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The Reaction Mechanism of Phenylalanine Hydroxylase. - a Question of Coordination

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The Reaction Mechanism of Phenylalanine Hydroxylase. - a Question of Coordination Teigen Κ. et al.: The Reaction Mechanism of Phenylalanine Hydroxylase 27 Pteridines Vol. 16, 2005, pp. 27 - 34 The Reaction Mechanism of Phenylalanine Hydroxylase. - A Question of Coordination. Knut Teigen1, Vidar R. Jensen2, Aurora Martinez1 'Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009-Bergen, Norway, ^Department of Chemistry, University of Bergen, Allégaten 41, 5007-Bergen, Norway Abstract Phenylalanine hydroxylase (PAH) is a non-heme iron and tetrahydrobiopterin-dependent enzyme that catalyzes the hydroxylation of L-phenylalanine to L-tyrosine using dioxygen as additional substrate. The cofactor tetrahy- drobiopterin accepts one of the oxygen atoms of dioxygen during catalysis and also seems to be involved in pre- reduction of the active site iron from the ferric to the activated ferrous form. Structures of the truncated form of PAH in complex with substrate and cofactor are available, but the oxygen binding site and the actual mechanism of electron transfer are uncertain. It is believed that dioxygen binds directly to the metal, where it is activated, and several reaction mechanisms involving end-on binding of 02 have been proposed based on both experimen- tal studies and quantum mechanical calculations. However, in this work we aimed to investigate the possibility of side-on binding of dioxygen to the iron. Furthermore, NMR and recent high-resolution crystallographic stud- ies also place the cofactor in closer proximity to the iron, challenging the mechanistic conclusions from earlier crystallographic and computational studies. In this paper we report preliminary results from a density functional theory (DFT) study of the coordination of dioxygen to a structural model of PAH based on a recent crystallo- graphic structure. These results are compared with existing computational and experimental data and their impli- cations for the mechanism of the PAH-reaction are discussed. Particular attention is paid to the binding-mode of dioxygen and the iron-cofactor distance. Key words: Phenylalanine hydroxylase. Reaction mechanism, 5,6,7,8-tetrahydrobiopterin, Density functional theory Abbreviations: AAH, aromatic amino acid hydroxylases; BH2. L-erythro-7,8-dihydrobiopterin; BH4, (6R)-L-erythro-5,6,7.8-tetrahydrobiopterin; DFT, density functional theory; DZV, double zeta valence; EFP, effective core potentials: L-Phe, L-phenylalanine; MD, molecular dynamics; MM, molecular mechanics; NLE, norleucine; PAH, phenylalanine hydroxylase; QM, quantum mechanics; RMS, root mean square: SCF, self consistent field; SCI-PCM, self-consistent iso- density polarized continuum model; SP, single-point; TH, tyrosine hydroxylase; THA, thienyl- alanine; TS, transition state; TZDP, triple zeta double polarization Introduction prior to the disruption of the dioxygen bond. Initial crystallographic studies showed that in the resting The reaction catalyzed by phenylalanine hydroxy- enzyme the oxidized iron is coordinated by two his- lase (PAH; phenylalanine 4-monooxygenase) (EC tidines (His285 and His290) and one glutamic acid 1.14.16.1) involves the incorporation of one of the (Glu330) in addition to three water molecules, forming oxygen atoms from dioxygen into the substrate L-Phe, a six-coordinate octahedral iron environment (3). converting it to L-Tyr, and the other into the cofactor Subsequently, the binding site for both the cofactor and BH4 (1, 2). The active site iron in the ferrous state is the amino acid substrate in PAH was first solved by a believed to be responsible for the activation of oxygen combination of NMR and molecular modelling (4) and Correspondence to: Knut Teigen. Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009-Bergen, Norway, Tel.: 1(47)55586328: Fax: +(47)55586360; e-mail: knul.teigen(S'biomed.uib.no Pteridines/Vol. 16/No. 1 28 Teigen Κ. et al.: The Reaction Mechanism of Phenylalanine Hydroxylase later by X-ray crystallography (5-8). cules. L-Phe was found to bind in the second coordi- Many experiments have been carried out in order to nation sphere of the iron (4). The crystal structure of delineate the catalytic mechanism of the aromatic the ferrous binary complex of the enzyme with BH4 amino acid hydroxylases (AAH), but little experimen- showed, however, that in this state the iron was six- tal evidence has been presented about the exact nature coordinate with three bound water molecules (6). of the reaction. Despite this, several mechanisms of Later, the complex with BH4 and a substrate analogue hydroxylation have been proposed for the AAH (1,2, was reported to have only one water molecule bound to 7, 9-13) although the actual catalytic mechanism, the ferrous iron and Glu330 coordinated in a bidentate including the chemical nature of the hydroxylating manner, giving rise to a fivc-coordinatc metal atom intermediate, is still not clear. It has been proven for (7). However, in a repeated study with higher resolu- the