Oxygen activation in mononuclear non-heme iron enzymes : requirement for the general mechanistic strategy and catalytic selectivity in sulfur transforming enzymes

Mononuclear non-heme iron (NHFe) enzymes catalyze a variety of reactions that are of pharmaceutical, industrial and environmental importance. A large number of these enzymes use a ferrous site to activate O2 for reaction with organic substrates, often requiring additional electrons from an outside source. These O2-activating NHFe enzymes can be divided into subclasses based on the cofactors used to supply these additional electrons. NHFe(II) enzymes generally possess a 2-His/1-(Asp/Glu) facial triad ligand set for binding of Fe(II), and they have been shown to utilize a general mechanistic strategy (GMS) for reaction with O2. The resting active sites of these enzymes (only Fe(II) bound) are six coordinate (6C) with the three protein derived ligands of the facial triad and three waters. When both cofactor and substrate are bound, the Fe(II) site loses a water ligand to become five coordinate (5C), opening a position for O2 to bind for reaction. NHFe(II) enzymes have d-d ligand field transitions in the near-IR region that are difficult to study because of their low absorption intensity and overlapping water and protein derived vibrations. A ferrous methodology using circular dichroism (CD), magnetic CD (MCD) and variable-temperature, variable-field (VTVH) MCD allows for determination of the active site geometric and electronic structures of these enzymes for development of functional insights. To extend these studies to the initial O2 binding step, NO is used as an unreactive analog of O2. The resulting {FeNO}7 (S = 3/2) complexes can be studied with absorbance, CD, MCD, VTVH MCD and electron paramagnetic resonance (EPR) to define the substrate interaction with the Fe(III) center that results from NO binding. These effects are used to gain insight into the experimentally inaccessible {FeO2}8 complexes with density functional theory calculations that are used to study the initial O2 activation steps. A major study of this thesis involves oxygen activation in deacetoxycephalosporin C synthase (DAOCS), a NHFe(II) enzyme that uses an alpha-ketoglutarate (aKG) cofactor to supply two electrons for its ring expansion of various penicillin substrates. A crystallographic study of DAOCS that did not observe simultaneous aKG and penicillin G (penG) binding to the same Fe(II) center led researchers to suggest a new sequential reaction for this enzyme, where reaction with aKG cofactor and O2 precedes substrate binding. Spectroscopic studies of the DAOCS Fe(II) site and its interaction with aKG and penG showed simultaneous binding of both is possible in solution. They further showed that the complex with aKG only was a mixture of 5C and 6C sites. A mixture of sites has not been observed in other enzymes of this class, which have been shown to remain as a single 6C site when aKG binds. This open coordination site allows for reaction with O2 in the absence of substrate, and this reaction was studied. Kinetic analysis of this reaction excludes the sequential reaction as a mechanistic possibility, because substrate binding cannot outpace the rapid decay of the intermediate that initiates the ring expansion. Comparison to the concerted reaction of the GMS, where both aKG and penG are bound before reaction with O2, shows that substrate binding activates the Fe site for a more kinetically efficient reaction with O2. This confirms the requirement for the general mechanistic strategy. The second major study of this thesis is on ETHE1, a member of a growing subclass of NHFe(II) enzymes that transforms sulfur containing substrates without a cofactor. ETHE1 dioxygenates glutathione persulfide (GSSH) to glutathione (GSH) and sulfite in a reaction that is similar to that of cysteine dioxygenase (CDO), but with monodentate (vs. bidentate in CDO) substrate coordination and a 2-His/1-Asp (vs. 3-His in CDO) ligand field. From MCD, GSS- binds directly to the iron active site causing coordination unsaturation to prime the site for O2 activation. {FeNO}7 complexes without and with GSSH were generated and spectroscopically characterized, and the new spectral features from persulfide binding to the Fe(III) were identified. Time-dependent density functional theory calculations were used to simulate the experimental absorbance spectra to determine the persulfide orientation in the active site (not known from crystallography). Comparison of these spectral features to those from monodentate cysteine binding in another enzyme of this subclass, isopenicillin N synthase (IPNS), shows that persulfide is a poorer donor than thiolate, but still results in an equivalent frontier molecular orbital (FMO) for reactivity. In IPNS, this reaction is an oxidative ring closure without incorporation of O2 atoms into the product. The persulfide dioxygenation reaction coordinate of ETHE1 was calculated, and while the initial steps are similar to the sulfur dioxygenation reaction coordinate of CDO, an additional hydrolysis step is required in ETHE1 to break the persulfide S-S bond. Unlike ETHE1 and CDO, which both oxygenate sulfur, IPNS oxidizes sulfur through an initial H-atom abstraction. Thus, the factors that control oxygenase vs. oxidase activity were evaluated. In general, sulfur oxygenation is thermodynamically favored and has a lower barrier for reactivity. However, in IPNS, second sphere residues in the active site pocket constrain the substrate raising the barrier for sulfur oxygenation relative to oxidation via H-atom abstraction.

Keywords: Chemistry.
Advisor: Solomon Edward I. 
Hodgson K. O. (Keith O.)
Waymouth Robert M.