The effect of substrate, dihydrobiopterin, and dopamine on the EPR spectroscopic properties and the midpoint potential of the catalytic iron in recombinant human phenylalanine hydroxylase

Phenylalanine hydroxylase (PAH) is a tetrahydrobiopterin (BH4) and non-heme iron-dependent enzyme that hydroxylates L-Phe to L-Tyr. The paramagnetic f erric iron at the active site of recombinant human PAH (hPAH) and its midpo int potential at pH 7.25 (E-m(Fe(III)/Fe(II))) were studied by EPR spectros copy. Similar EPR spectra were obtained for the tetrameric wild-type (wt-hP AH) and the dimeric truncated hpAH(Gly(103)-Gln(428)) corresponding to the "catalytic domain." A rhombic high spin Fe(III) signal with a g value of 4. 3 dominates the EPR spectra at 3.6 K of both enzyme forms. An E-m +207 +/- 10 mV was measured for the iron in wt-hPAH, which seems to be adequate for a thermodynamically feasible electron transfer from BH4 (E-m (quinonoid-BH2 /BH4) = +174 mV). The broad EPR features from g = 9.7-4.3 in the spectra of the ligand-free enzyme de creased in intensity upon the addition of L-Phe, whereas more axial type signals were observed upon binding of 7,8-dihydrob iopterin (BH2), the stable oxidized form of BH2, and of dopamine. Ah three ligands induced a decrease in the E-m value of the iron to +123 +/- 4 mV (L -Phe), +110 +/- 20 mV (BH2), and -8 +/- 9 mV (dopamine). On the basis of th ese data we have calculated that the binding affinities of L-Phe, BH2, and dopamine decrease by 28-, 47-, and 5040-fold, respectively, for the reduced ferrous form of the enzyme, with respect to the ferric form. Interestingly , an E-m value comparable with that of the ligand-free, resting form of wt- hPAH, i.e. +191 +/- 11 mV, was measured upon the simultaneous binding of bo th L-Phe and BH2, representing an inactive model for the iron environment u nder turnover conditions. Our findings provide new information on the redox properties of the active site iron relevant for the understanding of the r eductive activation of the enzyme and the catalytic mechanism.