined for a number of similar enzyme systems) to the time of transit through the aging line (26).

Mössbauer spectra were recorded on a spectrometer from WEB Research (Edina, MN) operating in the constant acceleration mode in a transmission geometry, equipped with a SVT-400 cryostat (Janis, Wilmington, MA). Spectra were collected at 4.2 K without externally applied magnetic field.

Data analyses

The program KaleidaGraph (Synergy Software, Reading, PA) was used to analyze single kinetic traces. Global analyses of the kinetic data were performed using the program KinTek Explorer Pro (KinTek Corp., Austin, TX) (27). Mössbauer data analysis was carried out using the program WMOSS (WEB Research); the details of the analysis have been described previously (21).

RESULTS

Rapid freeze-quench Mössbauer spectroscopy

Mössbauer spectroscopy was used to determine directly if an Fe(IV) species is an intermediate in the CvPheH reaction. An anaerobic solution containing concentrated CvPheH:57Fe(II), phenylalanine, and 6-MePH4 was reacted with an equal volume of an O2-saturated buffer solution, and the reaction was quenched by injection into 2-methylbutane at −150 °C. Mössbauer spectra of the samples quenched at various reaction times are shown in Figure 1. The spectrum of the reactant CvPheH:Fe(II):6-MePH4:Phe complex shows a broad quadrupole doublet with parameters typical of a high-spin Fe(II) (28). The asymmetry of the doublet suggests the presence of at least two different Fe(II) complexes that possibly arise from the conversion of a six-coordinate Fe(II) to a five-coordinate square-pyramidal Fe(II) site (10). The Mössbauer spectra of samples in which the reactant complex was exposed to O2 for 20 ms or 100 ms exhibit a distinct peak at ∼0.9 mm/s in addition to the contribution from the high-spin Fe(II) complexes. Because this peak has almost completely decayed after a reaction time of 400 ms, it is associated with a reaction intermediate. Analysis of the spectra of the 20-ms sample, in which the new peak has maximum intensity, and the anaerobic control reveals that the peak at ∼0.9 mm/s is the high-energy line of a quadrupole doublet with an isomer shift (δ) of 0.28 mm/s and quadrupole splitting parameter (|ΔEQ|) of 1.26 mm/s (Figure 2). These parameters are typical of high-spin Fe(IV) complexes (29). Deconvolution of the spectra reveals that the new iron species contributes (17 ± 3) % of the absorption at 20 ms and (13 ± 3) % at 100 ms. In the spectrum of the 400-ms sample, the upper limit of the new quadrupole doublet is 4% of the total intensity.

Figure 1.

Figure 1

Open in a new tab

4.2-K/zero-field Mössbauer spectra from the reaction of CvPheH:57Fe(II):Phe:6-MePH4 with O2 (concentrations of 0.8 mM CvPheH, 0.75 mM 57Fe(II), 2.5 mM 6-MePH4, 2.5 mM Phe and 0.95 mM O2 after mixing) in 100 mM HEPES, 50 mM NaCl (pH 7.2) at 5°C. The reaction times are indicated. The solid lines are quadrupole doublet simulations of the spectra of the Fe(IV) intermediate with the parameters quoted in the text.

Figure 2.

Figure 2

Open in a new tab

Determination of the Mössbauer parameters of the Fe(IV) intermediate observed in CvPheH. Top, 4.2-K/zero-field Mössbauer spectra of a sample of the CvPheH:57Fe(II):Phe:6-MePH4 complex before (solid line) and after exposure to O2 for 20 ms (vertical bars). Bottom, the difference spectrum (vertical bars) analyzed as a superposition of two quadrupole doublets (solid line) with the following parameters: δ(1) = 1.20 mm/s and ΔEQ(1) = 3.45 mm/s (−13% intensity; this component is assigned to the O2-reactive form of the enzyme) and δ(2) = 0.28 mm/s and ΔEQ(2) = 1.26 mm/s (+17% intensity; this component is assigned to the Fe(IV) intermediate).

