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The Development And Analysis Of Model Systems To Probe Proton Coupled Electron Transfer In Ribonucleotide Reductase Ia Of E Coli
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Book Synopsis The Development and Analysis of Model Systems to Probe Proton-Coupled Electron Transfer in Ribonucleotide Reductase Ia of E. Coli by : Bon Jun Koo
Download or read book The Development and Analysis of Model Systems to Probe Proton-Coupled Electron Transfer in Ribonucleotide Reductase Ia of E. Coli written by Bon Jun Koo and published by . This book was released on 2016 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Proton-coupled electron transfer (PCET) combines proton transfer with electron transfer to bypass high-energy intermediates. The ribonucleotide reductase (RNR) family of enzymes catalyzes the conversion of ribonucleotides to deoxynucleotides using amino acid radicals. The enzyme contains an efficient PCET pathway that transfers an electron and proton over a 35 Å distance across two subunits, the longest PCET pathway known in biology. The enzyme func-tions with very high fidelity, performing >105 turnovers before radical loss.
Book Synopsis Proton-coupled Electron Transfer in the Escherichia Coli Ribonucleotide Reductase by : Michelle Chia-yu Chang
Download or read book Proton-coupled Electron Transfer in the Escherichia Coli Ribonucleotide Reductase written by Michelle Chia-yu Chang and published by . This book was released on 2004 with total page 578 pages. Available in PDF, EPUB and Kindle. Book excerpt: (Cont.) Mutation of conserved tyrosines on the pathway, corresponding to Y356 and Y731, to phenylalanine effectively deactivates radical initiation. These results provide the first direct evidence of the radical transfer pathway of the class I RNRs and underscore the importance of aromatic amino acid radical intermediates for proton-coupled electron transfer in physiologically-relevant processes.
Book Synopsis Photoinitiated Proton-coupled Electron Transfer and Radical Transport Kinetics in Class la Ribonucleotide Reductase by : Arturo Alejandro Pizano
Download or read book Photoinitiated Proton-coupled Electron Transfer and Radical Transport Kinetics in Class la Ribonucleotide Reductase written by Arturo Alejandro Pizano and published by . This book was released on 2013 with total page 202 pages. Available in PDF, EPUB and Kindle. Book excerpt: Proton-coupled electron transfer (PCET) is a critical mechanism in biology, underpinning key processes such as radical transport, energy transduction, and enzymatic substrate activation. Ribonucleotide reductases (RNRs) rely on PCET to mediate the rate-limiting step in the synthesis of DNA precursors. E. coli class Ia RNR consists of two dimeric subunits: [alpha]2 contains the active site, while [beta]2 contains a stable diferric-tyrosyl radical cofactor. During turnover, transport occurs over 35 Ȧ, from Y122 in [beta]2 to C439 in [alpha]2) where an active-site thiyl radical mediates turnover. Radical transport is proposed to occur over a series of highly conserved redox-active amino acids, including Y356 in [beta]2,and Y731 and Y730 in [alpha]2 . This thesis examines three subject areas of PCET that pertain to RNR: Small-molecule model systems provide insights into tyrosine oxidation and radical generation. Under physiological conditions, tyrosine oxidation is accompanied by deprotonation and occurs by PCET. A critical factor in PCET reactions is the nature ofthe proton acceptor and the presence ofhydrogen bonding. In a modular model system, pyridyl-amino acid-methyl esters are appended to rhenium(I) tricarbonyl phenanthroline to yield rhenium-amino acid complexes. In dichloromethane solution, bases coordinate to tyrosine by hydrogen bonding. Emission kinetics reveal base-dependent oxidation by PCET. A photopeptide composed of the 19 C-terminal residues of [beta]2, fluorinated tyrosine in place of Y356, and a rhenium(I) bipyridine photooxidant enables photoinitated radical transport into [alpha]2. Transient absorption kinetics show rapid radical transport (105 s-1) that is only observed when both Y731 and Y730, are present, suggesting a critical role for the Y731-Y730, dyad for radical transport in RNR. An intact, photochemical [beta]2 enables studies of an [alpha]2:[beta]2 complex. A bromomethylpyridine rhenium(I) phenanthroline photooxidant labels a single surface-cysteine mutant of [beta]2 at position 355 to yield [Re]- [beta]2. Under flash-quench conditions, transient absorption reveals a tyrosine radical. [Re] -[beta]2 binds [alpha]2 and is capable of light-initiated substrate turnover. Transient emission quenching experiments reveal Y356 oxidation that is dependent on the presence of Y731 in [alpha]2. This result suggests that a Y356-Y731-Y730 triad mediates radical transport across the subunit interface and into [alpha]2.
