Direct coordination of pterin to FeII enables neurotransmitter biosynthesis in the pterin-dependent hydroxylases.

The pterin-dependent nonheme iron enzymes hydroxylate aromatic amino acids to perform the biosynthesis of neurotransmitters to maintain proper brain function. These enzymes activate oxygen using a pterin cofactor and an aromatic amino acid substrate bound to the FeII active site to form a highly reactive FeIV = O species that initiates substrate oxidation. In this study, using tryptophan hydroxylase, we have kinetically generated a pre-FeIV = O intermediate and characterized its structure as a FeII-peroxy-pterin species using absorption, Mössbauer, resonance Raman, and nuclear resonance vibrational spectroscopies. From parallel characterization of the pterin cofactor and tryptophan substrate-bound ternary FeII active site before the O2 reaction (including magnetic circular dichroism spectroscopy), these studies both experimentally define the mechanism of FeIV = O formation and demonstrate that the carbonyl functional group on the pterin is directly coordinated to the FeII site in both the ternary complex and the peroxo intermediate. Reaction coordinate calculations predict a 14 kcal/mol reduction in the oxygen activation barrier due to the direct binding of the pterin carbonyl to the FeII site, as this interaction provides an orbital pathway for efficient electron transfer from the pterin cofactor to the iron center. This direct coordination of the pterin cofactor enables the biological function of the pterin-dependent hydroxylases and demonstrates a unified mechanism for oxygen activation by the cofactor-dependent nonheme iron enzymes.

Nuclear Resonance Vibrational Spectroscopic Definition of the Facial Triad FeIV═O Intermediate in Taurine Dioxygenase: Evaluation of Structural Contributions to Hydrogen Atom Abstraction.

The α-ketoglutarate (αKG)-dependent oxygenases catalyze a diverse range of chemical reactions using a common high-spin FeIV═O intermediate that, in most reactions, abstract a hydrogen atom from the substrate. Previously, the FeIV═O intermediate in the αKG-dependent halogenase SyrB2 was characterized by nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT) calculations, which demonstrated that it has a trigonal-pyramidal geometry with the scissile C-H bond of the substrate calculated to be perpendicular to the Fe-O bond. Here, we have used NRVS and DFT calculations to show that the FeIV═O complex in taurine dioxygenase (TauD), the αKG-dependent hydroxylase in which this intermediate was first characterized, also has a trigonal bipyramidal geometry but with an aspartate residue replacing the equatorial halide of the SyrB2 intermediate. Computational analysis of hydrogen atom abstraction by square pyramidal, trigonal bipyramidal, and six-coordinate FeIV═O complexes in two different substrate orientations (one more along [σ channel] and another more perpendicular [π channel] to the Fe-O bond) reveals similar activation barriers. Thus, both substrate approaches to all three geometries are competent in hydrogen atom abstraction. The equivalence in reactivity between the two substrate orientations arises from compensation of the promotion energy (electronic excitation within the d manifold) required to access the π channel by the significantly larger oxyl character present in the pπ orbital oriented toward the substrate, which leads to an earlier transition state along the C-H coordinate.

Evaluation of a concerted vs. sequential oxygen activation mechanism in α-ketoglutarate-dependent nonheme ferrous enzymes.

Determining the requirements for efficient oxygen (O2) activation is key to understanding how enzymes maintain efficacy and mitigate unproductive, often detrimental reactivity. For the α-ketoglutarate (αKG)-dependent nonheme iron enzymes, both a concerted mechanism (both cofactor and substrate binding prior to reaction with O2) and a sequential mechanism (cofactor binding and reaction with O2 precede substrate binding) have been proposed. Deacetoxycephalosporin C synthase (DAOCS) is an αKG-dependent nonheme iron enzyme for which both of these mechanisms have been invoked to generate an intermediate that catalyzes oxidative ring expansion of penicillin substrates in cephalosporin biosynthesis. Spectroscopy shows that, in contrast to other αKG-dependent enzymes (which are six coordinate when only αKG is bound to the FeII), αKG binding to FeII-DAOCS results in ∼45% five-coordinate sites that selectively react with O2 relative to the remaining six-coordinate sites. However, this reaction produces an FeIII species that does not catalyze productive ring expansion. Alternatively, simultaneous αKG and substrate binding to FeII-DAOCS produces five-coordinate sites that rapidly react with O2 to form an FeIV=O intermediate that then reacts with substrate to produce cephalosporin product. These results demonstrate that the concerted mechanism is operative in DAOCS and by extension, other nonheme iron enzymes.

