Structure/function Correlations of Binuclear Non-heme Iron Enzymes and Their de Novo Models PDF Download
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Author: Rae Ana Snyder
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Languages : en
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Binuclear non-heme iron enzymes are pervasive in nature and catalyze a variety of biologically important reactions, including reactions relevant to the biosynthesis of DNA, fatty acid metabolism, the protection of pathogens from oxidative stress, cell signaling, iron storage, and many others. Elucidating the structures of their diiron active sites and the contributions of these structures to reactivity can provide molecular level insight into catalysis. The near IR circular dichroism (CD), magnetic circular dichroism (MCD), and variable temperature variable field (VTVH) MCD spectroscopies form a powerful methodology that allows for detailed structural understanding of the diiron active sites in these enzymes. Presented are studies that focus on myo-inositol oxygenase, an enzyme with significance to human health, and de novo designed diiron proteins that model native enzymes.
Author: Rae Ana Snyder
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Languages : en
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Book Description
Binuclear non-heme iron enzymes are pervasive in nature and catalyze a variety of biologically important reactions, including reactions relevant to the biosynthesis of DNA, fatty acid metabolism, the protection of pathogens from oxidative stress, cell signaling, iron storage, and many others. Elucidating the structures of their diiron active sites and the contributions of these structures to reactivity can provide molecular level insight into catalysis. The near IR circular dichroism (CD), magnetic circular dichroism (MCD), and variable temperature variable field (VTVH) MCD spectroscopies form a powerful methodology that allows for detailed structural understanding of the diiron active sites in these enzymes. Presented are studies that focus on myo-inositol oxygenase, an enzyme with significance to human health, and de novo designed diiron proteins that model native enzymes.
Author: Caleb Branson Bell
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Languages : en
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The foci of this dissertation are: 1) combined use of spectroscopies for mechanistic understanding of the oxygen reactions of various non-heme iron enzymes and related model complexes, and 2) the development of the recently described nuclear vibrational resonance spectroscopy (NRVS) coupled with density functional calculations (DFT) for characterization of non-heme iron enzyme intermediates. Binuclear non-heme iron enzymes are involved in many medically and industrially important processes such as DNA synthesis by ribonucleotide reductase (RNR), conversion of methane to methanol by methane monooxygenase (MMO), fatty acid desaturation by [Delta]9 desaturase, iron storage and homeostasis by ferritins, degradation of aromatic compounds by various bacterial monooxygenases (ToMO, T4MO, etc.) and antibiotic biogenesis by p-aminobenzoate N-oxygenase (AurF), etc. Interestingly, these diverse reactions typically begin with O2 reacting with a biferrous active site, coordinated by highly conserved protein ligands (ExxH motifs) in four [Alpha]-helix bundles. Moreover, spectroscopically and chemically similar intermediates can be detected in many of the enzyme systems. The best studied in this family are RNRs, where biferric peroxo intermediates (P and P'), and the high-valent Fe(III)Fe(IV) intermediate X have been stabilized and spectroscopically characterized in wt and numerous variants. De novo designed four [Alpha]-helix bundles have been synthesized (the ~140 amino acid dui ferri (DF) peptide family) and are good models for binuclear non-heme iron enzymes. These systems provide a protein environment and can be viewed as a bridge between inorganic model complexes and native proteins. The pseudo-symmetric single chain version (DFsc) coordinates two ferrous ions by two His and four Glu amino acid residues. Circular dichroism (CD), magnetic CD (MCD) and variable-temperature variable-field MCD (VTVH MCD) show that this "active site" in DFsc has a 4-coordinate and 5-coordinate (4C+5C) geometry that is weakly antiferromagnetically coupled (J [approximately equal to] --2 cm-1) indicative of [Mu]1,3 carboxylate bridges, highly similar to RNR biferrous structures. Extended x-ray absorption fine structure (EXAFS) data are consistent with this assignment and show that one terminal carboxylate residue coordinates in a bidentate fashion. Changes in the CD/MCD/VTVH MCD and EXAFS spectra in the Y51L and E11D variants show that the 4C site is proximal to (but not bound by) Y51 and the bidentate carboxylate is coordinated to the 5C iron. Open coordination positions on both irons allow for dioxygen to react rapidly with the biferrous site. The reaction of biferrous DFsc with dioxygen yields a 520 nm ([Epsilon] = [weak approximation to]1200 M-1cm-1) species with a formation rate of 2 s-1, again similar to