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This book highlights the many and varied catalytic activities of O2-dependent heme-iron enzymes, including monoxygenases and cytochrome P450, dioxygenases, oxidases and model heme systems required for postgraduate students and researchers in biochemistry and metallobiology.
Book Synopsis Dioxygen-dependent Heme Enzymes by : Masao Ikeda-Saito
Download or read book Dioxygen-dependent Heme Enzymes written by Masao Ikeda-Saito and published by Royal Society of Chemistry. This book was released on 2018-10-01 with total page 376 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book highlights the many and varied catalytic activities of O2-dependent heme-iron enzymes, including monoxygenases and cytochrome P450, dioxygenases, oxidases and model heme systems required for postgraduate students and researchers in biochemistry and metallobiology.
Aerobic organisms have evolved to utilise the intrinsic oxidising power of oxygen from the atmosphere. This so-called 'activation' of oxygen is often catalysed by a heme-containing enzyme. This book highlights the many and varied catalytic activities of O2-dependent heme–iron enzymes, including monoxygenases and cytochrome P450, dioxygenases, oxidases and model heme systems. Dioxygen-dependent Heme Enzymes will be a useful resource for postgraduate students and researchers in biochemistry and metallobiology working in, or moving into, research areas involving heme proteins.
Book Synopsis Dioxygen-dependent Heme Enzymes by : Masao Ikeda-Saito
Download or read book Dioxygen-dependent Heme Enzymes written by Masao Ikeda-Saito and published by Royal Society of Chemistry. This book was released on 2018-10-01 with total page 406 pages. Available in PDF, EPUB and Kindle. Book excerpt: Aerobic organisms have evolved to utilise the intrinsic oxidising power of oxygen from the atmosphere. This so-called 'activation' of oxygen is often catalysed by a heme-containing enzyme. This book highlights the many and varied catalytic activities of O2-dependent heme–iron enzymes, including monoxygenases and cytochrome P450, dioxygenases, oxidases and model heme systems. Dioxygen-dependent Heme Enzymes will be a useful resource for postgraduate students and researchers in biochemistry and metallobiology working in, or moving into, research areas involving heme proteins.
Book Synopsis Structure/function Correlations in Oxygen and Substrate Activating Mononuclear Non-heme Iron Enzymes by : Michael Lee Neidig
Download or read book Structure/function Correlations in Oxygen and Substrate Activating Mononuclear Non-heme Iron Enzymes written by Michael Lee Neidig and published by . This book was released on 2007 with total page 510 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Mononuclear Non-heme Iron Dependent Enzymes, Volume 703 focuses on methods for studying, characterizing, and leveraging the chemistry of mononuclear non-heme iron dependent enzymes. Chapters in this new release include Photoreduction for Rieske oxygenase chemistry, Insights into the Mechanisms of Rieske Oxygenases from Studying the Unproductive Activation of Dioxygen, Non-heme iron and 2-oxoglutarate enzymes catalyze cyclopropane and azacyclopropane formations, Obtaining precise metrics of substrate positioning in Fe(II)/2OG dependent enzymes using Hyperfine Sublevel Correlation Spectroscopy, Xe-pressurization studies for revealing substrate-entrance tunnels, and much more. Additional chapters cover A tale of two dehydrogenases involved in NADH recycling, Rieske oxygenases and/or their partner reductase proteins, Expression, assay and inhibition of 9-cis-epoxycarotenoid dioxygenase (NCED) from Solanum lycopersicum and Zea mays, Biocatalysis and non-heme iron enzymes, In vitro analysis of the three-component Rieske oxygenase cumene dioxygenase from Pseudomonas fluorescens IP01, Structure and function of carbazole 1,9a-dioxygenase, Characterization of a Mononuclear Nonheme Iron-dependent Mono-oxygenase OzmD in Oxazinomycin Biosynthesis, and much more. Provides detailed articles regarding how to study the structures and mechanisms of mononuclear non-heme iron dependent enzymes Guides readers on how to use partner proteins in non-heme iron enzyme catalysis Includes strategies to employ mononuclear non-heme iron enzymes in biocatalytic applications