Similar substoichiometric accumulation of the Fe(IV) intermediate was previously described for TyrH (12), TauD (21), and a prolyl-4-hydroxylase (30). For the latter two enzymes, it was possible to increase the fraction of the Fe(IV) by taking advantage of the large deuterium kinetic isotope effect of ∼50 for the C-H cleavage of the substrate (30–32). Such an approach is not practical for aromatic hydroxylation, because the deuterium kinetic isotope effects for this reaction are inverse at ∼0.9 for the aromatic amino acid hydroxylases (15–17). However, CvPheH will also catalyze the hydroxylation of cyclohexylalanine. This aliphatic hydroxylation reaction does exhibit a large deuterium kinetic isotope effect (∼15) (19), and amino acid hydroxylation is reported to be fully coupled to tetrahydropterin oxidation for this substrate (33). Similar rapid-quench Mössbauer experiments were carried out with deuterated cyclohexylalanine in an attempt to increase the amount of the Fe(IV) intermediate, but the intermediate could not be detected with this substrate. We attribute this to much slower formation of the Fe(IV) intermediate with this unnatural substrate (34). A similar reduced substrate triggering efficacy was also observed in the related Fe(II)- and α-ketoglutarate-dependent halogenases (35).

Chemical quench studies

The results shown in Figures 1 and 2 are consistent with the transient accumulation of an Fe(IV) species in the CvPheH reaction. To determine if the Fe(IV) intermediate is kinetically competent to be the hydroxylating intermediate, the time course of tyrosine formation was determined under the same conditions as were used to obtain the Mössbauer samples. An anaerobic solution of CvPheH:Fe(II) plus an excess of phenylalanine and 6-MePH4 was mixed with O2-containing buffer, and the reaction was quenched at times up to 300 ms by mixing with 5 M HCl. The amount of tyrosine produced at each time was then determined by HPLC. As shown in Figure 3, a stoichiometric amount of tyrosine was formed within the first 200–250 ms of the reaction. The data in Figure 3 could be fit by a single exponential increase in concentration with time to yield a rate constant of 10 ± 1.6 s−1 for the formation of tyrosine by CvPheH. This value is higher than the kcat value at 5 °C of ∼5 s−1, suggesting that formation of tyrosine is not rate-limiting for turnover by CvPheH. To more directly probe whether a slower subsequent step limits the reaction, the chemical quench analysis was repeated at a lower concentration of enzyme (100 µM), so that multiple turnovers were possible. The results are shown in Figure 4. There is a clear burst of tyrosine formation in the first 200 ms, followed by a slower linear phase. This result is consistent with hydroxylation being faster than a subsequent step, most likely the release of the product(s) from the active site.

Figure 3.

Figure 3

Open in a new tab

Time course for the formation of tyrosine during the reaction of CvPheH:Fe(II):Phe:6-MePH4 with O2 (concentrations of 0.8 mM CvPheH, 2.5 mM 6-MePH4, 2.5 mM Phe and 0.95 mM O2 after mixing) at 5 °C. The line is from a simulation of the data using the mechanism of Scheme 2 and values of k1 = 19 mM−1s−1, k2 = 42 s−1, and k3 = 6 s−1.

Figure 4.

Figure 4

Open in a new tab

Time course for the formation of tyrosine during the reaction of CvPheH:Fe(II):Phe:6-MePH4 with O2 (concentrations of 0.1 mM CvPheH, 2.5 mM 6-MePH4, 2.5 mM Phe and 0.95 mM O2 after mixing) at 5 °C. The line is from a simulation of the data using the mechanism of Scheme 2 and values of k1 = 19 mM−1s−1, k2 = 42 s−1, and k3 = 6 s−1.

Global analysis of the kinetics

A minimal kinetic mechanism consistent with the chemical reaction of PheH and the results presented here is shown in Scheme 2. In the experiments described here, the initial concentrations of phenylalanine and 6-MePH4 were in excess over the enzyme and 100 and 40 times the respective Km values, so that the binding of these two substrates to the enzyme in the first step of the reaction to form the ternary complex was assumed to be rapid and saturating.2 Similarly, in the absence of knowledge regarding which product is released first, the release of both products is incorporated into a single step.

