Author: Samuel Aaron Wilson

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Published: 2012

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In biological systems dioxygen serves two essential functions, one as a terminal electron acceptor, and two as a biosynthetic agent. The latter role will be primarily the focus of this thesis, which will look at the role of dioxygen in specific mononuclear iron metalloenzyme and biomimetic model systems. During enzymatic turnover, the use of dioxygen as a biosynthetic agent involves the binding of dioxygen and the formation of one or more iron-peroxo (Fe-OO) or hydroperoxo (Fe-OOH) intermediates. This is followed by the controlled cleavage of the oxygen-oxygen double bond, a highly energetically favorable and exothermic process, to form a high-valent iron-oxo intermediate. For many enzymatic systems, these iron-oxygen species and high-valent intermediates are represent a significant obstacle as they are often difficult to trap and isolate in pure form, making them very challenging to study. Thus, biomimetic model complexes offer an excellent way to understand the mechanisms for reactivity and how the enzyme may tune the ligand environment around the iron center in order to govern the electronic structure of many of these key intermediate species. Chapter 1 will introduce the fields of iron non-heme enzymes, heme enzymes, and biomimetic model studies that play a key role in understanding the enzyme systems that they represent. Chapter 1 will also introduce the methodology of X-ray absorption spectroscopy, a specialized spectroscopic technique that has been invaluable in understanding these difficult to study systems. Chapter 2 looks at the enzyme tyrosine hydroxylase, a pterin-dependent non-heme iron enzyme that utilizes dioxygen to catalyze the hydroxylation of L-tyr to L-DOPA in the rate-limiting step of catecholamine neurotransmitter biosynthesis. X-ray absorption spectroscopy (XAS) and variable-temperature-variable-field magnetic circular dichroism (VTVH MCD) spectroscopy are combined with single-turnover kinetic experiments to investigate the geometric and electronic structure of the wild-type tyrosine hydroxylase and two mutants, S395A and E332A, and their interactions with substrates. This research showns that all three forms of tyrosine hydroxylase undergo 6-coordinate (6C) → 5-coordinate (5C) conversion with tyr + pterin, consistent with the general mechanistic strategy established for O2-activating non-heme iron enzymes. When the FeII site is 6C, the two-electron reduction of O2 to peroxide by FeII and pterin is favored over individual one-electron reactions demonstrating that both a 5C FeII and a redox-active pterin are required for coupled O2 reaction. When the FeII is 5C, the O2 reaction is accelerated by at least 2 orders of magnitude. Comparison of the kinetics of wild-type tyrosine hydroxylase, which produces FeIV=O + 4a-OH-pterin, and the E332A mutant, which does not, shows that the E332 residue plays an important role in directing the protonation of the bridged FeII-OO-pterin intermediate in wild-type to productively form the FeIV=O intermediate, which is responsible for the hydroxylation of L-tyr to L-DOPA. Chapter 3 uses a combination of nuclear resonance vibrational spectroscopy (NRVS) and extended X-ray absorption fine structure spectroscopy (EXAFS) to define the natures of ferric (FeIIIBLM) and activated bleomycin (ABLM), an important glycopeptide anticancer drug capable of effecting single- and double-strand DNA cleavage, as (BLM)FeIII-OH and (BLM)FeIII([eta]1-OOH) species, respectively. These spectroscopically defined species are then used in a series of density functional theory (DFT) calculations to show that the direct H-atom abstraction by ABLM is the most thermodynamically favored reaction pathway. Chapter 4 reports the first high-resolution x-ray crystal structure of an side-on ferric peroxide species in a non-heme iron biomimetic complex, [FeIII(OO)(TMC)]+, and a series of spectroscopic studies which looks at the pathway of interconversion from a iron(III)-peroxo complex to a iron(III)-hydroperoxo complex, followed by the homolytic O-O bond cleavage to an iron(IV)-oxo intermediate species. This work is followed by a series of reactivity studies that show that the iron(III)-hydroperoxo complex is the most reactive of the three in the deformylation of aldehydes, and has a similar reactivity to the iron(IV)--oxo complex in the C--H bond activation of alkylaromatics. These three species represent the three most biologically relevant iron-oxygen intermediates, and have all been synthesized utilizing the same macrocyclic ligand, which has allowed for the elucidation of key differences at the iron center and its bonding interactions with dioxygen, while the ligand environment remains fixed. Chapter 5 focuses in more detail on the high-valent FeIV=O species with the spectroscopic characterization of a new iron-oxo complex [FeIV=O(BQEN)]2+. This non-heme iron(IV)-oxo complex is shown to activate the C-H bonds of both alkanes and alcohols via a hydrogen-atom (H-atom) abstraction mechanism. This work also presents evidence for the formation of an additional high-valent iron-oxo intermediate