Characterizing intermediates that govern reactivity in mononuclear nonheme iron enzymes

Iron enzymes are critical to the biosynthesis of neurotransmitters, natural products and antibiotics, the regulation of cellular oxygen levels and bioremediation. The α-ketoglutarate- (αKG)- and pterin-dependent enzymes are two classes of mononuclear nonheme iron enzymes that utilize a redox active 2-electron cofactor and an Fe(II) metal center to reduce molecular oxygen to form highly reactive Fe(IV)=O intermediates that catalyze their respective chemistries. Both of these enzyme classes have a 2-His/1-carboxylate facial triad motif, which leaves three cis sites available for cofactor, substrate and oxygen binding. These enzymes use a general mechanistic strategy, which ensures coordination saturation until both cofactor and substrate are bound in the active site before reacting with oxygen. This strategy is crucial as it prevents autooxidation and ensures that the oxidizing equivalents are used for substrate oxidation (i.e. coupled turnover). Additionally, this strategy is particularly important when only cofactor is bound to the active site, as all the reducing equivalents to produce the Fe(IV)=O are available. After these enzymes activate oxygen, identifying and characterizing reaction intermediates in these enzymes is critical to the elucidation of structure/function correlations that govern their reactivity. The focus of this thesis is to define the oxygen activation mechanisms of the αKG- and pterin-dependent enzymes. By leveraging site-selective spectroscopies, the structures of the different active sites and their intermediates have been correlated with their catalytic function. 1) Role of the Facial Triad Carboxylate in the αKG-dependent Enzymes FIH (Factor Inhibiting HIF [Hypoxia Inducible Factor]) is an αKG-dependent non-heme 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 oxygen activation and catalysis was evaluated by replacing the aspartate residue with glycine (D201G), alanine (D201A) and glutamate (D201E). Magnetic circular dichroism (MCD) spectroscopy showed that the (Fe(II))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 Fe(II)-water bonds for the sixth ligand compared to wt, and αKG bound D201E was found to be 5C, demonstrating that the facial triad aspartic acid residue plays an important role in the wt enzyme in ensuring that the (Fe(II)/αKG)FIH site remains 6C. Variable temperature, variable field (VTVH) MCD spectroscopy showed that all the αKG and CAD bound FIH variants, though 5C, have different ground state geometric and electronic structures than the wt enzyme, which impact their oxygen activation rates. Comparison of oxygen 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. 2) Evaluation of a Concerted vs Sequential Mechanism for Productive Chemistry Determining the requirements for efficient oxygen activation is key to understanding how enzymes maintain efficacy and mitigate unproductive, often detrimental, reactivity. For the αKG-dependent nonheme iron enzymes, both a concerted mechanism (both cofactor and substrate binding prior to reaction with oxygen) and a sequential mechanism (cofactor binding and reaction with oxygen precede substrate binding) have been previously 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. MCD spectroscopy shows that, in contrast to other αKG-dependent enzymes (which are 6-coordinate when only αKG is bound to the Fe(II)), αKG binding to Fe(II)-DAOCS results in ~45% 5-coordinate sites that, from Mössbauer spectroscopy, selectively react with oxygen relative to the remaining 6-coordinate sites. However, this reaction produces an Fe(III) species that does not catalyze productive ring expansion. Alternatively, simultaneous αKG and substrate binding to Fe(II)-DAOCS produces 5-coordinate sites that rapidly react with O2 to form an Fe(IV)=O intermediate that then reacts with substrate and produces cephalosporin product through an Fe(III)-OH and substrate radical species. Thus, these results demonstrate that the concerted mechanism is operative in DAOCS and, by extension, other nonheme iron enzymes. 3) Characterization of the Facial Triad Fe(IV)=O in the αKG-dependent enzymes and their Hydrogen Atom Abstraction Reactivity The αKG-dependent enzymes catalyze a diverse range of chemical reactions using a high-spin Fe(IV)=O intermediate, which performs a hydrogen atom abstraction (HAA) reaction with the substrate. Previously, the Fe(IV)=O intermediate in the halogenase SyrB2 has been structurally characterized and has a trigonal bipyramidal geometry with its substrate in a perpendicular orientation relative to the Fe-O bond. Taurine dioxygenase (TauD) is an αKG-dependent enzyme where an Fe(IV)=O intermediate was first characterized and has the taurine substrate positioned more along the Fe-O bond. By utilizing nuclear resonance vibrational spectroscopy (NRVS) with density functional theory (DFT) calculations, this study has defined the Fe(IV)=O intermediate in TauD as also having a trigonal bipyramidal geometry but with an aspartate residue replacing the equatorial halide in SyrB2. Using DFT generated square pyramidal, trigonal bipyramidal and six-coordinate Fe(IV)=O structures, we have evaluated the HAA reactivities of these geometries with two different substrate orientations (one orientation more along [σ-channel] and the other more perpendicular [π-channel] to the Fe-O bond). While Fe(IV)=O HAA reactivity along the σ-channel involves abstraction of a substrate α electron directly into dz2, reaction through the π-channel requires the promotion of an α electron from dπ* to dz2 before abstraction of the substrate α electron into dπ*, thus requiring additional energy to enable reactivity. Computational reaction coordinates for the three geometries and the two substrate orientations for the (TauD)Fe(IV)=O showed similar barriers for reactivity, i.e. all competent in performing HAA. The equivalence in reactivity between the two substrate orientations is due to the compensation of the promotion energy required to access the π channel by the increased polarization of the oxo-Fe π bond leading to an earlier transition state along the C-H coordinate. 4) Direct Coordination of Pterin to Iron Enables Oxygen Activation by 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 perform their chemistry by activating oxygen using a cofactor and substrate bound Fe(II) site to form highly reactive Fe(IV)=O species that initiate substrate oxidation. The consensus mechanism for reactivity in the pterin-dependent enzymes does not include the pterin cofactor directly interacting with the iron center, with much speculation over the undefined oxygen activation pathway. In this study, using tryptophan hydroxylase (TPH), we have generated a pre-Fe(IV)=O intermediate and characterized its structure as a Fe(II)-peroxy-pterin species using Mössbauer, resonance Raman and NRVS spectroscopies. Additionally, we have demonstrated that the pterin carbonyl is directly bound to the Fe(II) site, which is also the case in the pterin and tryptophan bound Fe(II)-TPH site before O2 reactivity. From reaction coordinate calculations, there is a 14 kcal/mol reduction in the oxygen activation barrier when the pterin carbonyl directly binds the Fe(II) 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

Keywords: Chemistry.
Advisor: Solomon Edward I. 
Kanan Matthew William
Stack T. (T. Daniel P.)