title: 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 alpha-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) O bond in the transition state leads to dominant Fe(III) O(*-) character, which significantly contributes to the reactivity with either the aromatic pi-system or the C H sigma-bond.
authors.affiliation: Department of Chemistry, Stanford University, CA 94306, USA.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Neidig
authors.forename: Andrea
authors.initials: A
authors.lastname: Decker
authors.forename: Oliver W
authors.initials: OW
authors.lastname: Choroba
authors.forename: Fanglu
authors.initials: F
authors.lastname: Huang
authors.forename: Michael
authors.initials: M
authors.lastname: Kavana
authors.forename: Graham R
authors.initials: GR
authors.lastname: Moran
authors.forename: Jonathan B
authors.initials: JB
authors.lastname: Spencer
authors.forename: Edward I
authors.initials: EI
authors.lastname: Solomon
mesh_terms: D015111:4-Hydroxyphenylpyruvate Dioxygenase / Q000737:chemistry; D002942:Circular Dichroism; D019295:Computational Biology; D006859:Hydrogen / Q000737:chemistry*; D019159:Nonheme Iron Proteins / Q000737:chemistry*; D013057:Spectrum Analysis; D013816:Thermodynamics
chemical_list: D019159:Nonheme Iron Proteins; D006859:Hydrogen; D015111:4-Hydroxyphenylpyruvate Dioxygenase