title: Model studies on p-hydroxybenzoate hydroxylase. The catalytic role of Arg-214 and Tyr-201 in the hydroxylation step.
abstract: A model C-(4a)-flavinhydroperoxide (FlHOOH) is described that contains the tricyclic isoalloxazine moiety, the C-4a-hydroperoxide functionality, and a beta-hydroxyethyl group to model the effect of the 2'-OH group of the ribityl side chain of native FADHOOH. The electronic structures of this reduced flavin (H(3)()Fl(red)()), its N1 anion (H(2)()Fl(red)()(-)()), oxidized flavin (HFl(ox)()), and FlHOOH have been fully optimized at the B3LYP/ 6-31+G(d,p) level of theory. This model C-4a-flavinhydroperoxide is used to describe the transition state for the key step in the paradigm aromatic hydroxylase, p-hydroxybenzoate hydroxylase (PHBH): the oxidation of p-hydroxybenzoate (p-OHB). The Tyrosine-201 residue in PHBH is modeled by phenol, and Arginine-214 is modeled by guanidine. Electrophilic aromatic substitution proceeds by an S(N)2-like attack of the aromatic sextet of p-OHB phenolate anion on the distal oxygen of FlHOOH 3. The transition structure for oxygen atom transfer is fully optimized [B3LYP/6-31+G(d,p)] and has a classical activation barrier of 24.9 kcal/mol. These data suggest that the role of the Tyr-201 is to orient the p-OHB substrate and to properly align it for the oxygen transfer step. Although the negatively charged phenolate oxygen does activate the C-3 carbon of p-OHB phenolate anion toward oxidation relative to ortho oxidation of the carboxylate anion, it appears that H-bonding the Tyr-201 residue to this phenolic oxygen stabilizes both the ground state (GS) and the transition state (TS) approximately equally and therefore plays only a minor role, if any, in lowering the activation barrier. Complexation of p-OHB with guanidine has only a modest effect upon the oxidation barriers. When the complex is in the form of a salt-bridge (10a), the barrier is only slightly reduced. When the TSs are placed in THF solvent (COSMO) with full geometry optimization, salt-bridge TS-A is slightly favored (DeltaDeltaE() = 2.3 kcal/mol).
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA.
authors.forename: Robert D
authors.initials: RD
authors.lastname: Bach
authors.forename: Olga
authors.initials: O
authors.lastname: Dmitrenko
mesh_terms: D015109:4-Hydroxybenzoate-3-Monooxygenase / Q000737:chemistry* / Q000378:metabolism*; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D004795:Enzyme Stability; D005415:Flavins / Q000737:chemistry / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism; D006900:Hydroxylation; D008956:Models, Chemical; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011789:Quantum Theory; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
chemical_list: D005415:Flavins; D014443:Tyrosine; D001120:Arginine; D006861:Hydrogen Peroxide; D015109:4-Hydroxybenzoate-3-Monooxygenase