DOCSLIB.ORG

Elucidation of Protein Interactions and the Re-Tuning of Porphyrin Electronics for Hydroxylases

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Elucidation of Protein Interactions and the Re-Tuning of Porphyrin Electronics for Hydroxylases University of South Carolina Scholar Commons Theses and Dissertations 2017 Elucidation Of Protein Interactions And The Re- Tuning Of Porphyrin Electronics For Hydroxylases Steven Charles Ratigan University of South Carolina Follow this and additional works at: https://scholarcommons.sc.edu/etd Part of the Chemistry Commons Recommended Citation Ratigan, S. C.(2017). Elucidation Of Protein Interactions And The Re-Tuning Of Porphyrin Electronics For Hydroxylases. (Doctoral dissertation). Retrieved from https://scholarcommons.sc.edu/etd/4482 This Open Access Dissertation is brought to you by Scholar Commons. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. ELUCIDATION OF PROTEIN INTERACTIONS AND THE RE-TUNING OF PORPHYRIN ELECTRONICS FOR HYDROXYLASES by Steven Charles Ratigan Bachelor of Arts The Citadel, 2005 Bachelor of Science University of South Carolina, 2009 Master of Arts The Citadel, 2012 Submitted in Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy in Chemistry College of Arts and Sciences University of South Carolina 2017 Accepted by: Thomas Makris, Major Professor F. Wayne Outten, Committee Member John Lavigne, Committee Member Zhengqing Fu, Committee Member Cheryl L. Addy, Vice Provost and Dean of the Graduate School © Copyright by Steven Charles Ratigan, 2017 All Rights Reserved. ii DEDICATION I dedicate this work to my family, who have always been there for me. Without their support, my time in academia would have been much more difficult. I would also like to thank Dr. David Donnell of The Citadel Dept. of Biology for teaching me everything I know about molecular biology, which proved to be a huge help early on in this program. iii ACKNOWLEDGEMENTS I would like to thank my mentor Dr. Makris for attempting to patiently deal with me for six years in this program. While I swore I would never be one of those people to sum things up with a generic quote, I’m going to do it anyway with “It was the best of times, it was the worst of times” - Charles Dickens I would also like to thank my fellow labmates. My early years were spent with two very interesting science personalities in Job Grant and Jason Hseih. Job nailed down a framework for analyzing high-valent intermediates in wild-type P450olet and Jason’s aminoacyl-CoA was the only batch anybody has been able to make since. Next came Jose Amaya who brought the chill to the lab and basically nailed down a framework for turnover analysis for my GC work. Courtney Wise, one of my former undergrads, followed Jose and dutifully wrote down, in excruciating detail, the procedures for doing anything in the lab. Finally, Olivia Manley arrived a little too late for knowledge to pass either way, but I think she’ll figure her stuff out. I would also like to thank Julia Bian, the only other undergrad that I mentored (besides Courtney) that it has been true honor to work with. iv ABSTRACT Non-Ribosomal Peptide Synthetases (NRPSs) and their exogenous tailoring partners have been heavily studied but not in the context of non-cognate systems. Orf78, a dinuclear iron β-hydroxylase from the lysobacter pathway and homologous to CmlA from the chloramphenicol pathway, is used to test affinities for one native and two non-native T-domains. Results indicate that there is enough difference between Type I and Type II NRPS systems to disfavor common recognition motifs. Additionally, the β-hydroxylase P450sky, from the skyllamycin biosynthetic pathway, is used in conjunction with the NRPS AT- domain NikP1AT from the nikkomycin biosynthetic pathway, in lieu of the homolog P450nikQ. The second portion of the thesis will discuss the creation of a sustainable source of biodiesel products are currently a goal of ‘green’ programs. The fatty acid decarboxylase P450olet uses the cheaply obtainable hydrogen peroxide as the oxidant to achieve a high-valent iron species. Substitution of the iron for manganese in the porphyrin scaffold raises the redox potential and forms a Mn(IV)-oxo complex that is too weak to perform C-H bond abstractions. Alternatively, substitution of the iron-protoporphyrin IX with iron- mesoporphyrin IX lowers the redox potential, increasing the amount of hydroxylated fatty acid at lower carbon chain lengths but also leads to a decrease in the efficiency in multiple turnover reactions. v TABLE OF CONTENTS DEDICATION ........................................................................................................... iii ACKNOWLEDGEMENTS ............................................................................................ iv ABSTRACT .............................................................................................................. v LIST OF TABLES ..................................................................................................... viii LIST OF FIGURES .................................................................................................... ix CHAPTER 1: A COMPARISON OF PCP DOMAINS AND THEIR RECOGNITION WITH A DINUCLEAR IRON HYDROXYLASE FROM THE LYSOBACTIN BIOSYNTHETIC PATHWAY .................................................................................................... 1 1.1 INTRODUCTION .......................................................................................... 1 1.2 MATERIALS AND METHODS ......................................................................... 6 1.3 RESULTS ................................................................................................. 13 1.4 DISCUSSION ............................................................................................ 