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Proton Coupled Electron Transfer In The Escherichia Coli Ribonucleotide Reductase
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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 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 High-field EPR and ENDOR Spectroscopy for Proton-coupled Electron Transfer Investigations in E.coli Ribonucleotide Reductase by : Tomislav Argirević
Download or read book High-field EPR and ENDOR Spectroscopy for Proton-coupled Electron Transfer Investigations in E.coli Ribonucleotide Reductase written by Tomislav Argirević and published by . This book was released on 2011 with total page 187 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis High-field EPR and ENDOR Spectroscopy for Proton-coupled Electron Transfer Investigations in E.coli Ribonucleotide Reductase by : Tomislav Argirevic
Download or read book High-field EPR and ENDOR Spectroscopy for Proton-coupled Electron Transfer Investigations in E.coli Ribonucleotide Reductase written by Tomislav Argirevic and published by . This book was released on 2011 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
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 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 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 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 Photochemical Ribonucleotide Reductase for the Study of Proton-coupled Electron Transfer by : Steven Y. Reece
Download or read book Photochemical Ribonucleotide Reductase for the Study of Proton-coupled Electron Transfer written by Steven Y. Reece and published by . This book was released on 2007 with total page 232 pages. Available in PDF, EPUB and Kindle. Book excerpt: (Cont.) To study the mechanism of this radical transport, we have developed photochemical RNRs wherein radical generation, transport, and enzymatic turnover can be initiated by UV-vis excitation of a peptide bound to the subunit containing the enzyme active site. This method allows us to observe Y*s competent for initiating turnover on the peptide bound to the protein subunit. Turnover assays with the wild-type and mutant proteins show that both the electron and proton move along a unidirectional pathway to affect radical transport in this subunit.
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 Structural Investigations of Class la Ribonucleotide Reductases by Electron Microscopy by : Gyunghoon Kang
Download or read book Structural Investigations of Class la Ribonucleotide Reductases by Electron Microscopy written by Gyunghoon Kang and published by . This book was released on 2019 with total page 178 pages. Available in PDF, EPUB and Kindle. Book excerpt: Ribonucleotide reductase (RNR) catalyzes the reduction of nucleotides to their 2'-deoxynucleotide counterparts. The class la RNR from Escherichia coli is composed of two homodimeric subunits [alpha]2 and [beta]2 that form an [alpha]2[beta]2 complex to perform nucleotide reduction. Chemistry is initiated by a thiyl-radical (C439·) in the active site of [beta]2 that is reversibly generated by a diferric-tyrosyl radical cofactor (Y122·) in [beta]2 by a series of proton-coupled electron transfer steps: Y122[beta] - [W48[beta]] - Y356[beta] - Y731[alpha] - Y730[alpha] - C439[alpha]. A high-resolution structure of the active [alpha]2[beta]2 complex has long eluded the field due to the weak and transient nature of the a2-P2 interaction. Previous studies revealed that perturbing radical transfer by incorporating unnatural amino acids along the transfer pathway, or by using mechanistic inhibitors that trap the radical in the active site, can extend the lifetime of the [alpha]2[beta]2 complex, allowing for structural studies. Here, we present our efforts to study the E. coli class la RNR [alpha]2[beta]2 complex, trapped using these different perturbation methods, using cryo-electron microscopy. The two [alpha]2[beta]2 structures presented here provide deeper insight into the structural dynamics of nucleotide reduction. We end with a brief discussion of class la RNR from T4 bacteriophage, which despite sharing high sequence identity to its host E. coli class la RNR, employs a very different mode of oligomeric regulation.
Book Synopsis Ribonucleotide Reductase by : Kristoffer Andersson
Download or read book Ribonucleotide Reductase written by Kristoffer Andersson and published by Nova Publishers. This book was released on 2008 with total page 236 pages. Available in PDF, EPUB and Kindle. Book excerpt: The subject of this book is the amazing enzyme ribonucleotide reductase (RNR), the enzyme responsible for the conversion of ribonucleotides to deoxyribonucleotides. The prerequisite for DNA-synthesis and DNA-repair in all living cells is the supply of the four deoxyribonucleotides. Such molecules result from the enzymatically difficult radical-induced reduction of ribonucleotides, a multistep chemical process catalyzed by RNR. RNR was the first enzyme in which the presence of an amino acid radical (a tyrosyl) in E. coli Class Ia RNR has been proven; since then several other biological amino acid radical species have been found on e.g. tryptophan, glycine, cysteine, lysine residues and on amino acid derived small cofactors like 2 tryptophanes in thryptophan-trypthanyl-radical or cysteine-tyrosyl-radical in other enzymes. As all known cellular life forms store their genetic information as DNA, RNR is likely to be found in all growing cells of every living organism, a fact that is confirmed by a rapidly increasing number of genomic screenings.
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 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 Oxidoreductases—Advances in Research and Application: 2012 Edition by :
Download or read book Oxidoreductases—Advances in Research and Application: 2012 Edition written by and published by ScholarlyEditions. This book was released on 2012-12-26 with total page 471 pages. Available in PDF, EPUB and Kindle. Book excerpt: Oxidoreductases—Advances in Research and Application: 2012 Edition is a ScholarlyEditions™ eBook that delivers timely, authoritative, and comprehensive information about Oxidoreductases. The editors have built Oxidoreductases—Advances in Research and Application: 2012 Edition on the vast information databases of ScholarlyNews.™ You can expect the information about Oxidoreductases in this eBook to be deeper than what you can access anywhere else, as well as consistently reliable, authoritative, informed, and relevant. The content of Oxidoreductases—Advances in Research and Application: 2012 Edition has been produced by the world’s leading scientists, engineers, analysts, research institutions, and companies. All of the content is from peer-reviewed sources, and all of it is written, assembled, and edited by the editors at ScholarlyEditions™ and available exclusively from us. You now have a source you can cite with authority, confidence, and credibility. More information is available at http://www.ScholarlyEditions.com/.
Book Synopsis Macromolecular Protein Complexes IV by : J. Robin Harris
Download or read book Macromolecular Protein Complexes IV written by J. Robin Harris and published by Springer Nature. This book was released on 2022-09-23 with total page 548 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book covers the latest findings of a wide variety of viral, prokaryotic and eukaryotic macromolecular protein complexes and builds upon the solid macromolecular foundations established by previous volumes of the Subcellular Biochemistry series. Thus, an almost encyclopaedic coverage of the broad field of protein complex structure and function has been established. The 17 interesting chapters included in this book have been organised into four sections: Soluble Protein Complexes, Membrane Protein Complexes, Fibrous Protein Complexes and Viral Protein Complexes. Significant topics present here are: Fatty Acid Synthase, the Fork Protection Complex, Ribonucleotide Reductase, the Kinetochore, G proteins, the FtsEX Complex, the Kainate Receptor, the Photosystem I-antenna, the Mycobacterial Arabinofuranosyltransferases, the the Bacterial Flagellum, the Actomyosin Complex, Motile Cilia, SLS Collagen Polymorphic Structures, and the Reovirus Capsid and Polymerase. Up-dates/expansion of chapter topics present in earlier volumes are now included in chapters here, e.g., those on Ferritin-like proteins and the Multi-tRNA Synthetase. The book is richly illustrated throughout, the result of an impressive integration of structural data from X-ray crystallography and cryo-electron microscopy. The functional aspects of protein-protein interactions are also given a high priority.
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.