Scheme 2.

Scheme 2

Open in a new tab

Minimal mechanism for the hydroxylation of phenylalanine by PheH.

The enzyme:phenylalanine:6-MePH4 complex reacts with oxygen to form the Fe(IV)=O species and the hydroxypterin; this reaction was treated as a single step in the absence of evidence for an intermediate. It was necessary to model this step explicitly as a second order reaction, because the complex was mixed with oxygen directly to start the reactions, and the concentrations of enzyme and oxygen were comparable in the experiments shown in Figures 1 and 3. The Fe(IV)=O reacts with the amino acid substrate to yield tyrosine, converting the iron back to the ferrous form. Finally, the products tyrosine and hydroxypterin dissociate from the enzyme. The results of all the analyses could be reasonably well simulated using this kinetic mechanism and a single set of rate constants, with k1 = 19 mM−1s−1, k2 = 42 s−1, and k3 = 6 s−1. The simulations are shown as the lines in Figures 3–5. A change in the value of any of the rate constants of >25% resulted in a visibly worse fit to the data.

Figure 5.

Figure 5

Open in a new tab

Time course for the Fe(IV) intermediate during turnover of CvPheH. The data are from Figure 1, and the line is from a simulation using the mechanism of Scheme 2 and values of k1 = 19 mM−1s−1, k2 = 42 s−1, and k3 = 6 s−1.

DISCUSSION

The results of the rapid-quench analyses presented here establish that an Fe(IV) species accumulates during the reaction of CvPheH. Its kinetics are consistent with the hypothesis that it is the oxygen donor for hydroxylation of the amino acid substrate. The Mössbauer spectral parameters for this Fe(IV) intermediate are similar to those previously reported for the Fe(IV) intermediates in TyrH (12) and in several Fe(II)- and α-ketoglutarate-dependent hydroxylases (21, 30) and halogenases (35, 38, 39). These all have high-spin (S = 2) Fe(IV) centers with similar spectroscopic parameters (isomer shifts of ∼ 0.3 mm/s, quadrupole splittings of −1.3 to −0.8 mm/s, axial zero-field splitting parameters of 10 – 15 cm−1, and small rhombic zero-field splitting parameters). For the extensively studied Fe(IV) intermediate from TauD, the presence of the ferryl group was conclusively shown by resonance Raman spectroscopy (20), X-ray absorption spectroscopy (22), and computational studies (40). The similar Mössbauer parameters of the Fe(IV) intermediates in CvPheH and TyrH and their demonstrated kinetic competency as the oxygen donors for aromatic hydroxylation support their assignment also as Fe(IV)=O species.

Previous studies of the reactions of CvPheH and the three eukaryotic aromatic amino acid hydroxylases have established that the iron centers in these enzymes have similar reactivities (17, 18). The similar Mössbauer parameters of the Fe(IV) intermediates in TyrH and CvPheH further confirm the similarities of the bacterial and eukaryotic enzymes. In contrast, the structures of the eukaryotic enzymes in the absence of both substrates show the glutamate ligand to the iron as monodentate (5, 41), while the structure of CvPheH shows a bidentate glutamate in the absence of substrates (3). A similar arrangement of a bidentate glutamate and two waters as iron ligands has been described for the PheH from Colwellia psychrerythraea 34H (42). For both TyrH and the eukaryotic PheH, the glutamate becomes a bidentate ligand only when both an amino acid substrate and a pterin are bound (10, 43, 44). The importance of this difference in the resting forms of the bacterial and eukaryotic enzymes is not clear. In all cases the iron site is hexacoordinate in the absence of substrate, with three waters as the remaining ligands in the eukaryotic enzymes and two waters in CvPheH. A productive reaction with oxygen by the pterin-dependent enzymes requires conversion of the iron to a pentacoordiante site, opening up a site for oxygen (10), so that the arrangement of ligands in the reactive ferryl enzyme is likely the same for both the eukaryotic and the prokaryotic enzymes. The difference in the type of interaction that the glutamate ligand forms with the iron in prokaryotic PheHs versus the eukaryotic enzymes in the absence of substrates may reflect relatively minor differences in the stability of the two interactions that can be affected by crystal packing forces. Alternatively, it may reflect tighter control of the reactivities of the iron in the larger eukaryotic enzymes in the absence of substrates.

