Reductive activation of phenylalanine hydroxylase and its effect on the redox state of the non-heme iron

Abstract

Phenylalanine hydroxylase undergoes an obligatory prereduction step in order to become catalytically active as shown by stopped-flow kinetics and by measuring tyrosine formation at limiting 6-methyltetrahydropterin levels. This initial step requires oxygen and involves conversion of 6-methyltetrahydropterin directly to the quinonoid form with or without phenylalanine. The EPR spectrum of the resting enzyme (geff = 9.4-8.7, 4.3 and geff = 6.7, 5.4) is consistent with two species possessing distinctively different ligand environments for the non-heme, high-spin Fe3+. The intensity of the geff N 4.3 feature is inversely proportional to the specific activity of the enzyme, whereas the intensity of the geff E 6.7-5.4 region correlates with the activity of the enzyme. The I n our attempts to understand the mechanism of action of phenylalanine hydroxylase (PAH) from rat liver, we (Gottschall et al., 1982) recently confirmed and extended an earlier observation by Fisher et al. (1972) that a tightly bound non-heme iron was necessary for the activity of the enzyme. We demonstrated that (1) the correct stoichiometry is one atom per subunit, (2) the enzymatic activity is proportional to the iron content, and (3) the iron can be removed from the enzyme and restored with nearly complete recovery of initial activity. In this paper we corroborate and expand the independent discovery (Marota & Shiman, 1984) that PAH is initially reduced to the catalytically active enzyme by the concomitant oxidation of 6-methyltetrahydropterin (6MPH4) directly to the quinonoid form in a stoichiometric reaction requiring oxygen but without the formation of reduced forms of oxygen such as superoxide and hydrogen peroxide, or of tyrosine. In this paper we (1) propose a two-step kinetic sequence for PAH activation and its catalytic turnover based on stopped-flow kinetic and complimentary product data, (2) define by EPR that the resting state of the activatable enzyme is associated with signals observed at geff = 6.7 and 5.4 consistent with high-spin Fe3+ (S = (3) show that the additional EPR signal observed at geff = 4.3 is associated with a form of the enzyme that is incapable of turnover, and (4) link the prereduction step to the conversion of PAH from an Fe3+ to an Fez+ state. Finally, we demonstrate that dithionite can substitute for 6MPH4 in the prereduction step and that the addition of one electron/subunit is sufficient to impart tightly coupled turnover. Experimental Procedures MaterialsDoubly distilled deionized water was used throughout. All reagents were of the highest grade commercially available. 0006-2960/84/0423-1295%01 SO10latter features are lost upon addition of phenylalanine under anaerobic or aerobic conditions. In the presence of ophenanthroline, the operation of the prereduction step results in nearly quantitative trapping of the iron in an Fez+ redox state. Dithionite can substitute for 6-methyltetrahydropterin in an anaerobic prereduction step, generating a catalytically active phenylalanine hydroxylase containing ...

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

“…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.…”

Effects of elemental sulfur on the metabolism of the deep-sea hyperthermophilic archaeon Thermococcus strain ES-1: characterization of a sulfur-regulated, non-heme iron alcohol dehydrogenase

“…Only three other enzymes that contain monomeric iron sites, phenylalanine monooxygenase (25), superoxide dismutase (8,70), and nitrile hydratase (31,67), are known to exist. The latter two enzymes and intradiol-type catecholic dioxygenases (37) contain a ferric center, while extradiol dioxygenases (76), lipoxygenases (17), phenylalanine monooxygenase (72), and ADH of Z. mobilis (6,69) contain high-spin ) and the dithionitereduced enzyme (predominantly Fe 2ϩ ) were equally active. The weak visible absorption of pure ADH presumably arises from the ferric form of the enzyme (17,37,46).…”

Phenylalanine Hydroxylase from Chromobacterium violaceum

“…The results indicated that DTT played a mechanistic role in addition to recycling the oxidized pterin to its reduced state during the catalytic turnover. Reductive activation by a cofactor or dithionite to form the catalytically active enzyme species was reported for the phenylalanine hydroxylase from rat liver (24,25). Spectroscopic studies linked the reductive activation to the conversion of iron bound to the enzyme from Fe(III) to Fe(II) (25).…”