Book Synopsis Mononuclear Non-heme Iron Dependent Enzymes by :
Download or read book Mononuclear Non-heme Iron Dependent Enzymes written by and published by Elsevier. This book was released on 2024-09-01 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Mononuclear Non-heme Iron Dependent Enzymes, Volume 703 focuses on methods for studying, characterizing, and leveraging the chemistry of mononuclear non-heme iron dependent enzymes. Chapters in this new release include Photoreduction for Rieske oxygenase chemistry, Insights into the Mechanisms of Rieske Oxygenases from Studying the Unproductive Activation of Dioxygen, Non-heme iron and 2-oxoglutarate enzymes catalyze cyclopropane and azacyclopropane formations, Obtaining precise metrics of substrate positioning in Fe(II)/2OG dependent enzymes using Hyperfine Sublevel Correlation Spectroscopy, Xe-pressurization studies for revealing substrate-entrance tunnels, and much more. Additional chapters cover A tale of two dehydrogenases involved in NADH recycling, Rieske oxygenases and/or their partner reductase proteins, Expression, assay and inhibition of 9-cis-epoxycarotenoid dioxygenase (NCED) from Solanum lycopersicum and Zea mays, Biocatalysis and non-heme iron enzymes, In vitro analysis of the three-component Rieske oxygenase cumene dioxygenase from Pseudomonas fluorescens IP01, Structure and function of carbazole 1,9a-dioxygenase, Characterization of a Mononuclear Nonheme Iron-dependent Mono-oxygenase OzmD in Oxazinomycin Biosynthesis, and much more. Provides detailed articles regarding how to study the structures and mechanisms of mononuclear non-heme iron dependent enzymes Guides readers on how to use partner proteins in non-heme iron enzyme catalysis Includes strategies to employ mononuclear non-heme iron enzymes in biocatalytic applications
MILS-15 provides an up-to-date review of the metalloenzymes involved in the activation, production, and conversion of molecular oxygen as well as the functionalization of the chemically inert gases methane and ammonia. Found either in aerobes (humans, animals, plants, microorganisms) or in anaerobes (so-called “impossible bacteria”) these enzymes employ preferentially iron and copper at their active sites, in order to conserve energy by redox-driven proton pumps, to convert methane to methanol, or ammonia to hydroxylamine or other compounds. When it comes to the light-driven production of molecular oxygen, the tetranuclear manganese cluster of photosystem II must be regarded as the key player. However, dioxygen can also be produced in the dark, by heme iron-dependent dismutation of oxyanions. Metalloenzymes Mastering Dioxygen and Other Chewy Gases is a vibrant research area based mainly on structural and microbial biology, inorganic biological chemistry, and environmental biochemistry. All this is covered in an authoritative manner in 7 stimulating chapters, written by 21 internationally recognized experts, and supported by nearly 1100 references, informative tables, and over 140 illustrations (many in color). MILS-15 provides excellent information for teaching; it is also closely related to MILS-14, The Metal-Driven Biogeochemistry of Gaseous Compounds in the Environment. Peter M. H. Kroneck is a bioinorganic chemist who is exploring the role of transition metals in biology, with a focus on functional and structural aspects of microbial iron, copper, and molybdenum enzymes and their impact on the biogeochemical cyles of nitrogen and sulfur. Martha E. Sosa Torres is an inorganic chemist, with special interests in magnetic properties of newly synthesized transition metal complexes and their reactivity towards molecular oxygen, applying kinetic, electrochemical, and spectroscopic techniques.