22 1.5 CONCLUSION ........................................................................................... 25 1.6 REFERENCES .......................................................................................... 26 CHAPTER 2: DETERMINATION OF THE ROLE OF A NON-RIBOSOMAL PEPTIDE SYNTHETASE IN THE RECOGNITION OF A NON-COGNATE TAILORING ENZYME .. 41 2.1 INTRODUCTION ....................................................................................... 41 2.2 MATERIALS AND METHODS ...................................................................... 51 2.3 RESULTS ................................................................................................ 60 2.4 DISCUSSION ........................................................................................... 65 2.5 CONCLUSION ............................................................................................ 6 vi 2.6 REFERENCES ......................................................................................... 69 CHAPTER 3: THE SUBSTITUTION OF MANGANESE-PROTOPORPHYRIN IX INTO CYTOCHROME P450OLET ............................................................................ 93 3.1 INTRODUCTION ....................................................................................... 93 3.2 MATERIALS AND METHODS ...................................................................... 97 3.3 RESULTS AND DISCUSSION .................................................................... 104 3.4 CONCLUSION ........................................................................................ 115 3.5 REFERENCES ....................................................................................... 115 CHAPTER 4: RE-TUNING THE REACTIVITY OF THE FERRYL-OXO INTERMEDIATES IN P450OLET THROUGH SUBSTITUTION WITH IRON-MESOPORPHYRIN IX ........... 137 4.1 Introduction ........................................................................................ 137 4.2 Materials and Methods ....................................................................... 145 4.3 Results ............................................................................................... 151 4.4 Discussion .......................................................................................... 157 4.5 Conclusion ......................................................................................... 159 4.6 References ......................................................................................... 160 LIST OF TABLES Table 1.1 Sequence identity of 3 cognate and 2 non-cognate PCPs for P450sky ................................................................................................... 40 Table 3.1 Rates of Mn(IV)-oxo formation reacted with differing concentrations of peracetic acid ......................................................................................... 133 Table 4.1 Kinetic rates associated with PPolet and MPolet for Cpd. I and Cpd. II decay ........................................................................................... 177 Table 4.2 Turnover data for MPolet and PPolet ................................................ 177 LIST OF FIGURES Figure 1.1 Diagrams of the hydroxylation reaction with P450sky and the lysobactin antibiotic…………………………………………….………...…...34 Figure 1.2 Diagrams of Orf78 endogenous ligands and the active-site structure ................................................................................................... 34 Figure 1.3 Size-exclusion chromatography plot of Orf78 .................................... 35 Figure 1.4 Reductive titration of Orf78 ................................................................ 35 Figure 1.5 Optical spectrum of Orf78 incubated with sodium azide .................... 36 Figure 1.6 EPR spectra of mixed-valent Orf78, dithionite saturation, power saturation,
Load more

Recommended publications
  • The Nature of O2 Activation by the Ethylene-Forming Enzyme 1-Aminocyclopropane-1-Carboxylic Acid Oxidase
    The nature of O2 activation by the ethylene-forming enzyme 1-aminocyclopropane-1-carboxylic acid oxidase Liviu M. Mirica and Judith P. Klinman* Departments of Chemistry and Molecular and Cell Biology, University of California, Berkeley, CA 94720 Contributed by Judith P. Klinman, December 10, 2007 (sent for review November 15, 2007) Ethylene is a plant hormone important in many aspects of plant studied extensively, and it generally is accepted that O2 activation growth and development such as germination, fruit ripening, and at the iron center is linked to oxidative decarboxylation of the senescence. 1-Aminocyclopropane-1-carboxylic acid (ACC) oxidase ␣KG, ultimately forming an FeIVAO species as the reactive (ACCO), an O2-activating ascorbate-dependent nonheme iron en- intermediate (14–17). This high-valent Fe/O2 species functions zyme, catalyzes the last step in ethylene biosynthesis. The O2 as a ‘‘generic’’ oxidant for a wide range of oxidation/oxygenation activation process by ACCO was investigated using steady-state reactions. kinetics, solvent isotope effects (SIEs), and competitive oxygen In contrast to the ␣KG-dependent enzymes, where ␣KG kinetic isotope effects (18O KIEs) to provide insights into the nature rather than substrate binds to the metal center, spectroscopic of the activated oxygen species formed at the active-site iron studies of ACCO have shown that ACC coordinates to the iron center and its dependence on ascorbic acid. The observed large 18O center via both its amino and carboxylate groups (10, 18).† This -KIE of 1.0215 ؎ 0.0005 strongly supports a rate-determining step finding implies a distinctly different mode for reductant inter formation of an FeIVAO species, which acts as the reactive inter- action with ACCO than for most other nonheme iron enzymes.