species, [FeV=O(BQEN)]3+, which exhibits high reactivity in oxidation reactions and fast oxygen exchange with H218O. This FeV=O species is proposed as a possible active oxidant in the catalytic oxidation of alkanes and alcohols. Chapter 6 takes a more detailed look at the role of the equatorial ligand in the tuning in the iron-oxo unit by comparing the reactivity differences between two S = 1 non-heme iron-oxo species, [FeIV=O(TBC)(CH3CN)]2+ and [FeIV=O(TMC)(CH3CN)]2+. TBC, 1,4,8,11-tetrabenzyl-1,4,8,11-tetraazacyclotetradecane, is a equatorially constrained cyclam ligand which exhibits a greater than two orders of magnitude reactivity increase over TMC for both H-atom abstraction and oxo-transfer reactions. In this study, the S = 1 ground states of [FeIV=O(TBC)(CH3CN)]2+ and [FeIV=O(TMC)(CH3CN)]2+ are first structurally defined using XAS. Next, this structural information is utilized in a series of DFT calculations to look at what structural differences are responsible for the reactivity differences between these two very similar complexes and the mechanistic reactivity differences between the S = 1 and S = 2 surface for the biologically relevant H-atom abstraction and oxo-transfer reactions. Chapter 7 considers the electronic structure of the Fe--O2 bond in oxy-hemoglobin and oxy-myoglobin which is a long-standing issue in the field of bioinorganic chemistry. Here, spectroscopic studies have been complicated by the highly delocalized electronic structure of the porphyrin and calculations require interpretation of multi-determinant wavefunctions of a highly covalent site. Iron L-edge X-ray absorption spectroscopy (XAS) is used with a valence bond configuration interaction (VBCI) multiplet model to directly probe the electronic structure of the iron in the biomimetic FeO2 heme complex [Fe(pfp)(1-MeIm)O2] (pfp = meso-tetra([alpha], [alpha], [alpha], [alpha]-o-pivalamidophenyl)porphyrin). This method allows separate estimates of [sigma]-donor, [pi]-donor, and [pi]-acceptor interactions through ligand to metal charge transfer (LMCT) and metal to ligand charge transfer (MLCT) mixing pathways. The L-edge spectrum of [Fe(pfp)(1-MeIm)O2] is further compared to those of [FeII(pfp)(1-MeIm)2], [FeII(pfp)], and [FeIII(tpp)(ImH)2]+ (tpp = meso-tetraphenylporphyrin) which have FeII S = 0, FeII S = 1 and FeIII S = 1/2 ground states, respectively. These serve as the expected references for the three contributions to the ground state of oxy-pfp. This FeO2 S = 0 site is found to have significant [sigma]-donation and a strong [pi]-interaction of the O2 with the iron.

Download or read book X-ray Absorption Spectroscopy of Heme and Non-heme Iron written by Samuel Aaron Wilson and published by . This book was released on 2012 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: In biological systems dioxygen serves two essential functions, one as a terminal electron acceptor, and two as a biosynthetic agent. The latter role will be primarily the focus of this thesis, which will look at the role of dioxygen in specific mononuclear iron metalloenzyme and biomimetic model systems. During enzymatic turnover, the use of dioxygen as a biosynthetic agent involves the binding of dioxygen and the formation of one or more iron-peroxo (Fe-OO) or hydroperoxo (Fe-OOH) intermediates. This is followed by the controlled cleavage of the oxygen-oxygen double bond, a highly energetically favorable and exothermic process, to form a high-valent iron-oxo intermediate. For many enzymatic systems, these iron-oxygen species and high-valent intermediates are represent a significant obstacle as they are often difficult to trap and isolate in pure form, making them very challenging to study. Thus, biomimetic model complexes offer an excellent way to understand the mechanisms for reactivity and how the enzyme may tune the ligand environment around the iron center in order to govern the electronic structure of many of these key intermediate species. Chapter 1 will introduce the fields of iron non-heme enzymes, heme enzymes, and biomimetic model studies that play a key role in understanding the enzyme systems that they represent. Chapter 1 will also introduce the methodology of X-ray absorption spectroscopy, a specialized spectroscopic technique that has been invaluable in understanding these difficult to study systems. Chapter 2 looks at the enzyme tyrosine hydroxylase, a pterin-dependent non-heme iron enzyme that utilizes dioxygen to catalyze the hydroxylation of L-tyr to L-DOPA in the rate-limiting step of catecholamine neurotransmitter biosynthesis. X-ray absorption spectroscopy (XAS) and variable-temperature-variable-field magnetic circular dichroism (VTVH MCD) spectroscopy are combined with single-turnover kinetic experiments to investigate the geometric and electronic structure of the wild-type tyrosine hydroxylase and two mutants, S395A and E332A, and their interactions with substrates. This research showns that all three forms of tyrosine hydroxylase undergo 6-coordinate (6C) → 5-coordinate (5C) conversion with tyr + pterin, consistent with the general mechanistic strategy established for O2-activating non-heme iron enzymes. When the FeII site is 6C, the two-electron reduction of O2 to peroxide