Book Synopsis Mechanistic Studies of Proton-coupled Electron Transfer in Aminotyrosine- and Fluorotyrosine- Substituted Class Ia Ribonucleotide Reductase by : Ellen Catherine Minnihan
Download or read book Mechanistic Studies of Proton-coupled Electron Transfer in Aminotyrosine- and Fluorotyrosine- Substituted Class Ia Ribonucleotide Reductase written by Ellen Catherine Minnihan and published by . This book was released on 2012 with total page 398 pages. Available in PDF, EPUB and Kindle. Book excerpt: Ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to 2'- deoxynucleotides in all organisms. The class Ia RNR from Escherichia coli is active as an a2p2 complex and utilizes an unprecedented mechanism of reversible proton-coupled electron transfer (PCET) to propagate a stable tyrosyl radical (Yi22-) in P2 over a distance of >35 A to an active site cysteine (C4 3 9) in a2 on each turnover. Generation of the cysteinyl radical (C4 3 9-) initiates active site nucleotide reduction. Radical propagation over 35 A by a pure tunneling mechanism would be too slow to support the observed turnover number. Instead, long-range, reversible PCET is proposed to occur by radical hopping along a specific pathway of redox-active amino acids: ... The details of this mechanism are kinetically masked in wild-type RNR, and mutation of any of these residues to another native amino acid inactivates the enzyme. Recent development of technology for the in vivo, site-specific incorporation of unnatural amino acids into proteins has provided the opportunity to systematically perturb the native PCET pathway by introduction of tyrosine analogues with modified redox potentials and/or pKas. This thesis focuses on 3-aminotyrosine (NH2Y) and fluorotyrosines (FnYs). NH2Y has a lower reduction potential than Y and, when incorporated at the three sites of transient Ye formation, generates a thermodynamic minimum and reduces kcat sufficiently to allow characterization of NH2Y. intermediates. A kinetic model for catalysis by NH2Y-RNRs has been proposed from the mechanistic studies described herein. Furthermore, the ability to generate NH2Y* on the pathway has afforded the first characterization of a kinetically stable c2p2 complex. FnYs span a wide range of solution pKas and reduction potentials and thus may be used to investigate both PT and ET events. The evolution of an orthogonal, polyspecific tRNA/tRNA synthetase pair for FnYs is reported. FnYs at positions 356, 730, and 731 have been used to measure the pH dependence of RNR activity, whereas FnY-s at position 122 of $2 have been used as radical initiators to begin mapping the relative thermodynamic landscape of the PCET pathway.
Book Synopsis Proton-Coupled Electron Transfer by : Sebastiao Formosinho
Download or read book Proton-Coupled Electron Transfer written by Sebastiao Formosinho and published by Royal Society of Chemistry. This book was released on 2011-12-07 with total page 169 pages. Available in PDF, EPUB and Kindle. Book excerpt: Proton-coupled electron transfer (PCET) is emerging as an important new class of reactions and, over the past decade, great strides have been made in our understanding of them. PCET reactions are studied in many branches of chemistry and are omnipresent in biological processes. This book covers recent developments from both the theoretical and experimental points of view. It concentrates on the importance of PCET in biological systems and for bioenergetic conversion. The oxidation of water in Photosystem II to produce oxygen, and the reduction of protons to hydrogen by hydrogenase, for energy storage gets particular emphasis. Chemical reactivity is currently explained in terms of several scientific principles. One of them is the bond-breaking-bond-forming process and is conceptually based on potential energy surfaces. Another incorporates the role of Franck-Condon factors resulting from the overlap of vibrational wavefunctions. A third, the so-called solvent reorganization, involves solvent configuration around a charged species. PCET brings together such concepts and links them to quantum mechanical tunnelling of the electron particle. This book uses personal accounts of experimental examples to provide additional insight on this important topic. It starts by presenting a general overview of the main theoretical approaches and experimental applications. The chapters then go on to cover topics including: the application of the Marcus Cross Relation; the solvation of ionic systems; experimental approaches in biological redox systems; metal ion-coupled electron transfer, and electrochemical concerted proton-electron transfers.