    [Show full text]
  • Active Site Structures of Deoxyhemerythrin and Oxyhemerythrin (Difference Electron Densities/Crystallography/Oxygenation Mechanism) RONALD E
    Proc. Nati. Acad. Sci. USA Vol. 82, pp. 713-716, February 1985 Biochemistry Active site structures of deoxyhemerythrin and oxyhemerythrin (difference electron densities/crystallography/oxygenation mechanism) RONALD E. STENKAMP*, LARRY C. SIEKER*, L. H. JENSEN*t, JOHN D. MCCALLUM, AND JOANN SANDERS-LOEHRt Departments of *Biological Structure and tBiochemistry, University of Washington, Seattle, WA 98195; and tDepartment of Chemistry, Portland State University, Portland, OR 97207 Communicated by Harry B. Gray, October 1, 1984 ABSTRACT The physiologically active forms of the non- his hiS his heme-iron, oxygen-transport protein hemerythrin have been studied by x-ray crystallographic techniques. At 3.9-A resolu- tion, a difference electron-density map between the deoxy form and met form (methemerythrin) of the protein suggests only small differences in the binuclear iron complexes. The coordination of the iron atoms appears to be the same in both asp the deoxy and met forms, one iron of the complexes being pen- tacoordinate, the other iron being hexacoordinate. The iron atoms appear to be somewhat farther apart in the deoxy form. A 2.2-A resolution study of oxyhemerythrin shows that dioxy- gen binds to one iron atom-the pentacoordinate one in the his met form of the protein, the same binding site found for azide FIG. 1. Structures of the binuclear iron complexes (1) in azido- in azidomethemerythrin. methemerythrin (Left) and methemerythrin (Right). The Fe atoms are bound to three histidine side chains, an aspartic acid, a glutamic The initial objective of the crystallographic structure deter- acid, and a /u-oxo bridging atom derived from the solvent.
    [Show full text]
  • Ligation of the Diiron Site of the Hydroxylase Component of Methane Monooxygenase ANELECTRON NUCLEAR DOUBLE RESONANCE STUDY*
    THEJOURNAL OF BIOLOGICAL CHEMISTRY VOl. 267, No 1, Issue of January 5, pp. 261-269,1992 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Ligation of the Diiron Site of the Hydroxylase Component of Methane Monooxygenase ANELECTRON NUCLEAR DOUBLE RESONANCE STUDY* (Received for publication, August 5, 1991) Michael P. HendrichSO, Brian G.FoxSlI, Kristoffer K. AnderssonS, Peter G.Debrunner]], and John D. Lipscomb$** From the $Department of Biochemistry, Medical School, University of Minnesota, Minneapolis, Minnesota 55455 and the 11 Department of Physics, University of Illinois, Urbana, Illinois 61801 Electron nuclear double resonance (ENDOR) spec- addition of methanol may result from binding toa site troscopy is used to probe the coordinationof the mixed away from the diiron cluster or from bulk solvent valence (Fe(II)*Fe(III)) diiron clusterof the methane effects on the protein structure. monooxygenase hydroxylase component("OH-) iso- lated from Methylosinus trichosporium OB3b. EN- DOR resonances are observed along the principal axis directions g, = 1.94 and g, = 1.76 from at least nine Soluble methane monooxygenase (EC 1.14.13.25, "0)' different protons and two differentnitrogens. The ni- isolated from Methylosinus trichosporium OB3b is composed trogens are stronglycoupled and appear tobe directly of three protein components(1): a 40-kDa reductase contain- coordinated to the cluster irons. The ratio of their ing FAD and a [2Fe-2S] cluster, a 16-kDa cofactorless com- superhyperfine couplingconstants is roughly 4:7, ponent B, anda 245-kDa hydroxylase with an ((~py)~subunit which equals the ratio of the spin expectation values structure containing two p-(H/R)-oxo-bridged dinuclear iron of the Fe(I1) and Fe(II1) in the ground state and suggestsclusters.' The function of the enzyme in vivo is to catalyze that atleast one nitrogenis coordinated to each ironof the 0; and NADH-dependent oxidationof methane to meth- the mixed valence cluster.