The Fe(IV) intermediate in CvPheH does not accumulate to more that 20% of the total iron present. The nearly stoichiometric formation of tyrosine occurs over the same time period as the Fe(IV) is seen (Figure 3) and more rapidly than a single turnover, as product release limits turnover, establishing that at least 90% of the enzyme is active under the conditions of these analyses. Consequently, the less than stoichiometric levels of the Fe(IV) complex are not due to low enzyme activity at the high (∼1 mM) concentrations used. Instead, the substoichiometric amount can be attributed to the rate constant for the decay of the Fe(IV) being comparable to the rate constant for its formation (32, 45). This conclusion is supported by the global analysis, in that k1[O2] ∼ k2. A similar, more rapid decay of the Fe(IV) complex has previously been described for TyrH (12) and a prolyl-4-hydroxylase (30).

Previous analyses of kinetic isotope effects in the CvPheH reaction suggested that formation of the hydroxylated amino acid is followed by a step that is ∼9-fold slower (17). The present results support that conclusion and identify product release as the most likely slow step. The value of k3, the rate constant for product release obtained from the global analysis, is ∼7-fold less than the value of k2, the rate constant for tyrosine formation, consistent with the previous analysis. Analyses of the single turnover kinetics of TyrH (11) and tryptophan hydroxylase (46) have established that product release is slower than hydroxylation also for those eukaryotic enzymes.

The single turnover kinetics of the eukaryotic TyrH and the present kinetic analysis of the CvPheH reaction make possible a comparison of the intrinsic rate constants for eukaryotic and prokaryotic amino acid hydroxylases. With TyrH, the rate constants for formation of the Fe(IV)=O intermediate and for hydroxylation of tyrosine (12) are within error of the values of k1 and k2 for CvPheH. This is consistent with similar reactivities of the iron center in the eukaryotic and prokaryotic enzymes, in support of a similar conclusion based on kinetic isotope effects and the contribution of tunneling to hydrogen abstraction by these enzymes (18).

In summary, the present study provides spectroscopic evidence for an Fe(IV) intermediate in the catalytic cycle of a bacterial aromatic amino acid hydroxylase with characteristics similar to those observed in the eukaryotic TyrH and other mononuclear non-heme-iron enzymes with facial 2-His 1-carboxylate triads (12, 21, 30). The similar spectroscopic properties and abilities to catalyze both aromatic and aliphatic hydroxylation suggest that the pterin-dependent and α-ketoglutarate dependent oxygenases represent two mechanisms for forming a common hydroxylating intermediate using a mononuclear iron center.

Footnotes

†

This work was supported by in parts by grants from the NIH (R01 GM047291 to PFF and R01 GM 69657 to JMB and CK), The Welch Foundation (AQ-1245 to PFF), and the National Science Foundation (CHE-724084 and MCB-642058 to JMB and CK).

1

Abbreviations: PheH, phenylalanine hydroxylase; CvPheH, Chromobacterium violaceum phenylalanine hydroxylase; TyrH, tyrosine hydroxylase; TauD, taurine:α-ketoglutarate dioxygenase; 6-MePH4, 6-methyltetrahydropterin.

2

While phenylalanine and the tetrahydropterin necessarily bind in separate steps, a mechanism involving two rapid binding steps is kinetically indistinguishable from one with a single rapid step. Moreover, there is some discrepancy as to the order of binding of substrates to CvPheH (36, 37); the mechanism of Scheme 2 is not meant to imply any order of binding of phenylalanine and the tetrahydropterin.

