Rat Liver Phenylalanine Hydroxylase, an Iron Enzyme

Abstract Phenylalanine hydroxylase that is essentially pure contains 1 to 2 moles of iron per mole of enzyme (assuming a molecular weight of 100,000). Electron spin resonance (ESR) studies have shown that the iron is present in the high spin ferric form. The addition of substrates (i.e. phenylalanine and dimethyltetrahydropterin) causes the signal for this form to disappear. The iron, 50 to 80%, can be removed by treatment of sucrose gradient purified enzyme with o-phenanthroline and cysteine; the loss of iron leads to the loss of enzymatic activity. The enzyme activity is restored by FeCl2 with half-maximum restoration achieved at 5 x 10-5 m FeCl2. Mercurous, nickelous, cobalt, manganese, cupric, chromium, cadmium, and zinc ions are ineffective in restoring activity to the apoenzyme (o-phenanthroline-treated enzyme). As judged by gel electrophoresis, the apoenzyme has the same charge and molecular weight as the holoenzyme. The apoenzyme, however, is much less stabile at 25° than the holoenzyme; FeCl2 restored the original stability to the apoenzyme. Furthermore, the apoenzyme has only three free cysteines per 50,000 molecular weight subunit, whereas the holoenzyme has five cysteines per subunit. The amino acid composition of the enzyme was also determined.


Phenylalanine
hydroxylase that is essentially pure contains 1 to 2 moles of iron per mole of enzyme (assuming a molecular weight of 100,000). Electron spin resonance (ESR) studies have shown that the iron is present in the high spin ferric form.
The addition of substrates (i.e. phenylalanine and dimethyltetrahydropterin) causes the signal for this form to disappear.
The iron, 50 to 80%, can be removed by treatment of sucrose gradient purified enzyme with o-phenanthroline and cysteine ; the loss of iron leads to the loss of enzymatic activity. The enzyme activity is restored by FeClz with half-maximum restoration achieved at 5 X 10e5 M FeCl*.
Mercurous, nickelous, cobalt, manganese, cupric, chromium, cadmium, and zinc ions are ineffective in restoring activity to the apoenzyme (o-phenanthroline-treated enzyme). As judged by gel electrophoresis, the apoenzyme has the same charge and molecular weight as the holoenzyme. The apoenzyme, however, is much less stabile at 25" than the holoenzyme; FeCl, restored the original stability to the apoenzyme.
Furthermore, the apoenzyme has only three free cysteines per 50,000 molecular weight subunit, whereas the holoenzyme has five cysteines per subunit. The amino acid composition of the enzyme was also determined.
Early evidence indicated that a metal might be involved in the conversion of phenylalanine to tyrosine by rat liver phenylalanine hydroxylase (1). Compounds capable of chelating metals drastically inhibited the hydroxylase.
Since it is now possible to obtain 95% pure phenylalanine hydroxylase (a), the metal content of this purified enzyme was analyzed.
We found that the enzyme contains iron and that the iron is essential for the hydroxylase activity. MATERIALS TPNH and crystalline catalase were obtained from Boehringer Mannheim Corp. Dihydropteridine reduct,ase was obtained from sheep liver and purified through the calcium phosphate gel step (3). Tetrahydrobiopterin was a gift from Dr. Long, Hoffmann-LaRoche.
Bathophenanthroline was obtained from Frederick Smith Chemical Co., Columbus, Ohio. Glucose dehydrogenase was prepared from beef liver (4) as prer-iousljr described.

ME'IHOI~S
Phenylalanine hydroxylase was assayed in a reaction mixture containing the following components (in micromoles) in a final volume of 1.0 ml: potassium phosphate, p1-I 6.8, 100; L-phcnylalanine, 2.0; TPNf, 0.25; glucose, 250; glucose dehydrogenase, 300 mg; dihydropteridine reductase, 100 pg; tetrahydropterin and hydroxylase, in concentrations indicated in the legends. The reaction mixture was routinely incubated 30 min at 25" and stopped by addition of 1 ml of 1 N perchloric acid. Tyrosine produced was determined fluorometrically by the nitrosonaphthol method (5). The enzyme was also assayed spectrophotometritally by measurement of the phenylalanine-dependent oxidation of TPNH (1).