by FeII and pterin is favored over individual one-electron reactions demonstrating that both a 5C FeII and a redox-active pterin are required for coupled O2 reaction. When the FeII is 5C, the O2 reaction is accelerated by at least 2 orders of magnitude. Comparison of the kinetics of wild-type tyrosine hydroxylase, which produces FeIV=O + 4a-OH-pterin, and the E332A mutant, which does not, shows that the E332 residue plays an important role in directing the protonation of the bridged FeII-OO-pterin intermediate in wild-type to productively form the FeIV=O intermediate, which is responsible for the hydroxylation of L-tyr to L-DOPA. Chapter 3 uses a combination of nuclear resonance vibrational spectroscopy (NRVS) and extended X-ray absorption fine structure spectroscopy (EXAFS) to define the natures of ferric (FeIIIBLM) and activated bleomycin (ABLM), an important glycopeptide anticancer drug capable of effecting single- and double-strand DNA cleavage, as (BLM)FeIII-OH and (BLM)FeIII([eta]1-OOH) species, respectively. These spectroscopically defined species are then used in a series of density functional theory (DFT) calculations to show that the direct H-atom abstraction by ABLM is the most thermodynamically favored reaction pathway. Chapter 4 reports the first high-resolution x-ray crystal structure of an side-on ferric peroxide species in a non-heme iron biomimetic complex, [FeIII(OO)(TMC)]+, and a series of spectroscopic studies which looks at the pathway of interconversion from a iron(III)-peroxo complex to a iron(III)-hydroperoxo complex, followed by the homolytic O-O bond cleavage to an iron(IV)-oxo intermediate species. This work is followed by a series of reactivity studies that show that the iron(III)-hydroperoxo complex is the most reactive of the three in the deformylation of aldehydes, and has a similar reactivity to the iron(IV)--oxo complex in the C--H bond activation of alkylaromatics. These three species represent the three most biologically relevant iron-oxygen intermediates, and have all been synthesized utilizing the same macrocyclic ligand, which has allowed for the elucidation of key differences at the iron center and its bonding interactions with dioxygen, while the ligand environment remains fixed. Chapter 5 focuses in more detail on the high-valent FeIV=O species with the spectroscopic characterization of a new iron-oxo complex [FeIV=O(BQEN)]2+. This non-heme iron(IV)-oxo complex is shown to activate the C-H bonds of both alkanes and alcohols via a hydrogen-atom (H-atom) abstraction mechanism. This work also presents evidence for the formation of an additional high-valent iron-oxo intermediate species, [FeV=O(BQEN)]3+, which exhibits high reactivity in oxidation reactions and fast oxygen exchange with H218O. This FeV=O species is proposed as a possible active oxidant in the catalytic oxidation of alkanes and alcohols. Chapter 6 takes a more detailed look at the role of the equatorial ligand in the tuning in the iron-oxo unit by comparing the reactivity differences between two S = 1 non-heme iron-oxo species, [FeIV=O(TBC)(CH3CN)]2+ and [FeIV=O(TMC)(CH3CN)]2+. TBC, 1,4,8,11-tetrabenzyl-1,4,8,11-tetraazacyclotetradecane, is a equatorially constrained cyclam ligand which exhibits a greater than two orders of magnitude reactivity increase over TMC for both H-atom abstraction and oxo-transfer reactions. In this study, the S = 1 ground states of [FeIV=O(TBC)(CH3CN)]2+ and [FeIV=O(TMC)(CH3CN)]2+ are first structurally defined using XAS. Next, this structural information is utilized in a series of DFT calculations to look at what structural differences are responsible for the reactivity differences between these two very similar complexes and the mechanistic reactivity differences between the S = 1 and S = 2 surface for the biologically relevant H-atom abstraction and oxo-transfer reactions. Chapter 7 considers the electronic structure of the Fe--O2 bond in oxy-hemoglobin and oxy-myoglobin which is a long-standing issue in the field of bioinorganic chemistry. Here, spectroscopic studies have been complicated by the highly delocalized electronic structure of the porphyrin and calculations require interpretation of multi-determinant wavefunctions of a highly covalent site. Iron L-edge X-ray absorption spectroscopy (XAS) is used with a valence bond configuration interaction (VBCI) multiplet model to directly probe the electronic structure of the iron in the biomimetic FeO2 heme complex [Fe(pfp)(1-MeIm)O2] (pfp = meso-tetra([alpha], [alpha], [alpha], [alpha]-o-pivalamidophenyl)porphyrin). This method allows separate estimates of [sigma]-donor, [pi]-donor, and [pi]-acceptor interactions through ligand to metal charge transfer (LMCT) and metal to ligand charge transfer (MLCT) mixing pathways. The L-edge spectrum of [Fe(pfp)(1-MeIm)O2] is further compared to those of [FeII(pfp)(1-MeIm)2], [FeII(pfp)], and [FeIII(tpp)(ImH)2]+ (tpp = meso-tetraphenylporphyrin) which have FeII S = 0, FeII S = 1 and FeIII S = 1/2 ground states, respectively. These serve as the expected references for the three contributions to the ground state of oxy-pfp. This FeO2 S = 0 site is found to have significant [sigma]-donation and a strong [pi]-interaction of the O2 with the iron.