REFERENCES

  • 1.Fitzpatrick PF. The aromatic amino acid hydroxylases. In: Purich DL, editor. Adv. Enzymol. Relat. Areas Mol. Biol. John Wiley & Sons, Inc.; 2000. pp. 235–294. [DOI] [PubMed] [Google Scholar]
  • 2.Eisensmith RC, Woo SLC. Phenylketonuria and the phenylalanine hydroxylase gene. Mol. Biol. Med. 1991;8:3–18. [PubMed] [Google Scholar]
  • 3.Erlandsen H, Kim JY, Patch MG, Han A, Volner A, Abu-Omar MM, Stevens RC. Structural comparison of bacterial and human iron-dependent phenylalanine hydroxylases: similar fold, different stability and reaction rates. J. Mol. Biol. 2002;320:645–661. doi: 10.1016/s0022-2836(02)00496-5. [DOI] [PubMed] [Google Scholar]
  • 4.Wang L, Erlandsen H, Haavik J, Knappskog PM, Stevens RC. Three-dimensional structure of human tryptophan hydroxylase and its implications for the biosynthesis of the neurotransmitters serotonin and melatonin. Biochemistry. 2002;41:12569–12574. doi: 10.1021/bi026561f. [DOI] [PubMed] [Google Scholar]
  • 5.Goodwill KE, Sabatier C, Marks C, Raag R, Fitzpatrick PF, Stevens RC. Crystal structure of tyrosine hydroxylase at 2.3 Å and its implications for inherited neurodegenerative diseases. Nat. Struct. Biol. 1997;4:578–585. doi: 10.1038/nsb0797-578. [DOI] [PubMed] [Google Scholar]
  • 6.Koehntop KD, Emerson JP, Que L., Jr The 2-His-1-carboxylate facial triad: a versatile platform for dioxygen activation by mononuclear non-heme iron(II) enzymes. J. Biol. Inorg. Chem. 2005;10:87–93. doi: 10.1007/s00775-005-0624-x. [DOI] [PubMed] [Google Scholar]
  • 7.Fitzpatrick PF. Mechanism of aromatic amino acid hydroxylation. Biochemistry. 2003;42:14083–14091. doi: 10.1021/bi035656u. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Loeb KE, Westre TE, Kappock TJ, Mitic N, Glasfeld E, Caradonna JP, Hedman B, Hodgson KO, Solomon EI. Spectroscopic characterization of the catalytically competent ferrous site of the resting, activated, and substrate-bound forms of phenylalanine hydroxylase. J. Am. Chem. Soc. 1997;119:1901–1915. [Google Scholar]
  • 9.Andersen OA, Stokka AJ, Flatmark T, Hough E. 2.0 Å Resolution crystal structures of the ternary complexes of human phenylalanine hydroxylase catalytic domain with tetrahydrobiopterin and 3-(2-thienyl)-L-alanine or L-norleucine: substrate specificity and molecular motions related to substrate binding. J. Mol. Biol. 2003;333:747–757. doi: 10.1016/j.jmb.2003.09.004. [DOI] [PubMed] [Google Scholar]
  • 10.Chow MS, Eser BE, Wilson SA, Hodgson KO, Hedman B, Fitzpatrick PF, Solomon EI. Spectroscopy and kinetics of wild-type and mutant tyrosine hydroxylase: Mechanistic insight into O2 activation. J. Amer. Chem Soc. 2009;131:7685–7698. doi: 10.1021/ja810080c. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Eser BE, Fitzpatrick PF. Measurement of intrinsic rate constants in the tyrosine hydroxylase reaction. Biochemistry. 2010;49:645–652. doi: 10.1021/bi901874e. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Eser BE, Barr EW, Frantom PA, Saleh L, Bollinger JM, Jr, Krebs C, Fitzpatrick PF. Direct spectroscopic evidence for a high-spin Fe(IV) intermediate in tyrosine hydroxylase. J. Am. Chem. Soc. 2007;129:11334–11335. doi: 10.1021/ja074446s. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Hillas PJ, Fitzpatrick PF. A mechanism for hydroxylation by tyrosine hydroxylase based on partitioning of substituted phenylalanines. Biochemistry. 1996;35:6969–6975. doi: 10.1021/bi9606861. [DOI] [PubMed] [Google Scholar]