    [Show full text]
  • BRION ALLEN BERMAN Studies of a Novel Hemerythrin-Like Protein From
    BRION ALLEN BERMAN Studies of a Novel Hemerythrin-like Protein from the Anaerobic, Hyperthermophilic Archaeon, Methanococcus jannaschii (Under the Direction of DONALD M. KURTZ) Hemerythrin (Hr) is a non-heme iron protein, which originally had only been found in marine invertebrates where it functions as an oxygen-transport or storage protein. Hrs typically contain an oxo-/hydroxo-bridged non-heme diiron site surrounded by a four-helical bundle protein fold. All Hrs have seven conserved amino acids that furnish iron ligands, namely five histidines, one aspartate and one glutamate. Subsequently, homologues have been found in other organisms. Through protein and nucleotide searches, a previously undescribed protein having the Hr-like sequence motif (MjHr), was discovered in the Methanococcus jannaschii genome. MjHr exhibits structural and spectral properties resembling those of previously characterized Hrs. This Hr-like protein is the first to be described from archaea. Unlike Hrs that function as O2- carrying proteins, MjHr is proposed to function as an O2 sensor or scavenger within this methanogenic anaerobe. INDEX WORDS: Hemerythrin, Methanococcus jannaschii, O2-binding, Stopped- flow spectrophotometry, Electron paramagnetic resonance (EPR), Circular dichroism (CD), Western-blot, Metalloprotein, Reconstitution, Overexpression, Protein Purification STUDIES OF A NOVEL HEMERYTHRIN-LIKE PROTEIN FROM THE ANAEROBIC, HYPERTHERMOPHILIC ARCHAEON, METHANOCOCCUS JANNASCHII by BRION ALLEN BERMAN B.S., The University of South Alabama, 1999 A Thesis Submitted to the Graduate Faculty of The University Georgia in Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE ATHENS, GEORGIA 2001 © 2001 Brion Allen Berman All Rights Reserved STUDIES OF A NOVEL HEMERYTHRIN-LIKE PROTEIN FROM THE ANAEROBIC, HYPERTHERMOPHILIC ARCHAEON, METHANOCOCCUS JANNASCHII by BRION ALLEN BERMAN Approved: August 7, 2001 Major Professor: Donald M.
    [Show full text]
  • Geometric and Electronic Structure/Function Correlations in Non-Heme Iron Enzymes Edward I
    Chem. Rev. 2000, 100, 235−349 235 Geometric and Electronic Structure/Function Correlations in Non-Heme Iron Enzymes Edward I. Solomon,* Thomas C. Brunold, Mindy I. Davis, Jyllian N. Kemsley, Sang-Kyu Lee, Nicolai Lehnert, Frank Neese, Andrew J. Skulan, Yi-Shan Yang, and Jing Zhou Department of Chemistry, Stanford University, Stanford, California 94305-5080 Received September 29, 1999 Contents tures of the active sites of non-heme iron enzymes and the contributions of these sites to molecular I. Introduction 235 mechanisms. Tables 1 and 2 present fairly complete II. Mononuclear Non-Heme Iron 237 lists of the presently known key classes of mono- A. Spectroscopic Methodology 237 nuclear and binuclear non-heme iron enzymes. As 1. FeII 237 indicated in the tables, these enzymes participate in 2. FeIII 243 a range of reactions as extensive as those found in 3. Quantum Chemical Calculations of 251 heme chemistry but are generally much less well Molecular Properties understood. For most of the enzymes one of the B. Substrate Activation 251 reactants is dioxygen. The uncatalyzed reactions of 1. Lipoxygenases 251 O2 with organic substrates are thermodynamically 2. Intradiol Dioxygenases 257 favorable but kinetically slow since they are spin 3. Comparison of Strategies 263 forbidden and the one-electron reduction potential of O is low. In the mononuclear non-heme iron en- C. O2 Activation 264 2 1. Extradiol Dioxygenases 264 zymes, these reactions are catalyzed either by a high- 2. Pterin-Dependent Hydroxylases 269 spin ferrous site which is involved in dioxygen activation or by a high-spin ferric site which activates 3.