Book Synopsis Towards New Model Systems for the Study of Proton-coupled Electron Transfer by : Jay Lee Yang
Download or read book Towards New Model Systems for the Study of Proton-coupled Electron Transfer written by Jay Lee Yang and published by . This book was released on 2009 with total page 46 pages. Available in PDF, EPUB and Kindle. Book excerpt: Two new model systems for the study of orthogonal proton-coupled electron transfer (PCET) have been developed. The first model system is based on Ru"(HzO)(tpy)(bpy) (tpy = 2,2';6',2"terpyridine, bpy = 2,2'-bipyridine) where methyl viologen (MV2 ) electron acceptors were appended to the ruthenium aqua complex through the bpy. Picosecond transient absorption measurements show that electron transfer from the excited state of the ruthenium complex to MV2+ occurs with [tau] -200 ps. Experiments performed in water and buffered solution at pH = 7 show no evidence of the loss of proton from the aqua ligand to the bulk solvent or to the phosphate buffer. A minor kinetic isotope effect for the rate of charge separation was found with kH/kD = 1.8 + 0.1 ps. Preliminary synthetic attempts of coupling the ET event to the PT event was accomplished by appending xanthene "Hangman" scaffolds to the 4' position of the tpy. The feasibility of modifying the xanthene scaffold to accommodate various hanging groups has been demonstrated. The second model system is based on Re'(CO)3(phen)(pyr) (phen = 1,10phenanthroline, pyr = pyridine) where tyrosine was appended to the rhenium complex through the axial pyr ligand. Unlike previous Re'(CO)3(bpy)(CN) (CN = cyanide) systems, substitution to the more rigid phen extended the lifetime of the excited state of the rhenium complex to 3.0 ls, which allowed PCET to occur from the tyrosine to the rhenium metal center and hydrogenbonded base in dichloromethane. This was inferred from substantial emission quenching of the rhenium-tyrosine complex through the titration of base (base = pyridine, imidazole, 2,4,6trimethylpyridine). Equilibrium constants measuring the extent of formation of the [rheniumtyrosine---base]+ species were found to correlate with the strength of the base based on aqueous pKa values.
Book Synopsis Proton-coupled Electron Transfer (PCET) Model Systems by : Ewuradjoa Gadzanku
Download or read book Proton-coupled Electron Transfer (PCET) Model Systems written by Ewuradjoa Gadzanku and published by . This book was released on 2013 with total page 84 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Kinetics and Dynamics Controlling Proton-coupled Electron Transfer in Ribonucleotide Reductase by : Lisa Olshansky
Download or read book Kinetics and Dynamics Controlling Proton-coupled Electron Transfer in Ribonucleotide Reductase written by Lisa Olshansky and published by . This book was released on 2015 with total page 310 pages. Available in PDF, EPUB and Kindle. Book excerpt: Proton-coupled electron transfer (PCET) reactions comprise a fundamental mechanism for energy transduction in nature. In catalyzing the conversion of ribonucleotides to deoxyribonucleotides, ribonucleotide reductase (RNR) performs reversible, long-range PCET over a pathway of redox active amino acids ([beta]-Y122 >/ [beta]-Y356 / [alpha]-Y731 / [alpha]-Y730 /
Book Synopsis Unraveling the Mechanistic Details of Proton Coupled Electron Transfer (PCET) Using Model Systems by : Afua Nti
Download or read book Unraveling the Mechanistic Details of Proton Coupled Electron Transfer (PCET) Using Model Systems written by Afua Nti and published by . This book was released on 2012 with total page 210 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Mechanistic Studies of the Class I Ribonucleotide Reductases from S. Cerevisiae and E. Coli by : Jie Ge
Download or read book Mechanistic Studies of the Class I Ribonucleotide Reductases from S. Cerevisiae and E. Coli written by Jie Ge and published by . This book was released on 2003 with total page 423 pages. Available in PDF, EPUB and Kindle. Book excerpt: (Cont.) The results of initial studies on the energetics of heterodimer formation using calorimetric techniques and site-directed mutagenesis are discussed in the context of the structures of Y2Y2, Y2Y4, and Y4Y4. As the discovery of a second R2 subunit is not unique to S. cerevisiae, the potential implication of the heterodimer in the regulation of deoxyribonucleotide synthesis is also considered. In the second part of this thesis, pre-steady state kinetics of the class I E. coli RNR is investigated using stopped-flow UV-visible spectroscopy and rapid chemical quench methods. These studies have led to a model in which the rate determining step in catalysis is a physical step prior to proton-coupled electron transfer (PCET), nucleotide reduction, and reverse PCET. These results provide a crucial first step in understanding radical initiation over 35 [angstroms].