catalytic cycle in mammalian PAH that the iron tion, the ferrous iron appeared to have no water mole- atom, which is in the ferric form in the isolated cules coordinated, rendering the iron in a four-coordi- enzyme, is prereduced to its ferrous form by the pterin nate configuration with the partially negatively cofactor prior to catalysis (1, 14). For tyrosine hydrox- charged keto-oxygen atom of the BH4 cofactor at a dis- ylase (EC 1.14.16.2, TH) it has also been shown that tance of 3.1 A from the iron (8). Thus, these recent the steady-state kinetic mechanism is sequential with crystallographic investigations indicate fewer, if any, the tetrahydropterin cofactor binding first, followed by coordinated water molecules, as well as a tighter asso- molecular oxygen and then the substrate (15). The ciation between iron and cofactor. Nevertheless, the steady-state kinetic mechanism of PAH seems to be coordination number of the iron prior to catalysis is not sequential as well, with some degree of randomness in completely clarified and in the present study we have the order of substrate addition ( 1, 14). For PAH from performed quantum chemical investigations based on the bacterium C. violaceum it has recently been report- DFT to investigate this issue. Our results are discussed ed that the mechanism is fully ordered, with the cofac- and compared to existing computational and experi- tor binding the active site first, L-Phe second and mental data. The implications of a different dioxygen dioxygen last ( 16). It seems clear for both enzymes binding mode and a tighter association of the cofactor that no product or intermediate is released prior to the on the reaction mechanism are discussed. binding of all the three substrates ( 1 ). Formation of a quaternaiy PAH-Fe(ll)BH4L-Phe02 complex also trims the iron atom for catalysis ( 1 7). The first observ- Methods able product of the pterin cofactor in the TH and PAH catalyzed reactions is a 4a-hydroxy-tetrahydropterin in The structure of the complexes of PAH with the which the oxygen atom in position 4a is derived from substrate and pterin cofactor was analyzed using the molecular oxygen (1, 10, 18). The other half of the Insightll software package and WebLabViewer (both oxygen molecule is found in the hydroxylated product from Accelrys Inc.). The quantum chemical geometry (L-Tyr). It has been postulated that a Felx=0 complex optimizations w ere performed using the three-parame- is an intermediate in the reaction mechanism (12). The ter hybrid density functional method of Becke (termed existence of FeIN-0 has been verified in model com- "B3LYP") (22) as implemented in the Gaussian 03 set pounds by a combination of Mössbauer and X-ray of programs (23). The unrestricted formulation was absorption spectroscopy ( 19, 20). Recent density func- used, i.e., "UB3LYP". Stationary points were opti- tional theory (DFT) studies confirmed that a reaction mized and characterized using algorithms involving mechanism for PAH based on an Fclv=0 intermediate analytic calculation of the first and second derivatives could be possible (11,21). In this mechanism, binding of the energy. Numerical integrations were performed of both substrate and cofactor triggers removal of one using the default "line" grid of Gaussian 03, consisting of the water molecules coordinated to the iron atom, of 75 radial shells and 302 angular points per shell, and and the water molecule is then replaced by dioxygen, the Gaussian 03 default values were chosen for the self thus forming an iron-peroxo-pterin intermediate. Upon consistent field (SCF) and geometry optimization con- heterolytic O-O bond cleavage, 4a-OH-BH4 is generat- vergence criteria. Thermochemical values were com- ed together with an 0H-Fe,v=0 intermediate, which in puted within the harmonic-oscillator, rigid-rotor, and turn is believed to be responsible for the hydroxylation ideal-gas approximations. The B3LYP-optimized of the aromatic amino acid. geometries were subjected to single-point (SP) energy and properties calculations. The SP calculations were Our group has investigated the binding of BH4 and performed using the "fine" grid described above and L-Phe to PAH by NMR (4). It was postulated that the the SCF procedure was converged to a RMS change of cofactor directly coordinates the active site iron, most the density matrix below 1.0-10 \ The basis sets that probably replacing one of the coordinated water mole- were used for the geometry optimizations (termed Pteridines/Vol. 16/Νυ. 1 Teigen Κ. et αι.: The Reaction Mechanism of Phenylalanine Hydroxylase 29 Table 1. Cofactor and substrate distances to the active site iron observed in different PAH complexes. Cofactor" and substrate'1' distances to the active site iron. The number of iron-coordinated water molecules"' are listed. Results obtained from NMR are commensurate with a model where one water molecule'1' is coordinated to the iron when both substrate and cofactor are present. Observed distance ranges0 from the MD simulations in (34) and unpublished results" from a 500 ps MD simulation of L-Phe in complex with hPAH are given.
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