    [Show full text]
  • Biochemical, Kinetic and Spectroscopic Characterizations Of
    BIOCHEMICAL, KINETIC AND SPECTROSCOPIC CHARACTERIZATIONS OF NON-HEME IRON OXYGENASE ENZYMES by BISHNU P SUBEDI Presented to the Faculty of the Graduate School of Science The University of Texas at Arlington in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY THE UNIVERSITY OF TEXAS AT ARLINGTON May 2015 Copyright © by Bishnu P Subedi 2014 All Rights Reserved ii Acknowledgements I would like to express my greatest appreciation and thanks to my advisor Professor Brad S Pierce for giving me the opportunity to work in his lab and providing valuable guidance and encouragement during the research period. I would also like to thank Professor Frank W Foss both for being a member in my thesis committee and a collaborator on MiaE project. My sincerest thanks goes to Professor Frederick MacDonnell for his valuable suggestions as a chair in my thesis committee. I also thank the Department of Chemistry and Biochemistry at The University of Texas at Arlington for supporting my graduate study, the UTA Shimadzu Center for Advanced Analytical Chemistry for the use of HPLC and LC-MS/MS instrumentation utilized in this work, and the UTA Center for Nanostructured Materials for the use of EPR facilities. I would also like to thank Professor Paul Lindahl and Dr. Mrinmoy Chakrabarti, Texas A&M University for their generous assistance in collecting Mössbauer spectra for MiaE, similarly Professor Brian Fox and Justin Acheson, University of Wisconsin, Madison for collaboration on MiaE structural study and Professor Tim Larson, Virginia Tech Department of Biochemistry for the IPTG inducible Azotobacter vinelandii CDO expression vector.
    [Show full text]
  • Cellular Iron Homeostasis
    BioIron 2017 Educational Session Cellular iron metabolism Kostas Pantopoulos Lady Davis Institute for Medical Research Division of Experimental Medicine Montreal, Quebec, Canada The problem of iron acquisition Iron is an abundant metal, but… In aqueous solutions, Fe2+ is readily oxidized to Fe3+ 3+ At neutral pH, Fe forms essentially insoluble Fe(OH)3 Iron’s bioavailability is limited Strategies for iron acquisition (1): direct iron transport 1. Reduction of Fe3+ to Fe2+ in the vicinity of the cell by plasma membrane-bound ferric reductases Frep Fre reductases 2. Direct uptake by low affinity (Km=40 mM) ferrous transporter Fet4p or Reoxidation by plasma membrane-bound ferroxidase Fet3p and internalization by permease Ftr1p in a high affinity transporting system (K =0.15 mM) Fet3 oxidase/Ftr1 permease m Strategies for iron acquisition (2): indirect iron uptake Many bacteria and lower eukaryotes secrete siderophores (low molecular weight Fe3+ chelators) Iron-loaded siderophores are internalized into the bacteria by binding to specific cell-surface receptors Deferroxamine, a bacterial siderophore, is widely employed for iron chelation therapy deferroxamine (desferral) Iron assimilation in higher organisms Both concepts: Direct iron transport by trans-membrane transporters, following reduction to soluble Fe2+ and Receptor-mediated uptake of iron, that is “captured” in form of Fe3+ by an iron-binding molecule have been conserved in higher organisms Direct iron transport is critical for dietary iron absorption Iron absorption from intestinal
    [Show full text]
  • II Msc Chemistry-18P
    | 79 3 Current Status and Mechanism of Action of Platinum-Based Anticancer Drugs Shanta Dhar and Stephen J. Lippard 3.1 Introduction 3.1.1 Platinum Chemotherapy and Cancer Chemotherapy, surgery, and radiation therapy are the main pillars of cancer treatment. The term ‘‘chemotherapy’’ refers to the use of any chemical agent to stop cancer cell proliferation. Chemotherapy has the ability to kill cancer cells at sites remote from the original cancer. Thus chemotherapy is referred to as sys- temic treatment. More than half of all people diagnosed with cancer receive che- motherapy. ‘‘Platinum chemotherapy’’ is the term used for cancer treatment where one of the chemotherapeutic drugs is a platinum derivative. The spectacular and first such platinum-based drug is cisplatin, cis-diamminedichloridoplati- num(II). Subsequently, the cisplatin relatives carboplatin and oxaliplatin were introduced to minimize side effects (Table 3.1). Platinum compounds have been the treatment of choice for ovarian, testicular, head and neck, and small cell lung cancer for the past 20 years. 