EPR and ¹H‐NMR spectroscopic studies on the paramagnetic iron at the active site of phenylalanine hydroxylase and its interaction with substrates and inhibitors

Abstract

The paramagnetic iron at the active site of highly purified, catalytically active phenylalanine hydroxylase was studied by EPR at 3.6 K and one-dimensional 'H-NMR spectroscopy at 293 K. The EPR-detectable iron of the bovine enzyme was found to be present as a high-spin form (S = 5j2) in different ligand field symmetries depending on medium conditions (buffer ions) and the presence of ligands known to bind at the active site. At 3.6 K and in phosphate buffer, the paramagnetic iron is coordinated in an environment of rhombic symmetry (g = 4.3), whereas Tris buffer favours an environment of axial ligand field symmetry (g = 6.7, 5.3 and 2.0). The latter axial type of signals resembles those observed at g = 7.0, 5.2 and 1.9 for the enzyme in phosphate buffer when L-noradrenaline is added as an active-site ligand (inhibitor). The same proportion of iron that coordinates to L-noradrenaline seems to be reduced by the pterin cofactor and participate in catalysis. Experimental evidence is presented that Tris inhibits the enzyme by interacting with the enzyme-bound ferric iron and decreases its rate of reduction by the tetrahydropterin cofactor. Preincubation with dithiothreitol also inhibits the enzyme activity and prevents the reduction of its catalytically active ferric iron by pterin cofactors as well as binding of catecholamines to the enzyme.'H-NM R spectroscopy revealed that the substrate (L-phenylalanine) and L-noradrenaline bind close to the paramagnetic iron, and that the catecholamine displaces the substrate from its binding at the active site. The results support our recently proposed model for the cooperative binding of inhibitor and substrate at the active site Eur. J. Biochem. 193, 211 -2191. Phenylalanine 4-monooxygenase (phenylalanine hydroxylase) is a tetrahydropterin-dependent enzyme that catalyses the formation of L-tyrosine from L-phenylalanine [l]. This 200-kDa tetrameric enzyme has non-heme iron coordinated at the active site, with a stoichiometry of one iron atom/ subunit in the fully reconstituted, active form [2, 31. In the original study on the native rat enzyme by EPR, only a signal at g = 4.3 was observed, corresponding to a high-spin Fe(I1I) ( S = 5/2) in an environment of predominant rhombic symmetry [4]. This signal was reported to disappear almost completely on addition of substrate and cofactor, indicating that the iron was involved in the catalytic process [4]. Also in later studies, the ferric iron has been found to be reduced to the ferrous state by the tetrahydropterin cofactor, which is oxidized to a quinonoid form [5,6]. By contrast, more recently it has been reported that the EPR spectrum of the resting rat enzyme exhibits signals at geff = 9.4-8.7, 4.3 (proposed to represent catalytically inactive iron) and g,,, = 6.7, 5.4 and 2 (catalytically active iron), consistent with the presence of two different populations of high-spin ferric iron in the enzyme 17, Correspondence to A. Martinez,

PAHdb 2003: What a locus-specific knowledgebase can do

“…Adjacent to the active site is a channel that is 16 Å long and 8 Å wide by which substrate may access the active site [Anderson and Flatmark, 2001]. As isolated, PAH crystals contain an active site Fe(III) iron atom [Martinez et al, 1991;Erlandsen et al, 1997] located 10 Å below the surface of the protein on the floor of the active site at the intersection of the channel and the active site pocket. The Fe(III) atom is coordinated to H 285, H 290, and one oxygen atom in E 330.…”

The incorporation of divalent metal ions into recombinant human tyrosine hydroxylase apoenzymes studied by intrinsic fluorescence and 1H‐NMR spectroscopy

“…Thus, the EPR measurements corroborate that the active, iron-reconstituted hTH contains iron in the ferrous form whereas, on addition of dopamine, the enzyme-bound iron is oxidized. The EPR spectrum of the high-spin ( S = 5/2) Fe(II1) in the catecholamine-enzyme complex closely matches that of tyrosine hydroxylase as isolated from bovine adrenal medulla [lo] and phaeochromocytoma cells [29] and that of phenylalanine hydroxylase in the presence of catecholamines [19,371, in which the axial type of signals have been associated with the catecholate-Fe(II1) complex. These catecholamine-enzyme complexes appear to represent inactive and biologically occurring forms of tyrosine hydroxylase [lo, 131.…”

“…The water proton relaxation rate of solutions of iron-reconstituted hTHl is much smaller than measured in solutions of the structurally and functionally related enzyme phenylalanine hydroxylase (Table 2), which is isolated with one high-spin (S = 5/2) Fe(II1) atom/subunit at the active site [19, 281. The paramagnetic effect on the water proton relaxation rate was eliminated by incubating phenylalanine hydroxylase with the reducing agent sodium dithionite (Table 2) [19], generating a diamagnetic form [19].…”

“…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.…”

“…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.…”