RNR (the Class Ia RNR from Escherichia coli has a dioxygen reaction rate of ~1 s-1, however the first species formed (intermediate P) has [Lambda]max = 700 nm). The resonance Raman (rR) spectrum obtained by excitation into the 520 nm feature in DFsc (and the E11D variant) proves this chromophore arises from a Tyr to ferric charge transfer (CT) transition. The 520 nm feature is lost by substitution of Y51 but not Y18, thus Y51 binds to the site after reaction with dioxygen. Subsequent binding of Y51 functions as an internal spectral probe of the dioxygen reaction and as a proton source that would promote loss of hydrogen peroxide. Coordination by a ligand that functions as a proton source could be a structural mechanism used by natural binuclear iron enzymes to drive their reactions past peroxo biferric level intermediates. RNR's can be divided into 3 major classes based on the radical generating machinery. Class I RNR's utilize a dimetal cofactor that reacts with dioxygen and can be subdivided into Classes Ia, Ib and Ic based on sequence homology and metal dependency. Class Ia enzymes are the best studied an present in higher organisms including human (host) while Class Ib enzymes are typically found in pathogens. CD, MCD and VTVH MCD data on biferrous loaded Class Ib RNR from Bacillus cereus allow assignment of the active site as 4C+5C in solution, resolving discrepancies from available crystal structures. Differences in the zero-field splitting parameters (D and E) and magnetic coupling extracted from fits to the VTVH MCD data can be ascribed to differences in the bridging carboxylate conformations. FeII loading, monitored by CD, shows cooperative binding with Kd 100 mM, significantly stronger that the metal binding in Class Ia. This provides the pathogen a competitive advantage relative to host in physiological, iron-limited environments Returning to Class Ia, the recently discovered intermediate P' notably lacks structural definition. This is mainly due to the lack of spectroscopic handles from which to obtain the needed experimental data. What is know, however, is that this species directly forms intermediate X and is directly derived from the well-defined intermediate P. Spectroscopically, P' has Mössbauer isomer shifts ([lowercase Delta] = 0.52 and 0.45 mm/s) that are significantly lower than the cis-[Mu]1,2 peroxo P ([lowercase Delta] = 0.63 mm/s) and lacks the ~700 nm peroxo to ferric CT suggesting some change in coordination mode or protonation may be involved in P -- P'. Comparisons of the reduced and oxidized crystal structures show differences in carboxylate coordination modes and water binding that must occur at some stage along the reaction coordinate. All of these potential structural perturbations were systematically incorporated into computational models of the intermediate site and correlated with experimental data using density functional theory (DFT). Two potential reaction pathways consistent with available experimental data were found. The first involves water addition to Fe1 of the cis-[Mu]-1,2 peroxo intermediate P causing opening of a bridging carboxylate to form intermediate P' which has an increased electron affinity and is activated for proton-coupled electron transfer to form the Fe(III)Fe(IV) intermediate X. While the second, more energetically favorable pathway, involves addition of a proton to a terminal carboxylate ligand in the site which increases the electron affinity and triggers electron transfer to form X. Vibrational characterization could, in principle, distinguish these pathways. However, the lack of a reasonably intense chromophore precludes rR experiments. The recently available method of nuclear vibrational resonance spectroscopy (NRVS) does not have these chromophoric constraints and can provide the needed vibrational data for P'--and many other "spectroscopically challenged" intermediates in non-heme iron biochemistry. The vibrations enhanced in NRVS are typically lower in energy and differ from those observed in rR, thus studies on well defined model complexes are needed prior to intermediate studies. A series of mononuclear Fe(IV)=O have been characterized by NRVS coupled with DFT calculations to define NRVS spectral assignments and set a foundation for vibrational characterization of non-heme iron enzyme intermediates. These studies show that the NRVS spectrum is rich in structural information. Of the four Fe(IV)=O models, supported by the 1, 4, 8, 11-tetramethyl-1,4,8,11-tetraazacyclotetradecane (TMC); N, N-bis(2-pyridylmethyl)-N-bis(2-pyridyl) methylamine (N4Py); N-benzyl-N, N', N'-tris(2-pyridylmethyl)-1,2-diaminoethane (BnTPEN); and 1,1,1-tris{2-[N(2)-(1,1,3,3-tetramethylguanidino)]ethyl}amine (TMG3tren) ligand sets, only the trigional bipyramidal geometry (relative to the 6C approximatly C4v geometry of TMC, N4Py and BnTPEN) enforced by the TMG3tren ligand affords a high-spin species. Isotope sensitive Fe-O stretches are observed for all complexes at 820 to 831 cm-1. However, at lower energy (
Author: Yeonju Kwak
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Languages : en
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Book Description