Geometric and Electronic Structural Contributions to Fe/O2 Reactivity.

While two classes of non-heme iron enzymes use ferric centers to activate singlet organic substrates for the spin forbidden reaction with 3O2, most classes use high spin ferrous sites to activate dioxygen. These FeII active sites do not exhibit intense absorption bands and have an integer spin ground state thus are mostly EPR inactive. We have developed new spectroscopic methodologies that provide geometric and electronic structural insight into the ferrous centers and their interactions with cosubstrates for dioxygen activation and into the nature of the intermediates generated in these reactions. First, we present our variable-temperature variable-field magnetic circular dichroism (VTVH MCD) methodology to experimentally define the geometric and electronic structure of the high spin ferrous active site. Then, we focus on using Nuclear Resonance Vibrational Spectroscopy (NRVS, performed at SPring-8) to define geometric structure and VTVH MCD to define the electronic structure of the FeIII-OOH and FeIV=O intermediates generated in O2 activation and the spin state dependence of their frontier molecular orbitals (FMOs) in controlling reactivity. Experimentally validated reaction coordinates are derived for the anticancer drug bleomycin in its cleavage of DNA and for an alpha- ketoglutarate dependent dioxygenase in its selective halogenation over the thermodynamically favored hydroxylation of substrate.

O2 Activation by Nonheme FeII α-Ketoglutarate-Dependent Enzyme Variants: Elucidating the Role of the Facial Triad Carboxylate in FIH.

FIH [factor inhibiting HIF (hypoxia inducible factor)] is an α-ketoglutarate (αKG)-dependent nonheme iron enzyme that catalyzes the hydroxylation of the C-terminal transactivation domain (CAD) asparagine residue in HIF-1α to regulate cellular oxygen levels. The role of the facial triad carboxylate ligand in O2 activation and catalysis was evaluated by replacing the Asp201 residue with Gly (D201G), Ala (D201A), and Glu (D201E). Magnetic circular dichroism (MCD) spectroscopy showed that the (FeII)FIH variants were all 6-coordinate (6C) and the αKG plus CAD bound FIH variants were all 5-coordinate (5C), mirroring the behavior of the wild-type ( wt) enzyme. When only αKG is bound, all FIH variants exhibited weaker FeII-OH2 bonds for the sixth ligand compared to wt, and for αKG-bound D201E this is either extremely weak or the site is 5C, demonstrating that the Asp201 residue plays an important role in the wt enzyme in ensuring that the (FeII/αKG)FIH site remains 6C. Variable-temperature, variable-field (VTVH) MCD spectroscopy showed that all of the αKG- and CAD-bound FIH variants, though 5C, have different ground-state geometric and electronic structures, which impair their oxygen activation rates. Comparison of O2 consumption to substrate hydroxylation kinetics revealed uncoupling between the two half reactions in the variants. Thus, the Asp201 residue also ensures fidelity between CAD substrate binding and oxygen activation, enabling tightly coupled turnover.

A chameleon catalyst for nonheme iron-promoted olefin oxidation.

We report the chameleonic reactivity of two nonheme iron catalysts for olefin oxidation with H2O2 that switch from nearly exclusive cis-dihydroxylation of electron-poor olefins to the exclusive epoxidation of electron-rich olefins upon addition of acetic acid. This switching suggests a common precursor to the nucleophilic oxidant proposed to Fe(III)-η(2)-OOH and electrophilic oxidant proposed to Fe(V)(O)(OAc), and reversible coordination of acetic acid as a switching pathway.