Book Synopsis Sustaining Life on Planet Earth: Metalloenzymes Mastering Dioxygen and Other Chewy Gases by : Peter M. H Kroneck
Download or read book Sustaining Life on Planet Earth: Metalloenzymes Mastering Dioxygen and Other Chewy Gases written by Peter M. H Kroneck and published by Springer. This book was released on 2015-02-23 with total page 362 pages. Available in PDF, EPUB and Kindle. Book excerpt: MILS-15 provides an up-to-date review of the metalloenzymes involved in the activation, production, and conversion of molecular oxygen as well as the functionalization of the chemically inert gases methane and ammonia. Found either in aerobes (humans, animals, plants, microorganisms) or in anaerobes (so-called “impossible bacteria”) these enzymes employ preferentially iron and copper at their active sites, in order to conserve energy by redox-driven proton pumps, to convert methane to methanol, or ammonia to hydroxylamine or other compounds. When it comes to the light-driven production of molecular oxygen, the tetranuclear manganese cluster of photosystem II must be regarded as the key player. However, dioxygen can also be produced in the dark, by heme iron-dependent dismutation of oxyanions. Metalloenzymes Mastering Dioxygen and Other Chewy Gases is a vibrant research area based mainly on structural and microbial biology, inorganic biological chemistry, and environmental biochemistry. All this is covered in an authoritative manner in 7 stimulating chapters, written by 21 internationally recognized experts, and supported by nearly 1100 references, informative tables, and over 140 illustrations (many in color). MILS-15 provides excellent information for teaching; it is also closely related to MILS-14, The Metal-Driven Biogeochemistry of Gaseous Compounds in the Environment. Peter M. H. Kroneck is a bioinorganic chemist who is exploring the role of transition metals in biology, with a focus on functional and structural aspects of microbial iron, copper, and molybdenum enzymes and their impact on the biogeochemical cyles of nitrogen and sulfur. Martha E. Sosa Torres is an inorganic chemist, with special interests in magnetic properties of newly synthesized transition metal complexes and their reactivity towards molecular oxygen, applying kinetic, electrochemical, and spectroscopic techniques.
Nonheme Iron Enzymes: Structures and Mechanisms, Volume 117, highlights new advances in the field, with this new volume presenting new and interesting chapters on the topics. Each chapter is written by an international board of authors. Targeted to a very wide audience of specialists, researchers and students Contains timely chapters written by well-renowned authorities in their field Includes a number of high quality illustrations, figures and tables
Book Synopsis Non-heme Iron Enzymes: Structures and Mechanisms by :
Download or read book Non-heme Iron Enzymes: Structures and Mechanisms written by and published by Academic Press. This book was released on 2019-09-28 with total page 134 pages. Available in PDF, EPUB and Kindle. Book excerpt: Nonheme Iron Enzymes: Structures and Mechanisms, Volume 117, highlights new advances in the field, with this new volume presenting new and interesting chapters on the topics. Each chapter is written by an international board of authors. Targeted to a very wide audience of specialists, researchers and students Contains timely chapters written by well-renowned authorities in their field Includes a number of high quality illustrations, figures and tables
Abstract : Computational chemistry methods have been extensively applied to investigate biological systems. This dissertation utilizes a multilevel computational approach to explore the dynamics and reaction mechanisms of two groups of enzymes belonging to non-heme Fe(II) and 2-oxoglutarate (2OG) dependent superfamily - histone lysine demethylases from class 7 and ethylene forming enzyme (EFE). Chapter 2 uncovers the role of conformational dynamics in the substrate selectivity of histone lysine demethylases 7A and 7B. The molecular dynamics (MD) simulations of the two enzymes revealed the importance of linker flexibility and dynamics in relative orientations of the reader (PHD) and the catalytic (JmjC) domains. Chapter 3 describes the use of combined quantum mechanics/molecular mechanics (QM/MM) and MD simulations to explore the reaction mechanism of histone lysine demethylases 7B (PHF8), including dioxygen activation, 2OG binding modes, and substrate demethylation steps. Importantly, the calculations imply the rearrangement of the 2OG C-1 carboxylate prior to dioxygen binding at a five-coordination stage in catalysis, highlighting the dynamic nature of the non-heme Fe-center. Chapter 4 develops a computational framework for identifying second coordination sphere (SCS) and especially long range (LR) residues relevant for catalysis through dynamic cross correlation analysis (DCCA) using the PHF8 as a model oxygenase and explores their effects on the rate determining hydrogen atom transfer step. The results from the QM/MM calculations suggest that DCCA can identify non-active site residues relevant to catalysis. Chapter 5 explores the unique catalytic mechanism of EFE. In particular, the study elucidates the atomic and electronic structure determinants that distinguish between ethylene formation and L-Arg hydroxylation reaction mechanisms in the EFE. The results indicated that synergy between the conformation of L-Arg and the coordination mode of 2OG directs the reaction toward ethylene formation or L-Arg hydroxylation. Chapter 6 demonstrates that applying an external electric field (EEF) along the Fe-O bond in the EFE·Fe(III)·OO.-·2OG·L-Arg complex can switch the EFE reactivity between L-Arg hydroxylation and ethylene generation. Overall, applying an EEF on EFE indicates that making the intrinsic electric field of EFE less negative and stabilizing the off-line binding of 2OG might increase ethylene generation while reducing L-Arg hydroxylation. Chapter 7 probes the role of the protein environment in modulating the dioxygen diffusion and binding and thus ultimately contributing to the diverging reactivities of PHF8 and EFE. Overall, the results of this dissertation together highlight the several catalytic strategies utilized by the non-heme Fe(II) and 2OG dependent enzymes for achieving their reaction outcomes. In the longer term, the results can be used to modulate the activities of these enzymes either through enzyme redesign or the generation of enzyme-selective inhibitors.