Binuclear non-heme iron enzymes catalyze various reactions including H-atom abstraction, desaturation, hydroxylation, and electrophilic aromatic substitution through O2 activation. In addition, they protect cells from oxidative stress and regulate iron levels in the cell. These enzymes utilize two irons and have common structural motif of 2-His / 4-carboxylate. Despite the enzymes' structural similarities, subtle changes at their active sites allow these enzymes to have different reactivities. Understanding the active site structures of these enzymes and the key mechanistic features related to these structures can provide a basis for potential applications: they could be drug inhibition targets to treat cancer, diabetes, and pathogenic diseases; they could work as biocatalysts; and they could carry out bioremediation reactions. In this dissertation, studies that examine three binuclear non-heme iron and Mn/Fe enzyme active sites (class Ic ribonucleotide reductase, ferritin variants, and bacterioferritin) and peroxo-bridged biferric model complexes are described. A combined spectroscopic methodology of nuclear resonance vibrational spectroscopy (NRVS), circular dichroism (CD), magnetic circular dichroism (MCD), and variable temperature, variable field (VTVH) MCD is used to probe geometric and electronic structures of Mn and Fe centers in protein active site and in model complexes.
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Category : Biomolecules
Languages : en
Pages : 1186
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Author: Sam P. De Visser
Publisher: Royal Society of Chemistry
ISBN: 1849731810
Category : Science
Languages : en
Pages : 463
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Book Description
Mononuclear iron containing enzymes are important intermediates in bioprocesses and have potential in the industrial biosynthesis of specific products. This book features topical review chapters by leaders in this field and its various sub-disciplines.
Author: William P. Jencks
Publisher: Courier Corporation
ISBN: 9780486654607
Category : Science
Languages : en
Pages : 866
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Book Description
Exceptionally clear coverage of mechanisms for catalysis, forces in aqueous solution, carbonyl- and acyl-group reactions, practical kinetics, more.
Author: H. Sund
Publisher: Springer Science & Business Media
ISBN: 3642694675
Category : Science
Languages : en
Pages : 318
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Author: Kelong Fan
Publisher: Frontiers Media SA
ISBN: 2889668215
Category : Science
Languages : en
Pages : 151
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Author: Eun Yeol Lee
Publisher: Springer Nature
ISBN: 3030232611
Category : Science
Languages : en
Pages : 283
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This book offers a comprehensive overview of the microbiological fundamentals and biotechnological applications of methanotrophs: aerobic proteobacteria that can utilize methane as their sole carbon and energy source. It highlights methanotrophs’ pivotal role in the global carbon cycle, in which they remove methane generated geothermally and by methanogens. Readers will learn how methanotrophs have been employed as biocatalysts for mitigating methane gas and remediating halogenated hydrocarbons in soil and underground water. Recently, methane has also attracted considerable attention as a potential next-generation carbon feedstock for industrial biotechnology, because of its abundance and low price. Methanotrophs can be used as biocatalysts for the production of fuels, chemicals and biomaterials including methanobactin from methane under environmentally benign production conditions. Sharing these and other cutting-edge insights, the book offers a fascinating read for all scientists and students of microbiology and biotechnology.
Author: Sue Pavord
Publisher: Cambridge University Press
ISBN: 1108548377
Category : Medical
Languages : en
Pages : 362
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Book Description
Understand the rapidly growing complexities of obstetric hematology and high-risk pregnancy management, with experts in the field. Now in its second edition, this comprehensive and essential guide focuses on providing the best support for patients and clinical staff, to prevent serious complications in pregnancy and the post-partum period for both mother and baby. Wide-ranging and detailed, the guide offers discussions on basic principles of best care, through to tackling lesser-known hematological conditions, such as cytopenias and hemoglobinopathies. Updated with color illustrations, cutting-edge research, accurate blood film reproductions, and practical case studies, the revised edition places invaluable advice into everyday context. This unique resource is essential reading for trainees and practitioners in obstetrics, anesthesia, and hematology, as well as midwives, nurses, and laboratory staff. Clarifying difficult procedures for disease prevention, the guide ensures safety when the stakes are high. Reflecting current evidence-based guidelines, the updated volume is key to improving pregnancy outcomes worldwide.