3.1.2 Palette of Current Platinum Chemotherapeutic Drugs During the last 30 years, over 700 FDA-approved drugs have entered into clinical practice. The success of cisplatin [1] has been the main impetus for the expansion of the family of platinum compounds. Carboplatin [2] and oxaliplatin [3] (Table 3.1) are registered worldwide and have been a major success in clinical practice. Nedaplatin [4] is used in Japan to treat head and neck, testicular, lung, ovarian, cervical, and non-small cell lung cancers. Heptaplatin [3, 4] is used in gastric cancer in South Korea.
    [Show full text]
  • Questions & Answers on Bioinorganic Chemistry (D Ray)
    Questions & Answers on Bioinorganic Chemistry (D Ray) 12. Explain the iron binding by transferrin. Ans. Iron, when taken up with the food and processed in the mouth is mostly present in Fe3+ form and thus gets into the gastrointestinal tract as Fe3+. In case of an intact milieu in the small intestines, ferric iron is reduced to its ferrous form (Fe2+). The Fe3+ ions are then taken up by 3+ apotransferrin (H2Tf); simultaneously, carbonate is coordinated to iron. Fe -Tf is the transport form for iron. The iron-loaded transferrin delivers iron to sites of potential use (e.g. incorporation into protoporphyrin IX and generation of haemoglobin), or stored in iron storage proteins (ferritins). The delivery of iron affords reduction from the ferric to the ferrous state; a reductant employed here is ascorbate (vitamin C). Transferrin is a glycoproteid of molecular weight 80 kDa (containing ca. 6% carbohydrate), having available two almost equivalent binding sites for iron(III), in the C- and N-teminal lobes, respectively. The pK (K = stability constant; see inset on p. 5) at pH 7.4 (the pH of blood) is -20.2. Transferrin is also an effective transporter for other tri- and divalent metal cations, and even for anions (e.g. vanadate). Since its loading capacity for iron commonly is only ca. 40%, other ions can be transported simultaneously. 13. Show and explain the iron storage protein ferritin. Ans. Ferritins (Fig. 4) are iron storage proteins, built up of a hollow protein sphere (apo-ferritin, M = 450 kDa, 24 subunits of 163 amino acids each) with an outer diameter of 130 and an inner diameter of 70 Å.
    [Show full text]
  • Biocatalytic Oxidations of Substrates Through Soluble Methane Monooxygenase from Methylosinus Sporium 5
    catalysts Article Biocatalytic Oxidations of Substrates through Soluble Methane Monooxygenase from Methylosinus sporium 5 Yeo Reum Park 1, Hee Seon Yoo 1, Min Young Song 1, Dong-Heon Lee 1 and Seung Jae Lee 1,2,* 1 Department of Chemistry and Institute for Molecular Biology and Genetics, Chonbuk National University, Jeonju 54896, Korea; [email protected] (Y.R.P.); [email protected] (H.S.Y.); [email protected] (M.Y.S.); [email protected] (D.-H.L.); 2 Department of Chemistry and Research Center for Bioactive Materials, Chonbuk National University, Jeonju 54896, Korea * Correspondence: [email protected]; Tel.: +82-63-270-3412; Fax: +82-63-270-3407 Received: 27 October 2018; Accepted: 21 November 2018; Published: 26 November 2018 Abstract: Methane, an important greenhouse gas, has a 20-fold higher heat capacity than carbon dioxide. Earlier, through advanced spectroscopy and structural studies, the mechanisms underlying the extremely stable C–H activation of soluble methane monooxygenase (sMMO) have been elucidated in Methylosinus trichosporium OB3b and Methylococcus capsulatus Bath. Here, sMMO components—including hydroxylase (MMOH), regulatory (MMOB), and reductase (MMOR)—were expressed and purified from a type II methanotroph, Methylosinus sporium strain 5 (M. sporium 5), to characterize its hydroxylation mechanism. Two molar equivalents of MMOB are necessary to achieve catalytic activities and oxidized a broad range of substrates including alkanes, alkenes, halogens, and aromatics. Optimal activities were observed at pH 7.5 for most substrates possibly because of the electron transfer environment in MMOR. Substitution of MMOB or MMOR from another type II methanotroph, Methylocystis species M, retained specific enzyme activities, demonstrating the successful cross-reactivity of M.