Book Synopsis MULTILEVEL COMPUTATIONAL INVESTIGATION INTO THE DYNAMICS AND REACTION MECHANISMS OF NON-HEME IRON AND 2-OXOGLUTARATE DEPENDENT ENZYMES by :
Download or read book MULTILEVEL COMPUTATIONAL INVESTIGATION INTO THE DYNAMICS AND REACTION MECHANISMS OF NON-HEME IRON AND 2-OXOGLUTARATE DEPENDENT ENZYMES written by and published by . This book was released on 2022 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Abstract : Computational chemistry methods have been extensively applied to investigate biological systems. This dissertation utilizes a multilevel computational approach to explore the dynamics and reaction mechanisms of two groups of enzymes belonging to non-heme Fe(II) and 2-oxoglutarate (2OG) dependent superfamily - histone lysine demethylases from class 7 and ethylene forming enzyme (EFE). Chapter 2 uncovers the role of conformational dynamics in the substrate selectivity of histone lysine demethylases 7A and 7B. The molecular dynamics (MD) simulations of the two enzymes revealed the importance of linker flexibility and dynamics in relative orientations of the reader (PHD) and the catalytic (JmjC) domains. Chapter 3 describes the use of combined quantum mechanics/molecular mechanics (QM/MM) and MD simulations to explore the reaction mechanism of histone lysine demethylases 7B (PHF8), including dioxygen activation, 2OG binding modes, and substrate demethylation steps. Importantly, the calculations imply the rearrangement of the 2OG C-1 carboxylate prior to dioxygen binding at a five-coordination stage in catalysis, highlighting the dynamic nature of the non-heme Fe-center. Chapter 4 develops a computational framework for identifying second coordination sphere (SCS) and especially long range (LR) residues relevant for catalysis through dynamic cross correlation analysis (DCCA) using the PHF8 as a model oxygenase and explores their effects on the rate determining hydrogen atom transfer step. The results from the QM/MM calculations suggest that DCCA can identify non-active site residues relevant to catalysis. Chapter 5 explores the unique catalytic mechanism of EFE. In particular, the study elucidates the atomic and electronic structure determinants that distinguish between ethylene formation and L-Arg hydroxylation reaction mechanisms in the EFE. The results indicated that synergy between the conformation of L-Arg and the coordination mode of 2OG directs the reaction toward ethylene formation or L-Arg hydroxylation. Chapter 6 demonstrates that applying an external electric field (EEF) along the Fe-O bond in the EFE·Fe(III)·OO.-·2OG·L-Arg complex can switch the EFE reactivity between L-Arg hydroxylation and ethylene generation. Overall, applying an EEF on EFE indicates that making the intrinsic electric field of EFE less negative and stabilizing the off-line binding of 2OG might increase ethylene generation while reducing L-Arg hydroxylation. Chapter 7 probes the role of the protein environment in modulating the dioxygen diffusion and binding and thus ultimately contributing to the diverging reactivities of PHF8 and EFE. Overall, the results of this dissertation together highlight the several catalytic strategies utilized by the non-heme Fe(II) and 2OG dependent enzymes for achieving their reaction outcomes. In the longer term, the results can be used to modulate the activities of these enzymes either through enzyme redesign or the generation of enzyme-selective inhibitors.