  • 14.Moran GR, Derecskei-Kovacs A, Hillas PJ, Fitzpatrick PF. On the catalytic mechanism of tryptophan hydroxylase. J. Am. Chem. Soc. 2000;122:4535–4541. [Google Scholar]
  • 15.Frantom PA, Fitzpatrick PF. Uncoupled forms of tyrosine hydroxylase unmask kinetic isotope effects on chemical steps. J. Am. Chem. Soc. 2003;125:16190–16191. doi: 10.1021/ja0383165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Pavon JA, Fitzpatrick PF. Insights into the catalytic mechanisms of phenylalanine and tryptophan hydroxylase from kinetic isotope effects on aromatic hydroxylation. Biochemistry. 2006;45:11030–11037. doi: 10.1021/bi0607554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Panay AJ, Fitzpatrick PF. Kinetic isotope effects on aromatic and benzylic hydroxylation by Chromobacterium violaceum phenylalanine hydroxylase as probes of chemical mechanism and reactivity. Biochemistry. 2008;47:11118–11124. doi: 10.1021/bi801295w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Pavon JA, Fitzpatrick PF. Intrinsic isotope effects on benzylic hydroxylation by the aromatic amino acid hydroxylases: evidence for hydrogen tunneling, coupled motion, and similar reactivities. J. Am. Chem. Soc. 2005;127:16414–16415. doi: 10.1021/ja0562651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Panay AJ, Fitzpatrick PF. Measurement of the intramolecular isotope effect on aliphatic hydroxylation by Chromobacterium violaceum phenylalanine hydroxylase. J. Am. Chem. Soc. 2010;132:5584–5585. doi: 10.1021/ja101563t. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Proshlyakov DA, Henshaw TF, Moterosso GR, Ryle MJ, Hausinger RP. Direct detection of oxygen intermediates in the non-heme Fe enzyme taurine/alpha-ketoglutarate dioxygenase. J Am Chem Soc. 2004;126:1022–1023. doi: 10.1021/ja039113j. [DOI] [PubMed] [Google Scholar]
  • 21.Price JC, Barr EW, Tirupati B, Bollinger JM, Jr, Krebs C. The first direct characterization of a high-valent iron intermediate in the reaction of an alpha-ketoglutarate-dependent dioxygenase: a high-spin Fe(IV) complex in taurine/alpha-ketoglutarate dioxygenase (TauD) from Escherichia coli. Biochemistry. 2003;42:7497–7508. doi: 10.1021/bi030011f. [DOI] [PubMed] [Google Scholar]
  • 22.Riggs-Gelasco PJ, Price JC, Guyer RB, Brehm JH, Barr EW, Bollinger JM, Jr, Krebs C. EXAFS spectroscopic evidence for an Fe=O unit in the Fe(IV) intermediate observed during oxygen activation by taurine:alpha-ketoglutarate dioxygenase. J. Am. Chem. Soc. 2004;126:8108–8109. doi: 10.1021/ja048255q. [DOI] [PubMed] [Google Scholar]
  • 23.Krebs C, Price JC, Baldwin J, Saleh L, Green MT, Bollinger JM., Jr Rapid freeze-quench Mossbauer spectroscopy: monitoring changes of an iron-containing site during a biochemical reaction. Inorg. Chem. 2005;44:742–757. doi: 10.1021/ic048523l. [DOI] [PubMed] [Google Scholar]
  • 24.Bollinger JM, Jr, Price JC, Hoffart LM, Barr EW, Krebs C. Mechanism of taurine: α-ketoglutarate dioxygenase (TauD) from Escherichia coli. Eur. J. Inorg. Chem. 2005;2005:4245–4254. doi: 10.1021/bi050227c. [DOI] [PubMed] [Google Scholar]
