Download or read book Probing the Heme Structure and Environment of Bacterial Heme Nitric Oxide OXygen Binding Domains with Resonance Raman Spectroscopy written by Rosalie Tran and published by . This book was released on 2010 with total page 242 pages. Available in PDF, EPUB and Kindle. Book excerpt: The Heme-Nitric oxide/OXygen binding (H-NOX) domain encompasses a family of proteins closely related (≤ 40% sequence identity) to the heme domain of soluble guanylate cyclase (sGC), the eukaryotic enzyme receptor for NO [1, 2]. sGC discriminates between NO and O2, and exclusively binds NO even in the presence of excess O2. Although some H-NOX domains have ligand binding properties that are identical to sGC, others additionally bind O2, including the atypical sGCs from Drosophila melanogaster and Caenorhabditis elegans [1, 3, 4]. The molecular basis of this ligand selectivity is not fully understood. In order to better understand the H-NOX domain, resonance Raman (RR) spectroscopy was employed in combination with isotopic substitution and site-directed mutagenesis to probe the heme pocket in Thermoanaerobacter tengcongensis (Tt H-NOX) as a model system for this family of heme proteins. Thus, the expression, purification, and RR characterization of Tt H-NOX and selected mutants were performed to elucidate the heme environmental properties that influence ligand binding. A striking feature of the O2-bound WT Tt H-NOX crystal structure is the presence of a distorted heme molecule [5]. RR investigation of Tt H-NOX proteins containing mutations at key conserved residues, Ile-5 and Pro-115, determined that the most dramatic heme conformational changes occurred in the O2-bound forms, and that the single P115A mutation generated a significantly relaxed chromophore. Decreased RR intensities were observed for several out-of-plane modes in the 400-750 cm-1 region known to be sensitive to ruffling and saddling deformations, as well as increased vibrational frequencies for the core heme skeletal modes. These changes demonstrated that the P115A heme conformation was considerably more relaxed than WT, with increased flexibility within the protein pocket that allowed for rapid sampling of alternate conformations. Another remarkable feature of this family is the spectroscopic observation of an unusually high [nu](C-O) frequency. To elucidate the interactions responsible for this property, mutations were made in Tt H-NOX to probe the distal pocket, the conserved Tyr-Ser-Arg (YxSxR) motif, and the heme-linkage site (His-102). RR spectra of these mutants indicated that H-bonding interactions between these residues and the heme significantly affected the CO bond by increasing the back-donation of the FeII d[pi] electrons into the CO [pi]* orbitals. The most significant change occurred upon disruption of the H-bonds between the YxSxR motif and the heme propionate groups, producing two dominant CO-bound heme conformations; one was structurally similar to WT, and the other conformer displayed [nu](C-O) downshifts of up to ̃70 cm-1. Based on these shifts, the most important factor contributing to the C-O stretching mode may be the neutralization of the negative charges on the propionate groups via the strictly conserved YxSxR motif. Finally, the role of the Tt H-NOX binding pocket in stabilizing the O2 complex was investigated. Evidence of H-bonds to the bound O2 was demonstrated in the RR spectra of Trp-9 and Tyr-140 mutants; the disruption of the H-bond network in these mutations increased [nu](Fe-O2) upon returning electron density to the Fe-O2 bond. In contrast, the addition of steric bulk to the H-NOX pocket decreased the [nu](Fe-O2) frequency relative to the WT protein. These shifts suggested that the bulky distal residues forced the H-NOX binding pocket into a more open position that increased the distance between Tyr-140 and the O2, thereby weakening this crucial H-bond. Thus, two distinct factors influence the [nu](Fe-O2) in Tt H-NOX: (i) electrostatic effects from H-bonding with Tyr-140, which increased the [nu](Fe-O2) frequency upon its removal; and (ii) steric effects which decreased [nu](Fe-O2) due to bond lengthening from a more loosely held O2 ligand. In summary, this work presents a comparative analysis of the heme environmental effects in Tt H-NOX. By directly probing the interactions between the heme chromophore and the protein pocket through a combination of RR spectroscopy and site-directed mutagenesis, this study may elucidate the biochemical properties of these H-NOX domains, and bring insight into the relationship between heme structure and protein function in this family.