Cysteine dioxygenase (CDO) is an non-heme mononuclear iron enzymes that catalyzes the O2-dependent oxidation of L-cysteine (Cys) to produce cysteine sulfinic acid (CSA). CDO controls cysteine levels in cells and is a potential drug target for some diseases such as Parkinson's and Alzhermer's. Several crystal structures of CDO have been determined and they reveal a ferrous iron active site coordinated by three histidine residues. This feature is divergent from the monoanionic 2-histidine-1-carboxylate coordination typically observed within the non-heme mononuclear iron super family of oxidase/oxygenase enzymes. Furthermore, within 3.3 Å of the CDO active site iron is an unusual covalently crosslinked cysteine-tyrosine pair (C93-Y157). To date, only 3 other enzymes have been identified with a similar Cys-Tyr post-transitional modification and the role of this modification in CDO is still unknown. Due to the lack of structural evidence of oxygen-bound intermediates, the mechanism of CDO remains unclear. In this work, a transient intermediate Fe III -superoxo was discovered by chemical rescue reaction and characterized using UV-vis, EPR and resonance Mossbauer. To probe the influence of second-sphere enzyme-substrate interaction, the steady-state kinetics and O2/CSA coupling were measured for wild-type CDO and selected active site variants (Y157F, C93A, H155A). In additional, using CN- as a probe, the influence of the C93-T157 pair to the active site is investigated on EPR. Key substrate-enzyme interaction was also investigated by substrate specificity of CDO. Selected thiol-containing compounds were incubated with CDO for steady-state kinetic analysis using NMR. LC-MS confirmed the presence of products and dioxygenase activity.
Book Synopsis Mechanistic Study of Cysteine Dioxygenase, a Non-heme Mononuclear Iron Enzyme by : Wei Li
Download or read book Mechanistic Study of Cysteine Dioxygenase, a Non-heme Mononuclear Iron Enzyme written by Wei Li and published by . This book was released on 2014 with total page 181 pages. Available in PDF, EPUB and Kindle. Book excerpt: Cysteine dioxygenase (CDO) is an non-heme mononuclear iron enzymes that catalyzes the O2-dependent oxidation of L-cysteine (Cys) to produce cysteine sulfinic acid (CSA). CDO controls cysteine levels in cells and is a potential drug target for some diseases such as Parkinson's and Alzhermer's. Several crystal structures of CDO have been determined and they reveal a ferrous iron active site coordinated by three histidine residues. This feature is divergent from the monoanionic 2-histidine-1-carboxylate coordination typically observed within the non-heme mononuclear iron super family of oxidase/oxygenase enzymes. Furthermore, within 3.3 Å of the CDO active site iron is an unusual covalently crosslinked cysteine-tyrosine pair (C93-Y157). To date, only 3 other enzymes have been identified with a similar Cys-Tyr post-transitional modification and the role of this modification in CDO is still unknown. Due to the lack of structural evidence of oxygen-bound intermediates, the mechanism of CDO remains unclear. In this work, a transient intermediate Fe III -superoxo was discovered by chemical rescue reaction and characterized using UV-vis, EPR and resonance Mossbauer. To probe the influence of second-sphere enzyme-substrate interaction, the steady-state kinetics and O2/CSA coupling were measured for wild-type CDO and selected active site variants (Y157F, C93A, H155A). In additional, using CN- as a probe, the influence of the C93-T157 pair to the active site is investigated on EPR. Key substrate-enzyme interaction was also investigated by substrate specificity of CDO. Selected thiol-containing compounds were incubated with CDO for steady-state kinetic analysis using NMR. LC-MS confirmed the presence of products and dioxygenase activity.