  • 25.Pavon JA, Fitzpatrick PF. Demonstration of a peroxide shunt in the tetrahydropterin-dependent aromatic amino acid monooxygenases. J. Am. Chem. Soc. 2009;131:4582–4583. doi: 10.1021/ja900128m. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Baldwin J, Krebs C, Ley BA, Edmondson DE, Huynh BH, Bollinger JM., Jr Mechanism of rapid electron transfer during oxygen activation in the R2 subunit of Escherichia coli ribonucleotide reductase. 1. Evidence for a transient tryptophan radical. J. Am. Chem. Soc. 2000;122:12195–12206. [Google Scholar]
  • 27.Johnson KA, Simpson ZB, Blom T. Global Kinetic Explorer: A new computer program for dynamic simulation and fitting of kinetic data. Anal. Biochem. 2009;387:20–29. doi: 10.1016/j.ab.2008.12.024. [DOI] [PubMed] [Google Scholar]
  • 28.Münck E. Aspects of 57Fe Mossbauer spectroscopy. In: Que L Jr, editor. Physical Methods in Bioinorganic Chemistry. Sausalito: University Science Books; 2000. pp. 287–319. [Google Scholar]
  • 29.Neese F. Theoretical spectroscopy of model-nonheme [Fe(IV)OL5]2+ complexes in their lowest triplet and quintet states using multireference ab initio and density functional theory methods. J. Inorg. Biochem. 2006;100:716–726. doi: 10.1016/j.jinorgbio.2006.01.020. [DOI] [PubMed] [Google Scholar]
  • 30.Hoffart LM, Barr EW, Guyer RB, Bollinger JM, Jr, Krebs C. Direct spectroscopic detection of a C-H-cleaving high-spin Fe(IV) complex in a prolyl-4-hydroxylase. Proc. Natl. Acad. Sci. USA. 2006;103:14738–14743. doi: 10.1073/pnas.0604005103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Price JC, Barr EW, Glass TE, Krebs C, Bollinger JM., Jr Evidence for hydrogen abstraction from C1 of taurine by the high-spin Fe(IV) intermediate detected during oxygen activation by taurine:α-ketoglutarate dioxygenase (TauD) J. Am. Chem. Soc. 2003;125:13008–13009. doi: 10.1021/ja037400h. [DOI] [PubMed] [Google Scholar]
  • 32.Bollinger JM, Jr, Krebs C. Stalking intermediates in oxygen activation by iron enzymes: motivation and method. J. Inorg. Biochem. 2006;100:586–605. doi: 10.1016/j.jinorgbio.2006.01.022. [DOI] [PubMed] [Google Scholar]
  • 33.Carr RT, Balasubramanian S, Hawkins PCD, Benkovic SJ. Mechanism of metal-independent hydroxylation by Chromobacterium violaceum phenylalanine hydroxylase. Biochemistry. 1995;34:7525–7532. doi: 10.1021/bi00022a028. [DOI] [PubMed] [Google Scholar]
  • 34.Panay AJ. PhD dissertation. College Station: Texas A&M University; 2010. Mechanistic, Structural and Functional Studies on Phenylalanine Hydroxylase from Chromobacterium violaceum, 139 pages. [Google Scholar]
  • 35.Matthews ML, Krest CM, Barr EW, Vaillancourt FH, Walsh CT, Green MT, Krebs C, Bollinger JM., Jr Substrate-triggered formation and remarkable stability of the C-H bond-cleaving chloroferryl intermediate in the aliphatic halogenase, SyrB2. Biochemistry. 2009;48:4331–4343. doi: 10.1021/bi900109z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Pember SO, Johnson KA, Villafranca JJ, Benkovic SJ. Mechanistic studies on phenylalanine hydroxylase from Chromobacterium violaceum. Evidence for the formation of an enzyme-oxygen complex. Biochemistry. 1989;28:2124–2130. doi: 10.1021/bi00431a024. [DOI] [PubMed] [Google Scholar]