To prepare the protein for iron determinations, it was wetashed with sulfuric and nitric acid, followed by perchloric acid. The iron content of the ashed protein was determined colorimet,rically; the complex of reduced iron with bat.hophenanthroline, was extracted into isoamyl alcohol and its absorbance at 535 nm was determined (6). For copper analysis, the enzyme n-as ashed in the same manner; the copper-bathocuproine complex was formed and its absorbance at 480 nm was determined (6). Protein was determined by the method of Lowry (7).
Sucrose gradients were made with a Beckman sucrose gradient former.
The light syringes contained 0.01 M Tris-HCl, pH 8.0, and the heavy syringes contained 26% (w/v) sucrose buffered with 0.01 M Tris-HCl, pH 8.0. Centrifugation was performed at 2" in a Beckman L2-65B ultracentrifuge in a SW 65 rotor. The enzyme samples for amino acid analysis were peak fractions from t,he G-200 Sephadex step (2), which were further purified by sucrose gradient centrifugation.
The peak tubes from the sucrose gradient were freed of salts and sucrose by pressure dialysis and the protein was lyophilized in a combustion tube. The samples were hydrolyzed for the indicated time with 5.7 N constant boiling HCl at 105" under a vacuum.
The amino acid analyses were performed with a Hitachi Perkin-Elmer KLA-3B amino acid analyzer equipped with an autosampler utilizing a zinc-ligand system. To determine the total cyst.ine and cysteine, the enzyme was oxidized with performic acid (8). The performic acid was formed by mixing hydrogen peroxide (30%) and formic acid (88%) in a 1: 10 ratio.
The reaction was carried out for 16 hours at 0" and excess performic acid was removed by lyophilization.
The rate for the cont,rol was 4.0 nmoles of TPNH oxidized per min. The tryptophan content of phenylalanine hydroxylase was determined from the absorbance of the protein at 280 nm and 294 nm in 0.1 N NaOH and from the tyrosine content determined by amino acid analysis (10). Tryptophan was also measured calorimetrically by treatment of the intact protein with the reagent of Spies and Chambers (11).
Electron spin resonance studies were carried out with a Varian V-4500 X band spectrometer.
A g-inch magnet was used. The field modulation was 100 kHz and was regulated by a Fieldial. The microwave frequency was measured by a cavity wave meter. The applied magnetic field, II, was calibrated by using a proton resonance line. The sample was placed in a quartz tube having an outer diameter of 5 mm. The sample was kept in liquid nitrogen during the measurements and was placed in a Joel cylindrical cavity. The modulation amplitude was 10 gauss. Curves of the first derivative were recorded at a microwave setting of 2.45 decibels.
No greater than 20% saturation of the g = 4.23 signal occurred at this setting and the degree of saturation was constant for all conditions studied.

Metal-chelating
Xfudies-Metal-chelating studies provided preliminary evidence suggesting that a metal might be involved in the reaction catalyzed by phenylalanine hydroxylase. As shown in Table I, a variety of chelators caused marked inhibition of phenylalanine hydroxylase. It is of interest to note that the degree of inhibition for a given concentration of o-phenanthroline was independent of the structure of the reduced pterin used as cofactor and that the nonchelating analogue of o-phenanthroline, m-phenanthroline, was not inhibitory. These latter results suggest that, at least in the case of o-phenanthroline, the inhibition is probably due to metal chelation and not due to an inhibition of cofactor binding.
The oxalate, and tiron suggest that hydrophobic interactions may aid the chelation process.
These studies showed that some component of the hydroxylating system is inhibited by certain metal chelators. Since the assay used included an enzymatic tetrahydropterin-regenerating system (i.e. TPNH and dihydropteridine reductase), it was important to show that it was the hydroxylase, rather than the reductase, that was being inhibited.
To prove this point, the hydroxylase was assayed in the absence of the enzymatic-regenerating system by direct measurement of the phenylalaninedependent oxidation of the tetrahydropterin (12). It was found that under these conditions, the extent of inhibition by o-phenanthroline and diethyldithiocarbamate was exactly the same as was previously found in the coupled assay system. Therefore, the inhibition by chelators illustrated in Table I is most likely due to inhibition of the hydroxylase. Metal Analysis-Since the foregoing evidence indicated that phenylalanine hydroxylase is a metallo-enzyme, and earlier evidence indicated that the metal might be iron (I), the purified enzyme was analyzed for iron. Our method for purifying the hydroxylase yields enzyme which is 90% pure by several criteria (2). Sucrose gradient centrifugation of this material yielded a single peak of protein and activity.
Iron analyses of fractions from the sucrose gradient demonstrated that a peak of iron corresponded with the activity and protein peak (Fig. 1). The spectrum of the complex of the bathophenanthroline and the metal from the enzyme was nearly identical to the complex of bathophenanthroline and iron (Fig. 2). Five determinations of the iron content of this sucrose gradient purified enzyme gave a mean of 1.4 (range 0.7 to 2.0) g atoms of iron per mole of 100,000'  2. Comparison of the spectrum of the bathophenanthroline-iron complex and of the complex of bathophenanthroline and the metal from phenylalanine hydroxylase. Phenylalanine hydroxylase, 1.35 mg, was purified by sucrose gradient centrifugation, ashed, and reacted with bathophenanthroline.
The blank was the o-phenanthroline reaction with an aliquot of dialysate from enzyme equal to the volume of enzyme analyzed. The hydroxylase (70% pure) was previously incubated in the complete reaction mixture without phenylalanine and tetrahydrobiopterin for 8 min and the reaction was started by addition of phenylalanine (1 mM final concentration) and tetrahydrobiopterin (0.01 mM). FeClz (2 mM) and DTT (1 mM) were added at indicated times. Consistent with this postulate is the finding that the activity of the hydroxylase can be increased by incubation of the enzyme with FeClz and dithiothreitol (Table II).
Under these conditions, FeClz activated the hydroxylase 50 to 100%.
One preparation of hydroxylase which had 1.1 g atoms of iron per 100,000 molecular weight, was activated 80% by treatment with FeClz and DTT.2 This was the degree of activation expected if the fully active enzyme contained 2.0 g atoms of iron per 100,000. This amount of iron would correspond to 1 iron per 50,000 molecular weight subunit of the enzyme.
It is of interest that when the FeCL and DTT were added at the beginning of the reaction in the presence of the substrates, no activation was seen. Furthermore, the added iron does not form a tight complex with the enzyme because precipitation of the enzyme-iron mixture with ammonium sulfate librium centrifugation studies gave a molecular u-eight of 88,000 (unpublished results).
In this paper we have averaged these different estimates to give the molecular weight of 100,000 for the hydroxylase dimer, and 50,000 for the monomer. led to the recovery of enzyme with the same low act'ivity as the untreated enzyme.
A second peak of iron appeared at the bottom of the gradient when the 90% pure enzyme was centrifuged for a shorter period (60,000 rpm for 1 hour).
The faster migrating iron peak had a sedimentation coefficient of 70 S aud migrated nearly the same as ferritin.
That the major impurity in 90% pure hydroxylase is ferritin is also indicated by the finding that its molecular weight and absorption spectrum are essentially the same as ferritin.
This impurity can be conveniently separated from the hydroxylase by sucrose gradient, centrifugation because its high iron content gives it a very high rate of sedimentation.
All subsequent studies were carried out with phenylalanine hydroxylase which had been purified to greater than 95y0 by sucrose gradient centrifugation.
An absorbance spectrum of this purified preparation of the hydroxylase (Fig. 3) revealed a peak at 280 nm and a 280 nm to 260 nm ratio of 1.6. There also was a small shoulder at 400 nm. This type of spectrum is often seen with non-heme iron proteins, such as pyrocatechase (13) and transferrin (14). It should be noted that the spectrum obtained for purified phenylalanine hydroxylase is different from those seen for non-heme iron proteins containing acid-labile sulfur (15). These latter proteins exhibit absorption peaks at 315 to 335 nm, 410 to 420 nm, and 450 to 460 nm.
Since several other hydroxylases have been shown to require copper for enzymatic activity (16, 17), purified phenylalanine hydroxylase was analyzed for copper. This analysis revealed that there are only 0.2 g atoms of copper per mole of 100,000 molecular weight hydroxylase.
ESR Xfudies-As another means of characterizing the metal in the enzyme, sucrose gradient purified phenylalanine hydroxylase was analyzed by ESR spectroscopy.
A dramatic signal was observed at g = 4.23 (Fig. 4) for the enzyme in 0.02 M Tris-HCI buffer, PI-I 6.8, in the presence of oxygen.
A signal at this g value is characteristic of a high-spin ferric ion (18). When phenylalanine and 6,7-dimethyltetrahydropterin, the other two substrates for the enzyme, were added, 90% of the signal at g = 4.23 disappeared (Fig. 4). This disappearance of t.he g = 4.23 signal could be due either to a reduction of the iron to the ferrous form, or a change from high to low spin state of Fe3+ due to a change in its ligand field. Forty percent of the signal reappeared when the reaction mixture was bubbled with oxygen. After these ESR measurements had been completed, the enzyme was assayed and still had 607; of its original activity. Therefore, after correcting for this loss in enzyme activity, 70% of the signal reappeared.
Preparation of Apoenzyme-The above studies demonstrated that iron is intimately associated with phenylalanine hydroxylase and that the iron may be involved in the catalytic activity of the enzyme.
Additional support for the idea that iron is an essential component of phenylalanine hydroxylase, was obtained by removal of most of the iron from the hydroxylase. By treatment of the sucrose gradient purified enzyme with o-phenanthroline and cysteine for 4 hours at O", it was possible to remove 50 to 80% of the iron (Table III).
In some experiments (see Experiment 3, Table III) the enzyme activity decreased to a greater extent than did t.he iron content, an indication that some o-phenant,hroline-iron complex might remain bound to the enzyme. An absorbance spectrum of the apoenzyme even after four ammonium sulfate precipitations revealed that some o-phenanthroline was still bound to the enzyme.
Addition of FeC& back to t,his apoenzyme gave nearly full restoration of enzyme activity.
Saturation with FeClz restored phenylalanine hydroxylase activity with hyperbolic kinetics.
A plot of the reciprocal of percentage control activity as a function of the reciprocal of the FeCL concentration revealed that 5 x 1OV FeCls gave maximum restoration of enzyme activity (Fig. 5). When a variety of metals were tested for their ability to restore the activity of the apoenzyme, only ferrous ions were effective (Table IV).
As can be seen, mercurous and cupric ions completely inactivated the apoenzyme.
Some of the physical characteristics of the apo-and holoenzyme were compared to see if the iron plays a structural role in the hydroxylase.
The apo-and holoenzyme gave identical patt,erns in disc gel electrophoresis, indicating that the molecular weight and charge of the hydroxylase are not affected by the absence of iron. The apoenzyme, however, was much more labile to incubation at 25" than the holoenzyme (Fig. 6). The apoenzyme had a half-life of less than 1 min at 25", whereas the holoenz~me's half-life was greater than 20 min. Addition of phenylalanine partially stabilized the apoenzyme. Addition of FeC12, with or without phenylalanine, conferred the same stability to the apoenzyme as exhibited by the holoenzyme. There were less free sulfhgdryl groups on the apo-than on the holoenzyme.
The denatured holoenzyme reacted twice as fast with DTNB and ['4C]NER4 as the apoenzyme.
The urea-denatured holoenzyme had 5.0 cysteines per 50,000 molecular weight by guest on March 23, 2020 http://www.jbc.org/ Downloaded from tracted) and holoenzyme (40yo pure) were previously incubated indicated times at 25" with or without 2 mM FeC& or 2 mM phenylalanine.
All samples were assayed in the presence of 2 mM FeC12.
The control for the holoenzyme was not previously incubated and had an activity of 45 nmoles per min per mg. The apoenzyme control had an activity of 50 nmoles per min per mg. The tetrahydrobiopterin concentration was 0.009 m&l.
subunit, whereas the apoenzyme had only 2.9 cysteines. APparently two of the apoenzyme's cysteines oxidize readily. The reducing agent DTT, however, did not prevent the loss of activity during incubation of the apoenzyme at 25". Thus, the oxidation of the two cysteines does not explain the increased lability of the apoenzyme.
Amino Acid Composition-The amino acid composition of the sucrose-gradient purified hydroxylase was analyzed (Table V). Ninety-two percent of the applied protein nitrogen was recovered in the amino acids. From t.he nitrogen analysis and amino acid composition the mass of the protein was calculated. Using this estimate of the protein concentration it was found that 1 mg per ml of the hydroxylase has an absorbance of 0.95 at 280 nm. Since there were 5 cysteines and 7 cystic acids per 50,000 molecular weight subunit, there must be one disulfide per subunit.. It should be noted that the amino acid composition is an average of the composition for two subunits of the same molecular weight (by SDS gel electrophoresis) but slightly different charge (by urea gel electrophoresi?).
From the amino acid composition, a specific volume of 0.72 was calculated. DISCUSSION These results demonstrate that the sucrose gradient purified phenylalanine hydroxylase, which is about 9570 pure, contains between 1 and 2 moles of iron per mole of enzyme (assuming 100,000 molecular weight).
Since the subunit molecular weight of the hydroxylase is 50,000 (a), it seems likely that there is 1 iron per subunit.
Iron values lower than 1 might be due t,o the loss of some of the iron during purification of t,he enzyme; this possibility is supported by our observations that incubation of the enzyme with FeCl, and DTT activates the enzyme 50 to 100%.
ESR studies revealed that the purified hydroxylase gives a signal at g = 4.23. A signal at g = 4.23 is attributed to a high spin ferric ion surrounded by an environment having a large component of rhombic symmetry (18). The high spin state of the d electrons of the ferric ion results from a small degree of splitting of the two groups of 3d orbitals.
It is likely that this small degree of splitting is due to a greater ionic, than covalent, binding between the iron and its ligands (18). Some of the iron might be present in another form giving a weaker signal. Since addition of the substrates caused the g = 4.23 signal to disappear, we have concluded that this iron is involved in the enzymatic catalysis.
The fact that removal of iron correlated with a loss of enzymatic activity further supports the postulate that the iron plays an important role in the hydroxylation reaction.
Furthermore, since of all metal ions t.ested, only iron restored the activity to the apoenzyme, the metal requirement is highly specific. This finding contrasts with the report that Pseudomonas phenylalanine hydroxylase is activated by mercuric, cadmium, cupric, and cuprous ions, as well as ferrous ions (19).
Although the present studies have established for the first time that a pterin-dependent hydroxylase is an iron enzyme, and that the iron is essential for the enzyme's catalytic activity, the precise mechanism by which the enzyme-bound metal participates in the reaction is not clear.
From previous studies on the role of metals in oxygenasecatalyzed reactions, it is apparent that dioxygenases require metals (20), but that not all monooxygenases do. In particular, a large group of FAD-dependent monooxygenases have been shown to contain neither iron nor copper (20). On the other hand, there have been many reports of monooxygenases, such as 2,5-diketocamphane-lactonizing enzyme (Zl), fatty acid CoAdesaturating enzyme (22), p-hydroxybenzoate hydroxylase (23), and bovine adrenal tyrosine hydroxylase (24), requiring Fe2+ for full activity.
It should be noted that with the last enzyme, it has been shown that the stimulation of the enzyme's activity by exogenous Fe2f is not due to the direct participation of the met.al in the hydroxylation reaction, but rather to the ability of Fe*+ to decompose H20?, and thereby protect the hydroxylase from inactivation by endogenously-generated peroxide (25). With that enzyme, catalase can substitute for Fe2+ (25).
Two quite distinct roles can be envisioned for the iron in phenylalanine hydroxylase: it could be involved in maintaining the structure of the actjive enzyme (e.g. by holding subunits together (as)), or it could be directly involved in the catalytic mechanism.
There is no evidence in favor of the idea that the function of iron in the hydrosylase is merely a structural one; the apoenzyme and the holoenzyme do not differ markedly in either molecular weight or net charge.
If the metal is involved in the catalytic functioning of the enzyme, it could take part in the activation of oxygen or, less likely, in the activation of phenylalanine or the tetrahydropterin. If it participates in oxygen activation, the metal could facilitate the transfer of electrons between the tetrahydropterin and oxygen. Such a role for the metal has been suggested for the copper-containing monooxygenase, dopamine P-hydroxylase, where it has been shown that the enzyme-bound copper undergoes a cycle of reduction (ascorbate serving as electron donor) and re-oxidation during the hydroxylation reaction (16, 27). Consistent with the demonstration that the reduced enzyme is an intermediate in the reaction is the finding that the mechanism of that hydroxylase is of the ping-pong type (28).
In contrast to the findings with dopamine /'3-hydroxylase, recent studies have revealed that all three subst.rates must be bound to phenylalanine hydroxylase before any detectable reaction occurs.4 Stoichiometric enzyme studies have demonstrated that there is no reduction of the enzyme in the absence of both the other two substrates.
Kinet,ic studies have shown that the phenylalanine hydroxylase mechanism is of the sequential, rather than ping-pong, type.4 The disappearance of the g = 4.23 ESR signal of phenylalanine hydroxylase on addition of 4 0. B. Fisher, and S. Kaufman, unpublished results.
DMPH4 and phenylalanine (Fig. 4) is consistent with reduction of Fe3+ to Fe2+. In light of the kinetic and stoichiometric enzyme studies, however, this reduction of iron may not be a distinct step, but a transient event occurring during a concerted mechanism.
Such a mechanism would be in accord with Hamilton's proposal that iron might form a link for the flow of electrons from the tetrahydropterin to oxygen (29). Alternatively, the disappearance of the g = 4.23 signal upon addition of the substrates may be due to a change of the Fe3f from a high to a low spin state caused by an alteration of the metal's ligand field. In this regard, it, should be noted that dioxygenase, pyrocatechase, also has an ESR signal characteristic of high spin Fe3+ (g = 4.28). With this enzyme, too, the signal disappears during catalysis, but this change is not believed to be due to reduction of t'he iron (13).
Studies with model hydroxylation systems have suggested that iron might be more directly involved in the activation of oxygen.
Oxotransition metals such as ferrate and manganate ions can catalyze the hydroxylation of olefins (30). Manganate ions, furthermore, promote the hydroxylation of aromatic cornpounds resulting in the NIH shift characteristic of the phenylalanine hydroxylase reaction (31). These findings have suggested that an osoiron (Fe = 0) species might be the active oxidant, in hydroxylation reactions (30). Further studies will be required to elucidate the role of the iron in the phenylalaninehydroxylase-catalyzed reaction.