Download or read book Characterization of a Bifunctional Globin Coupled Sensor Protein written by Dayna Patterson and published by . This book was released on 2021 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Heme proteins are found within all domains of life and play many key roles, including in diatomic gas sensing. Despite the large body of research on heme proteins, the molecular basis for the ability of the heme protein to modulate heme electronics and undergo conformational changes in response to gas binding to or the oxidation of the heme is still poorly understood. This is particularly true for the recently discovered class of heme proteins, known as globin coupled sensor (GCS) proteins. While evolutionarily related to mammalian globins, GCS proteins are found primarily in bacteria and archaea and serve as gas sensors that regulate activity of output domains within the same polypeptide chain. GCS proteins are involved in regulating bacterial biofilm formation (bacterial communities that form on surfaces and are recalcitrant to antibiotics), motility, and virulence. This dissertation presents the biochemical and structural characterization of DcpG, a diguanylate cyclase phosphodiesterase GCS protein from the bacterium Paenibacillus dendritiformis C454. DcpG is a bifunctional cyclic dimeric guanosine monophosphate (c-di-GMP) metabolic protein with GGDEF (Glu-Glu-Asp-Glu-Phe) and EAL (Glu-Ala-Leu) domains (synthesize and hydrolyze c-di-GMP, respectively) that are regulated by an N-terminal heme-bound sensor globin. C-di-GMP metabolic proteins are of great interest due to the role of c-di-GMP in controlling important bacterial phenotypes such as biofilm formation, motility, and virulence. However, the regulation of bifunctional c-di-GMP metabolic proteins is very poorly understood, despite putative homologs in thousands of bacterial genomes. The goal of the dissertation is to understand which gaseous ligand(s) regulate DcpG bifunctional enzyme activity and to probe its signal transduction mechanism. This first section of this dissertation reports the initial characterization of DcpG. DcpG bifunctional enzyme activity is differentially regulated by oxygen (O2) and nitric oxide (NO) in which this work highlights the first example, to our knowledge, of ligand binding to a heme sensor domain causing differential regulation of output domain activity. In addition, DcpG possesses unusual heme properties that include irreversible high midpoint potentials and rapid O2 and NO dissociation rates from the heme. By combining small angle X-ray scattering and negative stain electron microscopy data, the first structural model of a full-length globin coupled sensor protein was generated. These studies demonstrate for the first time that diatomic ligands (O2, NO, CO) can differentially modulate enzymatic domain activity within one heme sensor protein and allow bacteria to use a single protein to sense multiple gases within the environment. In the second section of the dissertation, the signal transduction mechanism within DcpG is probed. Enzyme kinetics, resonance Raman spectroscopy, and structural techniques are used to identify roles for heme-edge amino acids in modulating heme ligand binding and O2 dependent signal transduction. These studies identify an unusual heme-edge histidine residue that controls O2 dissociation, enzyme kinetics, and protein conformation. The last section of the dissertation focuses on establishing a high-throughput method to determine c-di-GMP specific phosphodiesterase activity of EAL domain containing enzymes. In addition, this section of the dissertation highlights future and ongoing work of DcpG characterization to further understand its structure and its role in vivo. In summary, this work has expanded our knowledge of GCS proteins and bifunctional c-di-GMP metabolic enzymes. This work suggest that DcpG may play a role in Paenibacillus dendritiformis C454 behavior in response to its gaseous environment. In addition, DcpG unusual heme properties play a role in its signal transduction mechanism, from gas sensing to bifunctional enzyme activation. Overall, this dissertation provides a foundation to understand of c-di-GMP enzymes that are activated by gaseous ligands.
Download or read book Characterization of the Nitric Oxide Sensing Domain of Soluable Guanylate Cyclase written by Yunde Zhao and published by . This book was released on 1999 with total page 474 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Cumulated Index Medicus written by and published by . This book was released on 2000 with total page 1796 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Hemoglobin and Oxygen Binding written by Chien Ho and published by North-Holland. This book was released on 1982 with total page 524 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Spectroscopic Probes of the Oxygen Binding Sites of Hemes and Hemeproteins written by Clyde Howard Barlow and published by . This book was released on 1974 with total page 155 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book The Chemistry of the Heme Domain of Nitric Oxide Synthase Toward a Detailed Mechanism of NG hydroxy l arginine Oxidation written by Joshua John Woodward and published by . This book was released on 2009 with total page 328 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Functional and Mechanistic Characterization of Bacterial Nitric Oxide Signaling Pathways written by Minxi Rao and published by . This book was released on 2016 with total page 96 pages. Available in PDF, EPUB and Kindle. Book excerpt: Nitric oxide (NO) is a well-established signaling molecule and cytotoxic agent in mammals. NO is synthesized by nitric oxide synthase (NOS) by macrophages at high concentrations as a key part of the host immune response, and at low concentrations in endothelial and neuronal cells as a signaling agent. In endothelial cells, the primary NO receptor is soluble guanylate cyclase (sGC), which contains a heme-nitric oxide/oxygen binding domain (H-NOX). Selective binding of NO to the H-NOX domain is responsible for activation of sGC. Thus, the mammalian NO signaling system involves NO synthesis by NOS, and NO sensing by the H-NOX domain of sGC. NOS and H-NOX proteins have also been identified in a number of bacterial species, including pathogens. Putative roles for bacterial NOS proteins include protection against oxidative stress and antibiotics, while bacterial H-NOX proteins have been shown to govern processes such as biofilm formation and bioluminescence via interactions with signaling proteins such as diguanylate cyclases (DGC) or histidine kinases (HK). Here, various aspects of NO signaling from three different organisms are characterized: the marine alphaproteobacterium Silicibacter sp. TrichCH4B; the soil-dwelling gammaproteobacterium Shewanella oneidensis, and the marine cyanobacterium Synechococcus sp. PCC 7335. This work and other recent studies seek to understand not only the diverse roles for NO in bacteria, but also the molecular mechanisms of bacterial NO signaling. Silicibacter sp. TrichCH4B is the first bacterial organism discovered to contain both an NOS and H-NOX, thus capable of both NO synthesis and sensing, analogous to mammalian systems. The H-NOX protein from Silicibacter is found in an operon adjacent to an HK, forming part of a two-component phospho-relay signaling network. The response regulator of the network was identified to be a diguanylate cyclase (DGC), which is inactivated upon phosphorylation and establishes the link between NO and intracellular cyclic-di-GMP levels, and consequently biofilm formation. It was also determined that Silicibacter NOS activity is stimulated by a signaling protein from an algal symbiont, Trichodesmium erythraeum, which is a major marine nitrogen fixer. Thus, in the presence of Trichodesmium, the increase in NOS activity results in Silicibacter biofilm formation and poising the two species for nutrient exchange, revealing a novel role for NO in interspecies communication and symbiosis. Given the diverse processes governed by NO/H-NOX signaling, it is crucial to understand the molecular mechanism by which H-NOX regulates HK autophosphorylation activity, the most common outcome of a NO-bound H-NOX. Here, the interaction and signal transduction between the H-NOX-HK signaling pair from Shewanella oneidensis are characterized. Binding kinetics measurements and analytical gel filtration revealed that NO-bound H-NOX has a tighter affinity for the HK, compared with H-NOX in the unliganded state, correlating binding affinity with kinase inhibition. Kinase activity assays with a panel of binding-deficient H-NOX mutants further reveal that while formation of the H-NOX-HK protein complex is required to stabilize the HK, H-NOX conformational changes upon NO binding are necessary for HK inhibition. Characterization of H-NOX proteins has led to an increased understanding of bacterial NO sensing. However, NO production in bacteria is less well-understood, and here the NOS protein from Synechococcus sp. PCC 7335 is characterized. Mammalian NOS proteins are comprised of a P450-like heme/oxidase domain responsible for catalysis, and a reductase domain responsible for electron transfer. While most bacterial NOS proteins discovered to date contain only the heme/oxidase domain, Synechococcus NOS contains both the oxidase and reductase domains, and additionally contains a predicted globin domain resembling bacterial flavohemoglobins. Spectroscopic and biochemical characterization of the globin indicated a possible role in redox communication in this novel class of bacterial NOS enzymes.
Download or read book cGMP Generators Effectors and Therapeutic Implications written by Harald H. H. W. Schmidt and published by Springer Science & Business Media. This book was released on 2008-12-17 with total page 581 pages. Available in PDF, EPUB and Kindle. Book excerpt: After the discovery of endogenous NO formation in the late '80s and the 1998 Nobel Prize in Physiology or Medicine, many researchers and physicians again became interested in the NO/sGC interaction and cGMP-dependent signaling. This book is an enthusiastic celebration of cyclic guanosine monophosphate (cGMP) and amply illustrates the importance of this field of science to patients and the way in which the field has evolved. It is exclusively devoted to this exciting and important signaling molecule, addressing all recent advances in understanding guanylate cyclase regulation, NO/sGC interactions, cGMP effector mechanisms and their pathophysiological and pharmacological implications. Particular attention will also be given to clinical applications of the novel cGMP-elevating drugs which are on the horizon, thus spanning the continuum from basic science to clinic.
Download or read book Structures of the Heme Chromophores in Cytochrome Oxidase written by Patricia Mary Callahan and published by . This book was released on 1983 with total page 476 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Dissertation Abstracts International written by and published by . This book was released on 2000 with total page 782 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Methods in Nitric Oxide Research written by Martin Feelisch and published by . This book was released on 1996-06-04 with total page 746 pages. Available in PDF, EPUB and Kindle. Book excerpt: This handbook describes methods for the measurement and investigation of nitric oxide. The topics discussed include the metabolism of substrates and cofactors, the detection of nitrogen oxides, the detection of nitrogen oxide synthase activity, and the detection of adducts and reaction products.
Download or read book 2 Oxoglutarate Dependent Oxygenases written by Christopher J Schofield and published by Royal Society of Chemistry. This book was released on 2015-05-06 with total page 508 pages. Available in PDF, EPUB and Kindle. Book excerpt: Since the discovery of the first examples of 2-oxoglutarate-dependent oxygenase-catalysed reactions in the 1960s, a remarkably broad diversity of alternate reactions and substrates has been revealed, and extensive advances have been achieved in our understanding of the structures and catalytic mechanisms. These enzymes are important agrochemical targets and are being pursued as therapeutic targets for a wide range of diseases including cancer and anemia. This book provides a central source of information that summarizes the key features of the essential group of 2-oxoglutarate-dependent dioxygenases and related enzymes. Given the numerous recent advances and biomedical interest in the field, this book aims to unite the latest research for those already working in the field as well as to provide an introduction for those newly approaching the topic, and for those interested in translating the basic science into medicinal and agricultural benefits. The book begins with four broad chapters that highlight critical aspects, including an overview of possible catalytic reactions, structures and mechanisms. The following seventeen chapters focus on carefully selected topics, each written by leading experts in the area. Readers will find explanations of rapidly evolving research, from the chemistry of isopenicillin N synthase to the oxidation mechanism of 5-methylcytosine in DNA by ten-eleven-translocase oxygenases.
Download or read book Cytochrome Complexes Evolution Structures Energy Transduction and Signaling written by William A. Cramer and published by Springer. This book was released on 2016-06-14 with total page 763 pages. Available in PDF, EPUB and Kindle. Book excerpt: An Introduction that describes the origin of cytochrome notation also connects to the history of the field, focusing on research in England in the pre-World War II era. The start of the modern era of studies on structure-function of cytochromes and energy-transducing membrane proteins was marked by the 1988 Nobel Prize in Chemistry, given to J. Deisenhofer, H. Michel, and R. Huber for determination of the crystal structure of the bacterial photosynthetic reaction center. An ab initio logic of presentation in the book discusses the evolution of cytochromes and hemes, followed by theoretical perspectives on electron transfer in proteins and specifically in cytochromes. There is an extensive description of the molecular structures of cytochromes and cytochrome complexes from eukaryotic and prokaryotic sources, bacterial, plant and animal. The presentation of atomic structure information has a major role in these discussions, and makes an important contribution to the broad field of membrane protein structure-function.
Download or read book The Smallest Biomolecules Diatomics and their Interactions with Heme Proteins written by Abhik Ghosh and published by Elsevier. This book was released on 2011-10-13 with total page 627 pages. Available in PDF, EPUB and Kindle. Book excerpt: This is not a book on NO biology, nor about hemoglobin, nor about heme-based sensors per se. Of course, it covers all these topics and more, but above all, it aims at providing a truly multidisciplinary perspective of heme-diatomic interactions. The overarching goal is to build bridges among disciplines, to bring about a meeting of minds. The contributors to this book hail from diverse university departments and disciplines – chemistry, biochemistry, molecular biology, microbiology, zoology, physics, medicine and surgery, bringing with them very different views of heme-diatomic interactions. The hope is that the juxtaposition of this diversity will lead to increased exchanges of ideas, approaches, and techniques across traditional disciplinary boundaries. The authors represent a veritable Who’s Who of heme protein research and include John Olson, Tom Spiro, Walter Zumft, F. Ann Walker, Teizo Kitagawa, W. Robert Scheidt, Pat Farmer, Marie-Alda Gilles-Gonzalez, and many other equally distinguished scientists. Extremely distinguished list of authors Multidisciplinary character – equally suitable for chemists and biochemists Covers the hottest topics in heme protein research: sensors, NO biology, new roles of hemoglobin, etc.
Download or read book Tetrapyrroles written by Martin Warren and published by Springer Science & Business Media. This book was released on 2009-12-21 with total page 406 pages. Available in PDF, EPUB and Kindle. Book excerpt: Excluding the biological polymers proteins, lipids and nucleic acids, modified tetrapyrroles are the biological molecules that have had the greatest impact on the evolution of life over the past 4 billion years. They are involved in a wide variety of fundamental processes that underpin central primary metabolism in all kingdoms of life, from photosynthesis to methanogenesis. Moreover, they bring colour into the world and it is for this reason that these compounds have been appropriately dubbed the ‘pigments of life’. To understand how and why these molecules have been so universally integrated into the life processes one has to appreciate the chemical properties of the tetrapyrrole scaffold and, where appropriate, the chemical characteristics of the centrally chelated metal ion. This book addresses why these molecules are employed in Nature, how they are made and what happens to them after they have finished their usefulness.
Download or read book Hemoglobin written by Jay F. Storz and published by . This book was released on 2019 with total page 258 pages. Available in PDF, EPUB and Kindle. Book excerpt: Provides a synthesis of our current understanding of hemoglobin (Hb) function and evolution, and illustrates how research on this protein has provided more general insights into mechanisms of protein evolution and biochemical adaptation.
