DOCSLIB.ORG

Enhancing the Nitrite Reductase Activity of Modified Hemoglobin: Bis-Tetramers and Their Pegylated Derivatives

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Enhancing the Nitrite Reductase Activity of Modified Hemoglobin: Bis-Tetramers and Their Pegylated Derivatives Enhancing the Nitrite Reductase Activity of Modified Hemoglobin: Bis-Tetramers and their PEGylated Derivatives by Francine Evelyn Lui A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Chemistry University of Toronto © Copyright by Francine E. Lui 2011 Abstract Enhancing the Nitrite Reductase Activity of Modified Hemoglobin: Bis-Tetramers and their PEGylated Derivatives Francine Evelyn Lui, Doctor of Philosophy, 2011 Department of Chemistry, University of Toronto The need for an alternative to red cells in transfusions has led to the creation of hemoglobin-based oxygen carriers (HBOCs). However, evaluations of all products tested in clinical trials have noted cardiovascular complications, raising questions about their safety that led to the abandonment of all those products. It has been considered that the adverse side effects come from the scavenging of the vasodilator – nitric oxide (NO) by the deoxyheme sites of the hemoglobin derivatives. Another observation is that HBOCs with lower oxygen affinity than red cells release oxygen prematurely in arterioles, triggering an unwanted homeostatic response. Since the need for such a product remains critical, it is important to understand the reactivity patterns that contribute to the observed complications. Various alterations of the protein have been attempted in order to reduce HBOC-induced vasoconstriction. Recent reports suggest that a safe and effective product should be pure, homogenous and have a high molecular weight along with appropriate oxygenation properties. While these properties are clearly important, vasodilatory features of hemoglobin through its nitrite reductase activity may also act as an in situ source of NO. It follows that HBOCs with an enhanced ability to produce NO from endogenous nitrite may serve to counteract vasoactivity associated with NO- scavenging by hemoglobin. ii Here we characterize the effects of different protein modifications on the nitrite reductase activity of hemoglobin. We produced a variety of HBOCs that include cross- linked tetramers, polyethylene glycol (PEG) conjugates and bis-tetramers of hemoglobin. We report that the rate of NO production strongly depends on the conformational state of the protein, with R-state stabilized proteins (PEG-Hbs), exhibiting the fastest rates. In particular, we found that PEGylated bis-tetramers of hemoglobin (BT-PEG) exhibit increased nitrite reductase activity while retaining cooperativity and stability. Animal studies of BT-PEG demonstrated that this material is benign: it did not cause significant increases in systemic blood pressure in mice, the major side effect associated with existing HBOCs. BT-PEG exhibits an enhanced nitrite reductase activity together with sample purity and homogeneity, molecular size and shape, and appropriate oxygenation properties, characteristics of a clinically useful HBOC. iii Acknowledgements Professor Ronald Kluger supervised and directed this research. The achievements in this research and my personal growth as a scientist are a result of his care, constant guidance and indispensible instruction. I truly thank him, both as a supervisor and as a mentor, for the memorable and irreplaceable time that has been my Ph.D. in the Kluger Lab. In addition, my committee members, the Kluger lab members, and many fellow classmates have also been central to my training as a graduate student. As well, the opportunity to collaborate with Dr. Warren Zapol at Massachusetts General Hospital, Harvard Medical School has been imperative to my research and understanding of physiological animal models. Jonathan S. Foot, Ying Yang and DongXin Hu from the Kluger lab as well as Binglan Yu and David Baron from the Zapol lab worked directly with me on this research and imparted much of their knowledge upon me. Maksims N. Volkovs wrote the program for the deconvolution of spectral data, a fundamental part of this research. Adelle Vandersteen and Sohyoung Her have been my chemistry comrades and will leave me with a lifetime’s worth of priceless memories. Scott Mundle, Raj Dhiman, Harini Kaluarachchi, Andrew Sydor and Kim Chan Chung all played crucial roles in this research, contributing their opinions and ideas, moulding and shaping the direction of my work. Finally and most importantly, I am especially grateful to my parents, sister and Maksims N. Volkovs. They were unquestionably the most essential contributors to this work, in their unwavering support and enthusiastic encouragement. They are the inspiration and motivation in my academic pursuits, and the source of any of my accomplishments in life. iv For M.N.V v Table of Contents Abstract _____________________________________________________________ ii Acknowledgements ____________________________________________________ iv Table of Contents ______________________________________________________ vi List of Tables _________________________________________________________ ix List of Schemes _______________________________________________________ x List of Schemes _______________________________________________________ x List of Figures ________________________________________________________ xi Abbreviations ________________________________________________________ xv Abbreviations ________________________________________________________ xv Statement of Authorship and Publication Status ____________________________ xvii Chapter 1 : HBOCs – Safety and Efficacy ___________________________________ 1 1.1 Blood, Transfusions, Hemoglobin, and Oxygen Delivery ..................................... 