Book Synopsis The Electron Transfer Properties of the R2 Protein of Ribonucleotide Reductase from Escherichia Coli by : Kathleen Elizabeth Silva
Download or read book The Electron Transfer Properties of the R2 Protein of Ribonucleotide Reductase from Escherichia Coli written by Kathleen Elizabeth Silva and published by . This book was released on 1995 with total page 390 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Mechanistic Investigations of the Radical Transport Pathway in Fluorotyrosine-substituted Class Ia Ribonucleotide Reductases by : Kanchana Ravichandran
Download or read book Mechanistic Investigations of the Radical Transport Pathway in Fluorotyrosine-substituted Class Ia Ribonucleotide Reductases written by Kanchana Ravichandran and published by . This book was released on 2016 with total page 326 pages. Available in PDF, EPUB and Kindle. Book excerpt: Ribonucleotide reductase (RNR) catalyzes the reduction of nucleotides to 2'- deoxynucleotides, providing the monomeric precursors for DNA replication and repair. The focus of this thesis is on the E. coli class la RNR that is composed of two homodimeric subunits ([alpha]a2 and [beta]2), which form an active [alpha]2[beta]2 complex. A stable diferric-tyrosyl radical (Y122*) in [beta]2 reversibly oxidizes an active site cysteine (C439*) in [alpha]2 via multiple proton-coupled electron transfer (PCET) steps through conserved aromatic amino acid residues: Y122* - [W48] - Y356 in [beta]2 to Y731 - Y730 - C439 in [alpha]2. The transient C439* is responsible for initiating nucleotide reduction. Long-range radical transport (RT) and nucleotide reduction are kinetically masked by rate-limiting protein conformational changes. Herein, the stable Y1228 is site-specifically replaced with a 2,3,5-trifluorotyrosyl radical (2,3,5-F3Y*) that modulates the driving force for RT. This 2,3,5-F3Y-substituted RNR perturbs PCET kinetics such that a radical intermediate (Y356*) can be observed and characterized. Rapid kinetic studies demonstrate that Y356* is kinetically and chemically competent for nucleotide reduction, and provide the first evidence for a pathway Yo that can complete the RNR catalytic cycle. Temperature and pH dependent studies show equilibration of the stable 2,3,5-F3Y* with the pathway radical intermediate, Y356*. These data are corroborated by similar experiments performed with 3,5-difluorotyrosine (3,5-F2Y) in place of Y356, which demonstrate equilibration of Y122*. with 3,5-F2Y*. These studies together provide insight into the thermodynamic landscape of the RT pathway. A model is proposed in which the RT pathway is thermodynamically uphill and driven forward by rapid irreversible water loss that occurs during nucleotide reduction. The 3,5-F2Y analog is further utilized to test the ability of E350, a conserved [beta]2 C-terminal tail residue, to function as the proton acceptor for Y356 or Y731 . A model is put forth in which E350 does not participate in proton transfer, but is involved in [alpha]2[beta]2 subunit interaction and in controlling radical initiation. Finally, an X-ray crystal structure of the active [alpha]2[beta]2 complex has remained elusive. Herein, Ni-NTA pull-down assays are presented, demonstrating that injection of a single electron into the diferric cluster site generates a stable [alpha]2[beta]2 complex. These studies afford the opportunity to structurally characterize the [alpha]2[beta]2 complex with the goal of understanding PCET across the [beta]a interface.