    [Show full text]
  • Bioiron: Origin, Chemical Properties and Evolution
    BioIron 2019: Iron Essentials for Clinicians and Scientists BioIron: origin, chemical properties and evolution Kostas Pantopoulos Lady Davis Institute for Medical Research McGill University Iron: a highly abundant transition metal The elements in haiku by Mary Soon Lee, sciencemag.org Produced in the universe by fusion in high mass stars and scattered into space to accumulate in rocky planets like earth Most abundant element on earth (crust and inner core) 4th most abundant element on earth’s crust Electron configuration and oxidation states Fe2+: ferrous iron Fe3+: ferric iron Fe4+: ferryl iron (unstable intermediate) Coordination chemistry Iron readily forms complexes with organic molecules, and this has immense biological implications iron-sulfur clusters heme iron-oxo centers Iron cofactors can be utilized by proteins for: A wide range of electron transfer biochemical reactions within a broad electrochemical potential (Eo from -400 to +400 mV) Various other structural and metabolic functions Functions of proteins with iron cofactors Heme oxygen binding (hemoglobin, myoglobin) electron transfer reactions (cytochromes) catalysis (oxidases, peroxidases, NO synthases) hemoglobin Iron-sulfur (Fe-S) clusters electron transfer reactions (ferredoxins, Rieske proteins) catalysis (aconitases, other dehydratases) activation of substrate (cyclooxygenase, lipoxygenase, other oxidases) transcriptional regulation (bacterial transcription factors FNR, SoxR) structure stabilization (bacterial endonuclease III) Iron-oxo (Fe-O-Fe) centers catalysis
    [Show full text]
  • Tertiary Structure of Myohemerythrin at Low Resolution (Hemerythrin/Oxygen Transport/Sipunculan Worm/Protein Structure/X-Ray Diffraction) WAYNE A
    Proc. Nat. Acad. Sci. USA Vol. 72, No. 6, pp. 2160-2164, June 1975 Tertiary Structure of Myohemerythrin at Low Resolution (hemerythrin/oxygen transport/sipunculan worm/protein structure/x-ray diffraction) WAYNE A. HENDRICKSON*, GERALD L. KLIPPENSTEINt, AND KEITH B. WARD* *Laboratory for the Structure of Matter, Naval Research Laboratory, Washington, D.C. 20375; and t Department of Biochemistry, University of New Hampshire, Durham, N.H. 03824 Communicated by I. M. Klotz, March 17, 1975 ABSTRACT X-ray diffraction studies have produced Many compounds seem to bind, but derivative crystals are a low resolution image and also located the iron atoms of a often prone to cracking and poor isomorphism. Nevertheless, monomeric hemerythrin from muscles of a sipunculan worm. These results reveal the course of the polypeptide five derivatives were found to be good enough at least for low- chain and some details of the active center. resolution phasing. X-ray diffraction data were measured by w-scans on a four- Oxygen transport in certain invertebrate animals is mediated circle diffractometer. CuKa radiation was used. Native data, by hemerythrin in erythrocytes of the coelomic fluid. He- including the Bijvoet pairs (hkl and hUl), were collected to merythrin usually occurs as an octamer of 108,000 molecular 2.8 A spacings. Derivative data were taken out to 3A spacings weight. It is a non-heme iron protein containing two iron from hkO, Okl, and hOl reflections and to 5.5 A spacings from atoms per subunit and it reversibly binds oxygen in the ratio Bijvoet pairs. Structure factor amplitudes were corrected for 1 02:2 Fe.
    [Show full text]