Spectroscopic and electronic structure studies of aromatic electrophilic attack and hydrogen-atom abstraction by non-heme iron enzymes

Abstract

(4-Hydroxy)mandelate synthase (HmaS) and (4-hydroxyphenyl)pyruvate dioxygenase (HPPD) are two ␣-keto acid dependent mononuclear non-heme iron enzymes that use the same substrate, (4-hydroxyphenyl)pyruvate, but exhibit two different general reactivities. HmaS performs hydrogen-atom abstraction to yield benzylic hydroxylated product (S)-(4-hydroxy)mandelate, whereas HPPD utilizes an electrophilic attack mechanism that results in aromatic hydroxylated product homogentisate. These enzymes provide a unique opportunity to directly evaluate the similarities and differences in the reaction pathways used for these two reactivities. An Fe II methodology using CD, magnetic CD, and variable-temperature, variable-field magnetic CD spectroscopies was applied to HmaS and compared with that for HPPD to evaluate the factors that affect substrate interactions at the active site and to correlate these to the different reactivities exhibited by HmaS and HPPD to the same substrate. Combined with density functional theory calculations, we found that HmaS and HPPD have similar substrate-bound complexes and that the role of the protein pocket in determining the different reactivities exhibited by these enzymes (hydrogen-atom abstraction vs. aromatic electrophilic attack) is to properly orient the substrate, allowing for ligand field geometric changes along the reaction coordinate. Elongation of the Fe IV AO bond in the transition state leads to dominant Fe III OO •؊ character, which significantly contributes to the reactivity with either the aromatic -system or the COH -bond. magnetic circular dichroism ͉ density functional calculations ͉ reaction coordinates

Spectroscopic and Quantum Chemical Studies on Low-Spin FeIVO Complexes: Fe−O Bonding and Its Contributions to Reactivity

“…Two different reaction channels were identified for Fe IV =O (S=2) enzyme intermediates in previous studies. 81,85,86 The first also involves the unoccupied β-spin d(xz/ yz) orbitals. 81 This is the case for the catalytic cycle of the non-heme iron enzyme 4-hydroxymandelate synthase (HmaS), which is believed to involve a high-spin Fe IV =O intermediate performing an H-atom abstraction reaction.…”

“…81, 88 Thus, in addition to the more horizontal approach and interaction with the β-spin d(xz/yz) orbitals as in S=1 complexes and geometrically constrained S=2 systems, 81,85 a vertical orientation of the substrate and interaction with the α-spin Fe-O σ* d(z 2 ) orbital, i.e. a σ-FMO mode, is possible for the electrophilic attack by Fe IV =O (S=2) species in enzymatic cycles, as calculated for the non-heme iron enzyme 4-hydroxyphenylpyruvate dioxygenase (HPDD) 81 and for an unconstrained substrate. 86 Thus, in the determination of σ-vs. π-reaction pathways of the Fe IV =O (S=2) species in different enzymatic cycles an investigation of the restrictions imposed by the protein pocket is required.…”

Role of Substrate Positioning in the Catalytic Reaction of 4-Hydroxyphenylpyruvate Dioxygenase—A QM/MM Study

“…7,8,14 The structure of the HMS-Co(II)-HMA complex shows that HMA is bound to the metal Figure 2: Active sites in X-ray crystal structures of Amycolatopsis orientalis HMS-Co(II)-HMA and Pseudomonas fluorescens HPPD-Fe(II)-acetate complexes (PDB codes: 2R5V and 1CJX, respectively). Residue names in red for the first shell metal ligands; residue names in blue for the amino acids hydrogen bonding to the first shell metal ligands; ovals mark residues substituted in the mutagenetic studies; hydrophobic residues in green.…”

“…15 In the resting form of HPPD the active site iron is six-fold coordinated (2-His-1Glu facial triad and three water molecules) as revealed by X-ray crystal structures (PDB:1SP8,1SP9) and spectroscopic studies. 14,18,19 Results of steady-state kinetics measurements indicated that HPP binds to Fe(II) prior to dioxygen, while CO 2 is the first product released from the active site, similarly to other α-keto acid dependent enzymes. [20][21][22] Respail and co-workers performed QM/MM calculations to test the possible binding modes of HPP in the HPPD active site.…”

“…[24][25][26] With respect to the hydroxylation reactions, for HPPD it was proposed that electrophilic attack on the aromatic ring of HPA leads to a high-spin Fe(III)/ring radical σ-complex. 8,9,14 The subsequent migration of the acetic acid side chain was found to proceed in two steps:…”