Book Synopsis Building Model Systems to Understand Proton-Coupled Electron Transfer in Heme by : Christina J. Hanson
Download or read book Building Model Systems to Understand Proton-Coupled Electron Transfer in Heme written by Christina J. Hanson and published by . This book was released on 2013 with total page 111 pages. Available in PDF, EPUB and Kindle. Book excerpt: Proton-Coupled Electron Transfer (PCET) is an important mechanistic motif in chemistry, which allows for efficient charge transport in many biological systems. We seek to understand how the proton and electron motions are coupled in a bidirectional system allowing for individual turning of the kinetics and thermodynamics. The target of interest is a biomimedic heme system allowing for a detailed mechanistic study of the formation of the oxidation states of heme, of particular interest the highly reactive Fe(IV)=O species. The bidirectional model is prepared using a hangman porphyrin with an axially coordinated to the metal center, and the electron transfer event is triggered by excitation of the porphyrin. The synthesis of this motif is discussed as well as initial studies into the binding of a coordinated electron acceptor to the metal center. In the future, the excited state of the acceptor will be used to trigger the electron transfer portion of the PCET event. To understand the signatures of different electron acceptors and binding to the metal center, a redox inactive zinc porphyrin is used as a model to allow for longer excited state lifetimes and well known transient signatures. Three diimide acceptors have been coordinated through a pyridine ring to the metal center of the porphyrin, and electron transfer was triggered both by excitation of the porphyrin and the acceptor. Lifetimes of the charge separated state were determined using picoseconds and nanosecond transient absorption. The acceptors are then coordinated to a symmetrical iron porphyrin in an attempt to understand the behavior of charge separation in the more complicated open d shell system. Spectroscopic data of both systems is shown.
Book Synopsis Regulation of Proton Coupled Electron Transfer from Amino Acids in Artificial Model Systems by : Martin Sjödin
Download or read book Regulation of Proton Coupled Electron Transfer from Amino Acids in Artificial Model Systems written by Martin Sjödin and published by . This book was released on 2004 with total page 65 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Cytochrome Oxidase by : Mårten Wikström
Download or read book Cytochrome Oxidase written by Mårten Wikström and published by . This book was released on 1981 with total page 216 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Expressed Protein Ligation by : Miquel Vila-Perelló
Download or read book Expressed Protein Ligation written by Miquel Vila-Perelló and published by Humana. This book was released on 2020-03-07 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book provides a comprehensive overview of Expressed Protein Ligation (EPL), detailing methods and protocols to generate site-specifically modified proteins. Chapters include an overview of the protein semi-synthesis field, as well as related areas that have contributed to the development of EPL such as protein splicing and peptide synthesis. Following the introductory chapters, the rest of the book guides readers through protocols to perform EPL reactions, methods to synthesize peptide thioesters and to perform peptide and protein ligations, label proteins inside living cells, protocols for the semi-synthesis of phorphorylated, glycosylated and ubiquitylated proteins, synthesis and assembly of assymetrically modified nucleosomes, use of ligation auxiliaries and synthesis of cyclic proteins, as well as novel desulfurization strategies and use of selective Cys side chain protection to obtain precisely modified proteins.Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Expressed Protein Ligation: Methods and Protocols will ensure successful implementation of protein semi-synthesis methods to further study the structure and function of proteins.
Book Synopsis Mitochondria and Anaerobic Energy Metabolism in Eukaryotes by : William F. Martin
Download or read book Mitochondria and Anaerobic Energy Metabolism in Eukaryotes written by William F. Martin and published by Walter de Gruyter GmbH & Co KG. This book was released on 2020-12-07 with total page 269 pages. Available in PDF, EPUB and Kindle. Book excerpt: Mitochondria are sometimes called the powerhouses of eukaryotic cells, because mitochondria are the site of ATP synthesis in the cell. ATP is the universal energy currency, it provides the power that runs all other life processes. Humans need oxygen to survive because of ATP synthesis in mitochondria. The sugars from our diet are converted to carbon dioxide in mitochondria in a process that requires oxygen. Just like a fire needs oxygen to burn, our mitochondria need oxygen to make ATP. From textbooks and popular literature one can easily get the impression that all mitochondria require oxygen. But that is not the case. There are many groups of organismsm known that make ATP in mitochondria without the help of oxygen. They have preserved biochemical relicts from the early evolution of eukaryotic cells, which took place during times in Earth history when there was hardly any oxygen avaiable, certainly not enough to breathe. How the anaerobic forms of mitochondria work, in which organisms they occur, and how the eukaryotic anaerobes that possess them fit into the larger picture of rising atmospheric oxygen during Earth history are the topic of this book.