MiaE is a non-heme diiron enzyme which catalyzes the O2-dependent hydroxylation of selected tRNA-nucleotides as part of a multienzyme posttranscriptional hypermodification pathway. This tRNA-modifying enzyme is postulated to signal O2- availability within the pathogenic bacteria, Salmonella typhimurium. Recombinant MiaE was cloned from Salmonella typhimurium genomic DNA, purified to homogeneity, and characterized by steady-state kinetics and spectroscopic techniques (UV-visible, Circular Dichroism, dual mode electron paramagnetic resonance (EPR), and Mössbauer) for comparison to other non-heme diiron enzymes. Remarkably, regardless of the substrate used in peroxide-shunt assays, hydroxylation of the terminal isopentenyl-C4-position was observed with > 97% E-stereoselectivity. The role of tRNA-protein macromolecular interactions on enzymatic reactivity and diiron site conformation was investigated studied using 17 nucleotide RNA oligomer corresponding to the anticodon stem and loop (ACSL) portion of substrate tRNAs. Steady-state reactions utilizing ACSL-substrates were investigated using a recombinant electron transfer chain. A variety of spectroscopic methods were employed to complement kinetic assays and observed structural perturbations within the protein-RNA secondary structure and diiron active site geometry. The tRNA-induced spectroscopic perturbations are reminiscent to be observed in the hydroxylase component of other monooxygenase enzymes upon binding their corresponding effector-protein. Thus, substrate-enzyme interactions may play a regulatory role in tRNA-hydroxylation for MiaE. Cysteine dioxygenase (CDO) is a non-heme mononuclear iron enzyme that catalyzes the O2-dependent oxidation of L-cysteine (L-Cys) to produce cysteine sulfinic acid (CSA). In contrast to mammalian CDO, all known bacterial CDO enzymes lack the Cys-Tyr post-translational modification. The relatively uncharacterized 'Gln-type' bacterial CDO enzymes offer a unique point of comparison to better understand the role of outersphere interactions in thiol dioxygense chemistry. In this work, the 'Gln-type' CDO enzyme was cloned from the soil bacteria Azotobacter vinelandii, purified to homogeneity, and characterized kinetically and spectroscopically for comparison to the Mm CDO enzyme. Remarkably, in steady-state assays using 3-mercaptopropionic acid (3-mpa), L-cysteine (cys), and cyteamine (ca), Av CDO exhibits nearly identical maximal velocity (kcat = v0/[E]) for each substrate (0.2
Book Synopsis Biochemical, Kinetic and Spectroscopic Characterizations of Non-heme Iron Oxygenase Enzymes by : Bishnu P. Subedi
Download or read book Biochemical, Kinetic and Spectroscopic Characterizations of Non-heme Iron Oxygenase Enzymes written by Bishnu P. Subedi and published by . This book was released on 2015 with total page 155 pages. Available in PDF, EPUB and Kindle. Book excerpt: MiaE is a non-heme diiron enzyme which catalyzes the O2-dependent hydroxylation of selected tRNA-nucleotides as part of a multienzyme posttranscriptional hypermodification pathway. This tRNA-modifying enzyme is postulated to signal O2- availability within the pathogenic bacteria, Salmonella typhimurium. Recombinant MiaE was cloned from Salmonella typhimurium genomic DNA, purified to homogeneity, and characterized by steady-state kinetics and spectroscopic techniques (UV-visible, Circular Dichroism, dual mode electron paramagnetic resonance (EPR), and Mössbauer) for comparison to other non-heme diiron enzymes. Remarkably, regardless of the substrate used in peroxide-shunt assays, hydroxylation of the terminal isopentenyl-C4-position was observed with > 97% E-stereoselectivity. The role of tRNA-protein macromolecular interactions on enzymatic reactivity and diiron site conformation was investigated studied using 17 nucleotide RNA oligomer corresponding to the anticodon stem and loop (ACSL) portion of substrate tRNAs. Steady-state reactions utilizing ACSL-substrates were investigated using a recombinant electron transfer chain. A variety of spectroscopic methods were employed to complement kinetic assays and observed structural perturbations within the protein-RNA secondary structure and diiron active site geometry. The tRNA-induced spectroscopic perturbations are reminiscent to be observed in the hydroxylase component of other monooxygenase enzymes upon binding their corresponding effector-protein. Thus, substrate-enzyme interactions may play a regulatory role in tRNA-hydroxylation for MiaE. Cysteine dioxygenase (CDO) is a non-heme mononuclear iron enzyme that catalyzes the O2-dependent oxidation of L-cysteine (L-Cys) to produce cysteine sulfinic acid (CSA). In contrast to mammalian CDO, all known bacterial CDO enzymes lack the Cys-Tyr post-translational modification. The relatively uncharacterized 'Gln-type' bacterial CDO enzymes offer a unique point of comparison to better understand the role of outersphere interactions in thiol dioxygense chemistry. In this work, the 'Gln-type' CDO enzyme was cloned from the soil bacteria Azotobacter vinelandii, purified to homogeneity, and characterized kinetically and spectroscopically for comparison to the Mm CDO enzyme. Remarkably, in steady-state assays using 3-mercaptopropionic acid (3-mpa), L-cysteine (cys), and cyteamine (ca), Av CDO exhibits nearly identical maximal velocity (kcat = v0/[E]) for each substrate (0.2
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.
Book Synopsis X-ray Absorption Spectroscopy of Heme and Non-heme Iron by : Samuel Aaron Wilson
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.