Spectroscopic and computational studies of dioxygen intermediates in non-heme iron enzymes and their models [electronic resource]

Non-heme iron enzymes play an important role in natural product and neurotransmitter biosynthesis, anticancer activity, and bioremediation. The ferrous non-heme iron (NHFe(II)) enzymes activate O2 for reaction with organic substrates. Understanding the nature of the activated O2 species in the NHFe(II) enzymes is important in elucidating their reaction mechanisms. O2 intermediates including Fe(IV)-oxo, Fe(III)-superoxo, and Fe(III)-hydroperoxo species have been trapped in several subclasses of NHFe(II) enzymes. Geometric and electronic structural definition of these intermediates provides significant insight into reactivity; however, in many cases, geometric structural data are not available by traditional spectroscopic methods. Nuclear resonance vibrational spectroscopy (NRVS) has recently emerged as an essential tool for structural definition of O2 intermediates in NHFe(II) enzymes. This technique involves probing vibrational sidebands of the Fe-57 nuclear transition using synchrotron radiation and is thus selective for iron sites, providing the complete set of vibrational normal modes involving iron displacement. Correlation to density functional theory (DFT) calculations allows for assignment of the iron vibrations in the NRVS spectrum and thus structural determination. In this thesis, NRVS is applied to three systems to provide insight into their structure and reactivity: 1) two high-spin Fe(III) models, a side-on Fe(III)-peroxo and an end-on Fe(III)-hydroperoxo, providing a set of spectroscopic handles to define peroxo protonation state and binding mode; 2) the high-spin Fe(III)-(hydro)peroxo intermediate in a Rieske dioxygenase; and 3) two Fe(III)-O2 intermediates in an extradiol dioxygenase. 1) Nuclear resonance vibrational spectroscopic definition of peroxy intermediates in non-heme iron sites: Fe(III)-(hydro)peroxy intermediates have been isolated in two classes of mononuclear non-heme Fe enzymes that are important in bioremediation: the Rieske dioxygenases and the extradiol dioxygenases. The binding mode and protonation state of the peroxide moieties in these intermediates are not well defined, due to a lack of vibrational structural data. The NRVS spectra of side-on Fe(III)-peroxy and end-on Fe(III)-hydroperoxy model complexes are presented and assigned using calibrated DFT calculations. DFT calculations are then used to define and understand the changes in the NRVS spectra that arise from protonation and from opening the Fe-O-O angle. Four spectroscopic handles are identified that enable definition of the binding mode and protonation state of Fe(III)-peroxy intermediates in mononuclear non-heme Fe enzymes. These structural differences are important in determining the frontier molecular orbitals available for reactivity. 2) NRVS studies of the peroxide shunt intermediate in a Rieske dioxygenase and its relation to the native Fe(II) O2 reaction: The Rieske dioxygenases are a major subclass of mononuclear non-heme iron enzymes that play an important role in bioremediation. Recently, a high-spin Fe(III)-(hydro)peroxy intermediate (BZDOp) has been trapped in the peroxide shunt reaction of benzoate 1,2-dioxygenase. Defining the structure of this intermediate is essential to understanding the reactivity of these enzymes. The NRVS data on BZDOp are presented, and its structure is assigned using these data coupled to experimentally calibrated DFT calculations. From this NRVS structure, the mechanism for the peroxide shunt reaction is defined. The relevance of the peroxide shunt to the native Fe(II)/O2 reaction is evaluated. For the native Fe(II)/O2 reaction, an Fe(III)-superoxo intermediate is found to react directly with substrate. This process, while uphill thermodynamically, is found to be driven by the highly favorable thermodynamics of proton coupled electron transfer, with an electron provided by the Rieske [2Fe-2S] center, at a later step in the reaction. These results offer important insight into the relative reactivities of Fe(III)-superoxo and Fe(III)-hydroperoxo species in nonheme Fe biochemistry. 3) NRVS definition of O2 intermediates in an extradiol dioxygenase with correlation to crystallography and reactivity: The extradiol dioxygenases are an important subclass of mononuclear non-heme Fe enzymes that catalyze the oxidative cleavage of catechols distal to their OH groups. These enzymes are important in bioremediation, and there has been significant interest in understanding how they activate O2. The extradiol dioxygenase homoprotocatechuate 2,3-dioxygenase (HPCD) provides an opportunity to study this process, as two O2 intermediates have been trapped and crystallographically defined using the slow substrate 4-nitrocatechol (4NC): a side-on Fe-O2-4NC species and a Fe-O2-4NC peroxy bridged species. Also with 4NC, two solution intermediates have been trapped in the H200N variant, where H200 provides a second-sphere hydrogen bond in the wild-type enzyme. While the electronic structures of these solution intermediates have been defined previously as Fe(III)-superoxo-catecholate and Fe(III)-peroxy-semiquinone, their geometric structures were unknown. NRVS is used to define the geometric structure of the H200N-4NC intermediates in HPCD. Parallel calculations are performed to define the electronic structures and protonation states of the wild-type HPCD-4NC intermediates, which are then correlated to the H200N intermediates through removal of H200 in silico. From this correlation, the nature of the initial active O2 intermediate is defined, and its reaction coordinate in generating the peroxy bridged species is evaluated. This provides insight into the relative reactivities of Fe(III)-superoxo and Fe(III)-hydroperoxo intermediates in non-heme Fe enzymes and into the role H200 plays in facilitating extradiol dioxygenation.

Keywords: Chemistry.
Advisor: Solomon Edward I. primary advisor. Thesis advisor
Hedman B. (Britt)
Hodgson K. O. (Keith O.)
Markland Thomas E.