  • 37.Volner A, Zoidakis J, Abu-Omar MM. Order of substrate binding in bacterial phenylalanine hydroxylase and its mechanistic implication for pterin-dependent oxygenases. J. Biol. Inorg. Chem. 2003;8:121–128. doi: 10.1007/s00775-002-0395-6. [DOI] [PubMed] [Google Scholar]
  • 38.Galonic DP, Barr EW, Walsh CT, Bollinger JM, Jr, Krebs C. Two interconverting Fe(IV) intermediates in aliphatic chlorination by the halogenase CytC3. Nature Chem. Biol. 2007;3:113–116. doi: 10.1038/nchembio856. [DOI] [PubMed] [Google Scholar]
  • 39.Galonic Fujimori D, Barr EW, Matthews ML, Koch GM, Yonce JR, Walsh CT, Bollinger JM, Jr, Krebs C, Riggs-Gelasco PJ. Spectroscopic evidence for a high-spin Br-Fe(IV)-oxo intermediate in the α-ketoglutarate-dependent halogenase CytC3 from Streptomyces. J. Am. Chem. Soc. 2007;129:13408–13409. doi: 10.1021/ja076454e. [DOI] [PubMed] [Google Scholar]
  • 40.Sinnecker S, Svensen N, Barr EW, Ye S, Bollinger JM, Jr, Neese F, Krebs C. Spectroscopic and computational evaluation of the structure of the high-spin Fe(IV)-oxo intermediates in taurine: α-ketoglutarate dioxygenase from Escherichia coli and its His99Ala ligand variant. J. Am. Chem. Soc. 2007;129:6168–6179. doi: 10.1021/ja067899q. [DOI] [PubMed] [Google Scholar]
  • 41.Andersen OA, Flatmark T, Hough E. High resolution crystal structures of the catalytic domain of human phenylalanine hydroxylase in its catalytically active Fe(II) form and binary complex with tetrahydrobiopterin. J. Mol. Biol. 2001;314:279–291. doi: 10.1006/jmbi.2001.5061. [DOI] [PubMed] [Google Scholar]
  • 42.Leiros HKS, Pey AL, Innselset M, Moe E, Leiros I, Steen IH, Martinez A. Structure of phenylalanine hydroxylase from Colwellia psychrerythraea 34H, a monomeric cold active enzyme with local flexibility around the active site and high overall stability. J. Biol. Chem. 2007;282:21973–21986. doi: 10.1074/jbc.M610174200. [DOI] [PubMed] [Google Scholar]
  • 43.Wasinger EC, Mitic N, Hedman B, Caradonna J, Solomon EI, Hodgson KO. X-Ray absorption spectroscopic investigation of the resting ferrous and cosubstrate-bound active sites of phenylalanine hydroxylase. Biochemistry. 2002;41:6211–6217. doi: 10.1021/bi0121510. [DOI] [PubMed] [Google Scholar]
  • 44.Andersen OA, Flatmark T, Hough E. Crystal structure of the ternary complex of the catalytic domain of human phenylalanine hydroxylase with tetrahydrobiopterin and 3-(2-thienyl)-L-alanine, and its implications for the mechanism of catalysis and substrate activation. J. Mol. Biol. 2002;320:1095–1108. doi: 10.1016/s0022-2836(02)00560-0. [DOI] [PubMed] [Google Scholar]
  • 45.Fierke CA, Hammes GG, Daniel LP. Transient kinetic approaches to enzyme mechanisms. Methods Enzymol. Volume. 1995;249:3–37. doi: 10.1016/0076-6879(95)49029-9. [DOI] [PubMed] [Google Scholar]
  • 46.Pavon JA, Eser BE, Huynh MT, Fitzpatrick PF. Single turnover kinetics of tryptophan hydroxylase: Evidence for a new intermediate in the reaction of the aromatic amino acid hydroxylases. Biochemistry. 2010;49:7563–7571. doi: 10.1021/bi100744r. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES