Eur. J. Biochem. 210,23-31 (1992) 0FEBS 1992
The incorporation of divalent metal ions into recombinant human tyrosine hydroxylase apoenzymes studied by intrinsic fluorescence and H-NMR spectroscopy Jan HAAVIK
'
Aurora MARTiNEZ ', Sigridur OLAFSDOTTIR ',Jacques MALLET' and Torgeir FLATMARK
Department of Biochemistry, University of Bergen, Norway Laboratoire de Neurobiologie Cellulaire and Moleculaire, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France
(Received June 18/August 31, 1992) - EJB 92 0858
Three isoforms of human tyrosine hydroxylase were expressed in Escherichiu coli and purified to homogeneity as the apoenzymes (metal-free). The apoenzymes exhibit typical tryptophan fluorescence emission spectra when excited at 250 - 300 nm. The emission maximum (342 nm) was not shifted by the addition of metal ions, but reconstitution of the apoenzymes with Fe(I1) at pH 7-9 reduced the fluorescence intensity by about 35%, with an end point at 1.0 iron atom/enzyme subunit. The fluorescence intensity of purified bovine adrenal tyrosine hydroxylase, containing 0.78 mol tightly bound iron/mol subunit, was reduced by only 6% on addition of an excess amount of Fe(I1). Other divalent metal ions [Zn(II), Co(II), Mn(II), Cu(11) and Ni(II)] also reduced the fluorescence intensity of the human enzyme by 12-30% when added in stoichiometric amounts. The binding of Co(I1) at pH 7.2 was also found to affect its 'H-NMR spectrum and this effect was reversed by lowering the pH to 6.1. The quenching of the intrinsic fluorescence of the human isoenzymes by Fe(I1) was reversed by the addition of metal chelators. However, the addition of stoichiometric amounts of catecholamines, which are potent feedback inhibitors of tyrosine hydroxylase, to the iron-reconstituted enzyme, prevented the release of iron by the metal chelators. Fluorescence quenching, nuclear magnetic relaxation measurements and EPR spectroscopy all indicate that the reconstitution of an active holoenzyme from the isolated apoenzyme, with stoichiometric amounts of Fe(I1) at neutral pH, occurs without a measurable change in the redox state of the metal. However, on addition of dopamine or suprastoichiometric amounts of iron, the enzyme-bound iron is oxidized to a high-spin Fe(II1) ( S = 5/2) form in an environment of nearly axial symmetry, thus providing an explanation for the inhibitory action of the catecholamines
Tyrosine hydroxylase is an iron- and tetrahydropterindependent enzyme which catalyses the rate-limiting reaction in the biosynthesis of catecholamines [l]. The human enzyme is present as four isoforms, generated by alternative splicing of pre-mRNA, and is a tetramer composed of four identical subunits (the mass of the subunits ranging over 5 5 5 5 3 58 521 Da for the different isoforms) [2 - 41. We have recently expressed three of these isozymes (hTH1, hTH2 and hTH4) in Escherichiu coli and shown that the purified apoenzymes (metal-free) bind stoichiometric amounts of iron and zinc with relatively high affinity at pH 5.4-6.5 [S]. The incorporation of Fe(1I) results in a rapid and up to 40-fold increase in activity Correspondence to J . Haavik, Department of Biochemistry, University of Bergen, N-5009 Bergen, Norway Fax: f 4 7 5 20 64 00. Abbreviations. hTHl - hTH4, human tyrosine hydroxylase isozymes 1-4; apo-hTH1 -apo-hTH4, apoenzymes of the human tyrosine hydroxylase isozymes. Enzyrne.r. Tyrosine 3-monooxygenase or tyrosine hydroxylase (EC 1.14.16.2), phenylalanine 4-monooxygenase or phenylalanine hydroxylase (EC 1.14.16.1).
[ 51. However, the kinetics and stoichiometry of metal binding at physiological pH is not known. Based on the deduced amino acid sequence of human tyrosine hydroxylase, it is known that the enzyme contains three tryptophan residues (i.e. Trp165, Trp232 and Trp371 in hTH1) [2]. With the exception of Trp371, which is lacking in tryptophan hydroxylase [6], these tryptophan residues are conserved in the mammalian tetrahydropteridine-dependent hydroxylases sequenced so far [6 - 91. We have previously described the intrinsic fluorescence spectra of bovine tyrosine hydroxylase [lo] and rat phenylalanine hydroxylase [I 11. In the present study it is shown that the tryptophan fluorescence of the apo-form of human tyrosine hydroxylase is partially quenched by added Fe(I1) and other divalent metal ions, and that fluorescence quenching can be used to monitor the reversible binding of metal ions to the apoenzyme at physiological pH values. Furthermore, the effects of the metal incorporation on the 'H-NMR spectrum of the apoenzyme and on the relaxation rate of water protons have been studied in order to obtain further information on the reconstitution process and the redox state of the iron in the holoenzyme.
24 MATERIALS AND METHODS Materials All the reagents used, including metal salts [(NH,),Fe(SO,),, FeSO,, ZnSOa, CuSO,, CoCl,, MnCI,, NiC12, A1(N03)3 Ga(N03)3, CaC12, BaCI,, MgCl,, FeCl,, VOSO,, SrCI,, HgC12, CdCI2 and AgN03] were of analytical grade (E. Merck, Darmstadt, FRG). Sheep liver dihydropteridine reductase was from Sigma Chemical Co. (St. Louis, MO). Human tyrosine hydroxylase isoforms 1, 2 and 4 were expressed in E. coli and purified to homogeneity as previously described [5, 121. The purified preparations of hTH1, hTH2 and hTH4 used in the present study contained 0.02 k 0.01, 0.02 k 0.01 and 0.09 f 0.02 (mean f SD, II = 3 or 4) atoms iron/subunit, respectively, as determined by atomic absorption spectrometry [lo]. Bovine tyrosine hydroxylase was purified from the cytosolic fraction of adrenal medullary extracts as described [lo]. Enzyme prepared using this procedure typically contains 0.6 - 0.9 atom tightly bound iron and 0.3 - 0.4 molecule catecholamine/enzyme subunit [lo, 13, 141. The preparations of bovine tyrosine hydroxylase used in the present study contained 0.78 0.12 atom iron/ enzyme subunit. Rat liver phenylalanine hydroxylase was purified by the method (procedure 11D) of Shiman and coworkers [15]. The concentration of purified protein was estimated using an absorption coefficient of A;&, = 10.0 cmpl for phenylalanine hydroxylase [15] and 10.4 cm-' for tyrosine hydroxylase [lo]. The phenylalanine hydroxylase preparations contained 1.I f 0.07 atom iron/enzyme subunit. The buffers were passed through a column of Chelex-100 ionexchange resin (Bio-Rad Laboratories, Richmond, CA, USA) before use. Fluorescence measurements Fluorescence spectroscopy was performed using a PerkinElmer LS-50 luminescence spectrometer with the cuvettes (1 x 1 cm path-length) kept at 20°C and with maximal stirring. A typical sample contained 0.1 - 1.O pM tyrosine hydroxylase subunit in 20 mM Na-Hepes and 0.15 M NaCI, pH 7.50. The anaerobic experiments were performed in solutions which had been subjected to repeated evacuation and flushing with argon (99.99%). The fluorescence measurements were corrected to eliminate contributions from dilutions ( < 5%), buffer blanks and the inner filter effect of the added dipicolinic acid. The correction for inner filter effect was performed according to the equation FobaE F,,,, (10p(Aex+Aem)~2), where Fobsand F,,,, are the observed and corrected fluorescence intensities, and A,, and A,, are the absorbances at the excitation and emission wavelengths, respectively [16,17]. No further corrections were used. Stock solutions of Fe(I1) ammonium sulfate (1 mM) were prepared in water and used within 30 min. The quantum yields of hTHl and hTH2 were determined using free tryptophan as a reference and assuming a quantum yield of 0.13 for L-tryptophan in water [18]. For these determinations, the absorbance of enzyme solutions at 296 nm was < 0.05.
400-MHz 'H-NMR spectroscopy Longitudinal relaxation rates of water protons were measured on enzyme samples in 20 mM potassium phosphate and 0.2 M KC1, pH 7.25, prepared from double-distilled water. The longitudinal relaxation times (T1,obs) were measured at a probe temperature of 293 K by using a standard inversionrecovery sequence (180"-~-90"),recycle delay 2 5 x TI. When
indicated, relaxation rates of the residual water signal were measured in enzyme samples prepared in ,H20 (99.8%), containing 20 mM potassium phosphate and 0.2 M KCl of pH* 7.2- 7.4 (uncorrected value in 'H20). Deuteration of the samples was performed as described [19]. The samples, with a final volume of approximately 0.4 ml (0.1 -0.5 mM enzyme subunit, assuming a subunit mass of 60 kDa), were centrifuged in an Eppendorf microfuge and the supernatant transferred to NMR tubes 5 mm in diameter. The 'H-NMR spectra were recorded at a probe temperature of 293 K on a Bruker AM-400 WB spectrometer using internal deuterium lock, selective presaturation of the residual water signal and standard Fourier-transformation techniques (see figure legends for parameters). The pH* of t h e N M R samples was measured by using a Radiometer PHM84 pH meter with an Ingold electrode and represents the uncorrected value in D 2 0 . The pH was adjusted by the addition of diluted DCl and NaOD or deuterated potassium phosphate buffer of appropriate pH. EPR spectra acquisition Spectra were recorded on a Bruker ER 200 D-SRC Xband spectrometer equipped with an Oxford Instruments ESR 10 helium flow cryostat (see figure legends for acquisition parameters). All samples (250 p1 each) were prepared individually in EPR tubes (internal diameter = 3 mm) and frozen in liquid nitrogen. Assay of tyrosine hydroxylase activity The activity of tyrosine hydroxylase was measured either by the method of Reinhard et al. [20] or by a modification of the procedure of Shiman et al. [21]. RESULTS AND DISCUSSlON Fluorescence spectra of human and bovine tyrosine hydroxylase As shown in Fig. 1, human and bovine tyrosine hydroxylase have similar intrinsic fluorescence excitation and emission spectra. When excited at 295 nm, the emission maxima were found to be at 342 nm for human and at 340 nm for bovine tyrosine hydroxylase. These emission maxima are typical values for partially solvent-exposed tryptophan residues (type-I1 tryptophan fluorescence). Using an excitation wavelength of 296 nm and measuring the emission between 305- 500 nm, the quantum yield was estimated to be 0.11 for both apo-hTH1 and apo-hTH2 at pH 7.5 and 20°C. In the presence of 8 M urea, the fluorescence intensity of the human apoenzymes decreased by 22% and the emission maximum was shifted from 342 nm to 353 nm, i.e. to a value expected for a highly denatured protein [ 161. Similar spectroscopic changes have been observed for the bovine adrenal enzyme [lo]. Since tyrosine hydroxylase contains three tryptophan residues, it is likely that the fluorescence is predominantly due to this amino acid, although it also contains 15 tyrosine residues [2] and weak tyrosinate fluorescence emission at 340 nm has been reported for some proteins [22]. A striking difference between the human and bovine tyrosine hydroxylase is that at a similar protein concentration, the fluorescence intensity of the bovine enzyme is about 30% less than that of the human enzyme (Fig. 1). Thus, assuming that all of the three conserved tryptophan residues are intact, the intrinsic fluorescence of the bovine enzyme seems to be par-
25
Wavelength (nm) Fig. 1. Fluorescence excitation (A) and emission (B) spectra of tyrosine hydroxylase (hTHZ). The spectra of hTH2 before (curve 1) and after (curve 2) addition of a stoichiometric amount of Fe(I1) are shown. For comparison, the spectra of bovine tyrosine hydroxylase is shown before (curve 3) and after (curve 4) addition of a stoichiometric amount of Fe(I1). Both enzymes were present at a concentration of 1 pM subunit in 20 mM Na-Hepes and 150 mM NaCl, pH 7.50. The spectra were obtained at 20°C using an emission wavelength of 340 nm and slit of 5 nm (A) and an excitation wavelength of 295 nm and slit of 2.5 nm (B).
tially quenched. The position of the emission maxima (340 342 nm), the relatively broad half-width (55 - 58 nm), the high quantum yield and the negative change in quantum yield on urea denaturation all indicate that the fluorescent tryptophan residues of human and bovine tyrosine hydroxylase are located on the surface of the proteins [23]. Protein structure analysis according to Emini et al. [24] indicates a high probability of surface localization of Trp165 and Trp232, while Trp371 seems to be located in a more hydrophobic environment. Quenching of tyrosine hydroxylase fluorescence by added metal ions
The preparations of bovine tyrosine hydroxylase used in the present study contained 0.78 f 0.12 (SD) atom iron/enzyme subunit. In contrast, we have recently reported that the human recombinant enzyme, purified by a similar procedure, contains only 0.02-0.1 atom iron/enzyme subunit [5]. Thus, it was of interest to examine whether the presence of iron influenced the fluorescence spectra of the enzymes. As shown in Figs 1 and 2, the addition of stoichiometric amounts of Fe(I1) to apo-hTH2 caused a reduction in the fluorescence emission by 33.7 0.5% (n = 4), without changing the shapes of the excitation or emission spectra (Fig. 1). The observed extent of decreased quenching with increasing temperatures (10-37°C) (data not shown), the tight association of the metal with the apoenzyme and the defined stoichiometry, all indicate that this quenching is of a static type and may be due either to contact quenching or to a conformational effect of metal binding [25, 261. Since we still lack structural information on the relative positions of tryptophan residues in the enzyme and the iron-binding site, it is difficult to determine the relative contribution of each process to the quenching. It seems plausible, however, that at least one of the solventexposed tryptophans of hTH is located close to the iron-
binding site in the three-dimensional structure of the protein. The extent of quenching and stoichiometry of metal binding was similar for human TH isoforms 1, 2 and 4, although the maximal fluorescence quenching varied between 33 - 40% for different enzyme preparations. Since the enzyme preparations showing the highest fluorescence quenching also had the highest specific activity, this variation may be related to small differences in purity or content of denatured enzyme species in the individual batches. Under similar assay conditions, the fluorescence emission of bovine tyrosine hydroxylase was reduced by only 6% on the addition of Fe(I1) (Fig. 1B). However, when corrections were made for the iron already present in the bovine enzyme (0.78 0.12 atom iron/subunit), the extent of fluorescence quenching observed by added Fe(I1) was similar for the human and bovine enzymes. In the presence of atmospheric dioxygen, Fe(I1) is gradually oxidized to Fe(II1) in protein-free buffered solutions at pH 7.5. It was therefore of interest to check if the metal was incorporated to the apo-hTH as Fe(I1) and kept in that state, or as Fe(II1) originated from chemical oxidation in the buffer, or from oxidation after the metal is bound. For this purpose, the solutions of Fe(l1) were prepared under anaerobic conditions in the presence of ascorbate (28 mM). The rate of fluorescence quenching was affected by neither deaeration nor the presence of ascorbate. Furthermore, stoichiometric amounts of added Fe(II1) did not have any effect on the fluorescence spectrum of human apo-hTH2 (Table 1). Addition of 1 pM of the natural cofactor, (6RI-tetrahydrobiopterin to 1 pM solution of iron-reconstituted hTHl increased the fluorescence intensity of the holoenzyme by 6%. Conversely, addition of stoichiometric amounts of HzOz reduced the fluorescence intensity of the holoenzyme by l6%, without changing the shape of the emission spectra. Since these effects were not observed on the apoenzyme or enzyme prepared with either Zn(I1) or Co(Il), they can probably be explained by a change in the redox state of the enzyme-bound iron [27]. These effects are qualitatively similar to the effects reported for rat liver phenylalanine hydroxylase [28] and rat pheochromocytoma tyrosine hydroxylase [27], although much less pronounced. Since tyrosine hydroxylase and phenylalanine hydroxylase contain tryptophan residues at similar positions, these differences may reflect differences in iron-binding sites or the initial redox state of the iron in the two enzymes. Phenylalanine hydroxylase is isolated with the iron largely in the ferric, high-spin state [19] (and this work). Although tyrosine hydroxylase isolated from bovine adrenal medulla and from rat phaeochromocytoma cells has been shown to contain both Fe(I1) and Fe(II1) [lo, 291, the redox state of the catalytically active iron species is not established. However, it has been postulated that Fe(I1) is the active form [27]. Preliminary studies using Mijssbauer spectroscopy have also shown that hTHl reconstituted with 57Fe(lI)at pH 7.50 contains high-spin Fe(1I) (E. Bill, personal communication). Other divalent metal ions [Zn(ll), Co(II), Mn(I1) and Ni(II)] also quenched the fluorescence intensity by 12 - 30%, with distinct end points at 1.0 atom/enzyme subunit of hTH2 (Fig. 2 and Table 1). However, the fluorescence intensity by added Cu(I1) (Fig. 2), Hg(I1) and Cd(I1) continued to decrease even at concentrations 10 times above the enzyme concentration; the molecular basis for the fluorescence quenching by these metals may therefore be distinct from that of Fe(I1). As shown in Table 1, the extent of fluorescence quenching produced by divalent metal ions was maximal for metal ions with cation radii between 0.07 -0.08 nm. Monovalent metal ions [Na(I), K(1) and Ag(I)] and trivalent ions [Al(III), Fe(II1)
) .
c .-
u)
k
+. c .-
1.0
a,
2 0.9 0 u)
a,
$ 0 8
-
I
z a,
0.7
m
a,
U
.->o
x
-
CI
a l x 0
i c 0
60
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0
50
1
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2
4
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[MeZ+] ( P W
Fig.2. Changes in the fluorescence quenching of human tyrosine hydroxylase on titration with Fe(I1) (H), Co(I1) (V), Cu(I1) (0) and Zn(I1) (0).Increasing amounts of either Fe(I1) ammonium sulfate, ZnS04 or Cu(II)S04 (1 mM stock solutions) were added to 1 pM apo-hTH2. Titration with Co(I1) was performed by adding CoC1, to 0.5 pM apo-hTH1. The fluorescence intensity was measured after it had stabilized (30 s). The excitation and emission wavelengths were 295 nm and 340 nm, with slit widths of 5 nm and 10 nm, respectively. Other conditions as in Fig. 1.
Table 1. Quenching of intrinsic fluorescence of hTH2 by divalent and trivalent metal ions. The end point of the fluorescence quenching was measured after addition of 0.5 pM metal salt to 0.5 pM apo-hTH2 (subunit concentration). The effective cation radii are given for sixcoordinated metal ions [38].
Compound
5 Time (rnin)
Decrease in fluorescence intensity
Cation radius
%
nm
32 31 20 19 13 12 10" 9.1
The incorporation of divalent metal ions into recombinant human tyrosine hydroxylase apoenzymes studied by intrinsic fluorescence and H-NMR spectroscopy Jan HAAVIK
'
Aurora MARTiNEZ ', Sigridur OLAFSDOTTIR ',Jacques MALLET' and Torgeir FLATMARK
Department of Biochemistry, University of Bergen, Norway Laboratoire de Neurobiologie Cellulaire and Moleculaire, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France
(Received June 18/August 31, 1992) - EJB 92 0858
Three isoforms of human tyrosine hydroxylase were expressed in Escherichiu coli and purified to homogeneity as the apoenzymes (metal-free). The apoenzymes exhibit typical tryptophan fluorescence emission spectra when excited at 250 - 300 nm. The emission maximum (342 nm) was not shifted by the addition of metal ions, but reconstitution of the apoenzymes with Fe(I1) at pH 7-9 reduced the fluorescence intensity by about 35%, with an end point at 1.0 iron atom/enzyme subunit. The fluorescence intensity of purified bovine adrenal tyrosine hydroxylase, containing 0.78 mol tightly bound iron/mol subunit, was reduced by only 6% on addition of an excess amount of Fe(I1). Other divalent metal ions [Zn(II), Co(II), Mn(II), Cu(11) and Ni(II)] also reduced the fluorescence intensity of the human enzyme by 12-30% when added in stoichiometric amounts. The binding of Co(I1) at pH 7.2 was also found to affect its 'H-NMR spectrum and this effect was reversed by lowering the pH to 6.1. The quenching of the intrinsic fluorescence of the human isoenzymes by Fe(I1) was reversed by the addition of metal chelators. However, the addition of stoichiometric amounts of catecholamines, which are potent feedback inhibitors of tyrosine hydroxylase, to the iron-reconstituted enzyme, prevented the release of iron by the metal chelators. Fluorescence quenching, nuclear magnetic relaxation measurements and EPR spectroscopy all indicate that the reconstitution of an active holoenzyme from the isolated apoenzyme, with stoichiometric amounts of Fe(I1) at neutral pH, occurs without a measurable change in the redox state of the metal. However, on addition of dopamine or suprastoichiometric amounts of iron, the enzyme-bound iron is oxidized to a high-spin Fe(II1) ( S = 5/2) form in an environment of nearly axial symmetry, thus providing an explanation for the inhibitory action of the catecholamines
Tyrosine hydroxylase is an iron- and tetrahydropterindependent enzyme which catalyses the rate-limiting reaction in the biosynthesis of catecholamines [l]. The human enzyme is present as four isoforms, generated by alternative splicing of pre-mRNA, and is a tetramer composed of four identical subunits (the mass of the subunits ranging over 5 5 5 5 3 58 521 Da for the different isoforms) [2 - 41. We have recently expressed three of these isozymes (hTH1, hTH2 and hTH4) in Escherichiu coli and shown that the purified apoenzymes (metal-free) bind stoichiometric amounts of iron and zinc with relatively high affinity at pH 5.4-6.5 [S]. The incorporation of Fe(1I) results in a rapid and up to 40-fold increase in activity Correspondence to J . Haavik, Department of Biochemistry, University of Bergen, N-5009 Bergen, Norway Fax: f 4 7 5 20 64 00. Abbreviations. hTHl - hTH4, human tyrosine hydroxylase isozymes 1-4; apo-hTH1 -apo-hTH4, apoenzymes of the human tyrosine hydroxylase isozymes. Enzyrne.r. Tyrosine 3-monooxygenase or tyrosine hydroxylase (EC 1.14.16.2), phenylalanine 4-monooxygenase or phenylalanine hydroxylase (EC 1.14.16.1).
[ 51. However, the kinetics and stoichiometry of metal binding at physiological pH is not known. Based on the deduced amino acid sequence of human tyrosine hydroxylase, it is known that the enzyme contains three tryptophan residues (i.e. Trp165, Trp232 and Trp371 in hTH1) [2]. With the exception of Trp371, which is lacking in tryptophan hydroxylase [6], these tryptophan residues are conserved in the mammalian tetrahydropteridine-dependent hydroxylases sequenced so far [6 - 91. We have previously described the intrinsic fluorescence spectra of bovine tyrosine hydroxylase [lo] and rat phenylalanine hydroxylase [I 11. In the present study it is shown that the tryptophan fluorescence of the apo-form of human tyrosine hydroxylase is partially quenched by added Fe(I1) and other divalent metal ions, and that fluorescence quenching can be used to monitor the reversible binding of metal ions to the apoenzyme at physiological pH values. Furthermore, the effects of the metal incorporation on the 'H-NMR spectrum of the apoenzyme and on the relaxation rate of water protons have been studied in order to obtain further information on the reconstitution process and the redox state of the iron in the holoenzyme.
24 MATERIALS AND METHODS Materials All the reagents used, including metal salts [(NH,),Fe(SO,),, FeSO,, ZnSOa, CuSO,, CoCl,, MnCI,, NiC12, A1(N03)3 Ga(N03)3, CaC12, BaCI,, MgCl,, FeCl,, VOSO,, SrCI,, HgC12, CdCI2 and AgN03] were of analytical grade (E. Merck, Darmstadt, FRG). Sheep liver dihydropteridine reductase was from Sigma Chemical Co. (St. Louis, MO). Human tyrosine hydroxylase isoforms 1, 2 and 4 were expressed in E. coli and purified to homogeneity as previously described [5, 121. The purified preparations of hTH1, hTH2 and hTH4 used in the present study contained 0.02 k 0.01, 0.02 k 0.01 and 0.09 f 0.02 (mean f SD, II = 3 or 4) atoms iron/subunit, respectively, as determined by atomic absorption spectrometry [lo]. Bovine tyrosine hydroxylase was purified from the cytosolic fraction of adrenal medullary extracts as described [lo]. Enzyme prepared using this procedure typically contains 0.6 - 0.9 atom tightly bound iron and 0.3 - 0.4 molecule catecholamine/enzyme subunit [lo, 13, 141. The preparations of bovine tyrosine hydroxylase used in the present study contained 0.78 0.12 atom iron/ enzyme subunit. Rat liver phenylalanine hydroxylase was purified by the method (procedure 11D) of Shiman and coworkers [15]. The concentration of purified protein was estimated using an absorption coefficient of A;&, = 10.0 cmpl for phenylalanine hydroxylase [15] and 10.4 cm-' for tyrosine hydroxylase [lo]. The phenylalanine hydroxylase preparations contained 1.I f 0.07 atom iron/enzyme subunit. The buffers were passed through a column of Chelex-100 ionexchange resin (Bio-Rad Laboratories, Richmond, CA, USA) before use. Fluorescence measurements Fluorescence spectroscopy was performed using a PerkinElmer LS-50 luminescence spectrometer with the cuvettes (1 x 1 cm path-length) kept at 20°C and with maximal stirring. A typical sample contained 0.1 - 1.O pM tyrosine hydroxylase subunit in 20 mM Na-Hepes and 0.15 M NaCI, pH 7.50. The anaerobic experiments were performed in solutions which had been subjected to repeated evacuation and flushing with argon (99.99%). The fluorescence measurements were corrected to eliminate contributions from dilutions ( < 5%), buffer blanks and the inner filter effect of the added dipicolinic acid. The correction for inner filter effect was performed according to the equation FobaE F,,,, (10p(Aex+Aem)~2), where Fobsand F,,,, are the observed and corrected fluorescence intensities, and A,, and A,, are the absorbances at the excitation and emission wavelengths, respectively [16,17]. No further corrections were used. Stock solutions of Fe(I1) ammonium sulfate (1 mM) were prepared in water and used within 30 min. The quantum yields of hTHl and hTH2 were determined using free tryptophan as a reference and assuming a quantum yield of 0.13 for L-tryptophan in water [18]. For these determinations, the absorbance of enzyme solutions at 296 nm was < 0.05.
400-MHz 'H-NMR spectroscopy Longitudinal relaxation rates of water protons were measured on enzyme samples in 20 mM potassium phosphate and 0.2 M KC1, pH 7.25, prepared from double-distilled water. The longitudinal relaxation times (T1,obs) were measured at a probe temperature of 293 K by using a standard inversionrecovery sequence (180"-~-90"),recycle delay 2 5 x TI. When
indicated, relaxation rates of the residual water signal were measured in enzyme samples prepared in ,H20 (99.8%), containing 20 mM potassium phosphate and 0.2 M KCl of pH* 7.2- 7.4 (uncorrected value in 'H20). Deuteration of the samples was performed as described [19]. The samples, with a final volume of approximately 0.4 ml (0.1 -0.5 mM enzyme subunit, assuming a subunit mass of 60 kDa), were centrifuged in an Eppendorf microfuge and the supernatant transferred to NMR tubes 5 mm in diameter. The 'H-NMR spectra were recorded at a probe temperature of 293 K on a Bruker AM-400 WB spectrometer using internal deuterium lock, selective presaturation of the residual water signal and standard Fourier-transformation techniques (see figure legends for parameters). The pH* of t h e N M R samples was measured by using a Radiometer PHM84 pH meter with an Ingold electrode and represents the uncorrected value in D 2 0 . The pH was adjusted by the addition of diluted DCl and NaOD or deuterated potassium phosphate buffer of appropriate pH. EPR spectra acquisition Spectra were recorded on a Bruker ER 200 D-SRC Xband spectrometer equipped with an Oxford Instruments ESR 10 helium flow cryostat (see figure legends for acquisition parameters). All samples (250 p1 each) were prepared individually in EPR tubes (internal diameter = 3 mm) and frozen in liquid nitrogen. Assay of tyrosine hydroxylase activity The activity of tyrosine hydroxylase was measured either by the method of Reinhard et al. [20] or by a modification of the procedure of Shiman et al. [21]. RESULTS AND DISCUSSlON Fluorescence spectra of human and bovine tyrosine hydroxylase As shown in Fig. 1, human and bovine tyrosine hydroxylase have similar intrinsic fluorescence excitation and emission spectra. When excited at 295 nm, the emission maxima were found to be at 342 nm for human and at 340 nm for bovine tyrosine hydroxylase. These emission maxima are typical values for partially solvent-exposed tryptophan residues (type-I1 tryptophan fluorescence). Using an excitation wavelength of 296 nm and measuring the emission between 305- 500 nm, the quantum yield was estimated to be 0.11 for both apo-hTH1 and apo-hTH2 at pH 7.5 and 20°C. In the presence of 8 M urea, the fluorescence intensity of the human apoenzymes decreased by 22% and the emission maximum was shifted from 342 nm to 353 nm, i.e. to a value expected for a highly denatured protein [ 161. Similar spectroscopic changes have been observed for the bovine adrenal enzyme [lo]. Since tyrosine hydroxylase contains three tryptophan residues, it is likely that the fluorescence is predominantly due to this amino acid, although it also contains 15 tyrosine residues [2] and weak tyrosinate fluorescence emission at 340 nm has been reported for some proteins [22]. A striking difference between the human and bovine tyrosine hydroxylase is that at a similar protein concentration, the fluorescence intensity of the bovine enzyme is about 30% less than that of the human enzyme (Fig. 1). Thus, assuming that all of the three conserved tryptophan residues are intact, the intrinsic fluorescence of the bovine enzyme seems to be par-
25
Wavelength (nm) Fig. 1. Fluorescence excitation (A) and emission (B) spectra of tyrosine hydroxylase (hTHZ). The spectra of hTH2 before (curve 1) and after (curve 2) addition of a stoichiometric amount of Fe(I1) are shown. For comparison, the spectra of bovine tyrosine hydroxylase is shown before (curve 3) and after (curve 4) addition of a stoichiometric amount of Fe(I1). Both enzymes were present at a concentration of 1 pM subunit in 20 mM Na-Hepes and 150 mM NaCl, pH 7.50. The spectra were obtained at 20°C using an emission wavelength of 340 nm and slit of 5 nm (A) and an excitation wavelength of 295 nm and slit of 2.5 nm (B).
tially quenched. The position of the emission maxima (340 342 nm), the relatively broad half-width (55 - 58 nm), the high quantum yield and the negative change in quantum yield on urea denaturation all indicate that the fluorescent tryptophan residues of human and bovine tyrosine hydroxylase are located on the surface of the proteins [23]. Protein structure analysis according to Emini et al. [24] indicates a high probability of surface localization of Trp165 and Trp232, while Trp371 seems to be located in a more hydrophobic environment. Quenching of tyrosine hydroxylase fluorescence by added metal ions
The preparations of bovine tyrosine hydroxylase used in the present study contained 0.78 f 0.12 (SD) atom iron/enzyme subunit. In contrast, we have recently reported that the human recombinant enzyme, purified by a similar procedure, contains only 0.02-0.1 atom iron/enzyme subunit [5]. Thus, it was of interest to examine whether the presence of iron influenced the fluorescence spectra of the enzymes. As shown in Figs 1 and 2, the addition of stoichiometric amounts of Fe(I1) to apo-hTH2 caused a reduction in the fluorescence emission by 33.7 0.5% (n = 4), without changing the shapes of the excitation or emission spectra (Fig. 1). The observed extent of decreased quenching with increasing temperatures (10-37°C) (data not shown), the tight association of the metal with the apoenzyme and the defined stoichiometry, all indicate that this quenching is of a static type and may be due either to contact quenching or to a conformational effect of metal binding [25, 261. Since we still lack structural information on the relative positions of tryptophan residues in the enzyme and the iron-binding site, it is difficult to determine the relative contribution of each process to the quenching. It seems plausible, however, that at least one of the solventexposed tryptophans of hTH is located close to the iron-
binding site in the three-dimensional structure of the protein. The extent of quenching and stoichiometry of metal binding was similar for human TH isoforms 1, 2 and 4, although the maximal fluorescence quenching varied between 33 - 40% for different enzyme preparations. Since the enzyme preparations showing the highest fluorescence quenching also had the highest specific activity, this variation may be related to small differences in purity or content of denatured enzyme species in the individual batches. Under similar assay conditions, the fluorescence emission of bovine tyrosine hydroxylase was reduced by only 6% on the addition of Fe(I1) (Fig. 1B). However, when corrections were made for the iron already present in the bovine enzyme (0.78 0.12 atom iron/subunit), the extent of fluorescence quenching observed by added Fe(I1) was similar for the human and bovine enzymes. In the presence of atmospheric dioxygen, Fe(I1) is gradually oxidized to Fe(II1) in protein-free buffered solutions at pH 7.5. It was therefore of interest to check if the metal was incorporated to the apo-hTH as Fe(I1) and kept in that state, or as Fe(II1) originated from chemical oxidation in the buffer, or from oxidation after the metal is bound. For this purpose, the solutions of Fe(l1) were prepared under anaerobic conditions in the presence of ascorbate (28 mM). The rate of fluorescence quenching was affected by neither deaeration nor the presence of ascorbate. Furthermore, stoichiometric amounts of added Fe(II1) did not have any effect on the fluorescence spectrum of human apo-hTH2 (Table 1). Addition of 1 pM of the natural cofactor, (6RI-tetrahydrobiopterin to 1 pM solution of iron-reconstituted hTHl increased the fluorescence intensity of the holoenzyme by 6%. Conversely, addition of stoichiometric amounts of HzOz reduced the fluorescence intensity of the holoenzyme by l6%, without changing the shape of the emission spectra. Since these effects were not observed on the apoenzyme or enzyme prepared with either Zn(I1) or Co(Il), they can probably be explained by a change in the redox state of the enzyme-bound iron [27]. These effects are qualitatively similar to the effects reported for rat liver phenylalanine hydroxylase [28] and rat pheochromocytoma tyrosine hydroxylase [27], although much less pronounced. Since tyrosine hydroxylase and phenylalanine hydroxylase contain tryptophan residues at similar positions, these differences may reflect differences in iron-binding sites or the initial redox state of the iron in the two enzymes. Phenylalanine hydroxylase is isolated with the iron largely in the ferric, high-spin state [19] (and this work). Although tyrosine hydroxylase isolated from bovine adrenal medulla and from rat phaeochromocytoma cells has been shown to contain both Fe(I1) and Fe(II1) [lo, 291, the redox state of the catalytically active iron species is not established. However, it has been postulated that Fe(I1) is the active form [27]. Preliminary studies using Mijssbauer spectroscopy have also shown that hTHl reconstituted with 57Fe(lI)at pH 7.50 contains high-spin Fe(1I) (E. Bill, personal communication). Other divalent metal ions [Zn(ll), Co(II), Mn(I1) and Ni(II)] also quenched the fluorescence intensity by 12 - 30%, with distinct end points at 1.0 atom/enzyme subunit of hTH2 (Fig. 2 and Table 1). However, the fluorescence intensity by added Cu(I1) (Fig. 2), Hg(I1) and Cd(I1) continued to decrease even at concentrations 10 times above the enzyme concentration; the molecular basis for the fluorescence quenching by these metals may therefore be distinct from that of Fe(I1). As shown in Table 1, the extent of fluorescence quenching produced by divalent metal ions was maximal for metal ions with cation radii between 0.07 -0.08 nm. Monovalent metal ions [Na(I), K(1) and Ag(I)] and trivalent ions [Al(III), Fe(II1)
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Fig.2. Changes in the fluorescence quenching of human tyrosine hydroxylase on titration with Fe(I1) (H), Co(I1) (V), Cu(I1) (0) and Zn(I1) (0).Increasing amounts of either Fe(I1) ammonium sulfate, ZnS04 or Cu(II)S04 (1 mM stock solutions) were added to 1 pM apo-hTH2. Titration with Co(I1) was performed by adding CoC1, to 0.5 pM apo-hTH1. The fluorescence intensity was measured after it had stabilized (30 s). The excitation and emission wavelengths were 295 nm and 340 nm, with slit widths of 5 nm and 10 nm, respectively. Other conditions as in Fig. 1.
Table 1. Quenching of intrinsic fluorescence of hTH2 by divalent and trivalent metal ions. The end point of the fluorescence quenching was measured after addition of 0.5 pM metal salt to 0.5 pM apo-hTH2 (subunit concentration). The effective cation radii are given for sixcoordinated metal ions [38].
Compound
5 Time (rnin)
Decrease in fluorescence intensity
Cation radius
%
nm
32 31 20 19 13 12 10" 9.1