1 1.2 Hemoglobin-Based Oxygen Carriers (HBOCs)..................................................... 3 1.3 HBOC-Induced Vasoconstriction: Hypotheses ..................................................... 4 1.4 Chemical Approaches that Deal with HBOC-induced Vasoconstriction .............. 6 1.5 Future Prospects: Understanding HBOC-induced Vasoconstriction ..................... 9 1.6 ChemBioChem Review: Reviving Artificial Blood: Meeting the Challenge of Dealing with NO Scavenging by Hemoglobin .......................................................... 11 1.7 Purpose of Thesis ................................................................................................. 21 Chapter 2 : PEG-Hemoglobins and Nitrite Reductase Activity __________________ 23 2.1 Introduction .......................................................................................................... 23 2.2 Biochemistry (ACS) Article: Polyethylene Glycol Conjugation Enhances the Nitrite Reductase Activity of Native and Cross-Linked Hemoglobin ....................... 24 2.3 Detailed ................................................................................................................ 33 vi 2.2.1 Chemical Modification of Hemoglobin: Cross-linking and PEGylation ...... 33 2.2.2 Analytical Methods: HPLC, SDS-PAGE, CD spectroscopy, Oxygen Binding ................................................................................................................................ 35 2.2.3 Kinetic Measurements .................................................................................. 36 2.2.4 Deconvolution of Spectra ............................................................................. 37 2.4 Supplementary Information ................................................................................. 38 2.4.2 CD Spectroscopy .......................................................................................... 38 2.4.2 Kinetic Measurements: Nitrite Reductase Activity ...................................... 39 2.5 Summary .............................................................................................................. 42 Chapter 3 : Enhancing Nitrite Reductase Activity – PEGylated Bis-Tetramers _____ 44 3.1 Introduction .......................................................................................................... 44 2.2 Biochemistry (ACS) Article: Enhancing Nitrite Reductase Activity of Modified Hemoglobin: Bis-tetramers and Their PEGylated Derivatives .................................. 46 3.3 Detailed Experimental Methods ......................................................................... 55 3.3.1 General Methods ........................................................................................... 55 3.3.2 Chemical Modification of Hemoglobin: Cross-linking and PEGylation ...... 55 3.3.3 Analytical Methods: HPLC, SDS-PAGE, CD spectroscopy, Oxygen Binding ................................................................................................................................ 57 3.3.4 Kinetic Measurements .................................................................................. 59 3.4 Supplementary Information ................................................................................. 59 3.4.1 Kinetic Measurements .................................................................................. 59 3.5 Summary .............................................................................................................. 61 Chapter 4 : Physiological Responses of Bis-Tetramers and their PEGylated Derivatives ___________________________________________________________________ 65 4.1 Introduction .......................................................................................................... 65 4.2 Experimental ........................................................................................................ 66 4.2.1 General Methods and
Load more

Recommended publications
  • Cytochrome C Nitrite Reductase (Ccnir) Does Not Disproportionate Hydroxylamine to Ammonia and Nitrite, Despite a Strongly Favorable Driving Force
    Marquette University e-Publications@Marquette Physics Faculty Research and Publications Physics, Department of 4-2014 Shewanella oneidensis Cytochrome c Nitrite Reductase (ccNiR) Does Not Disproportionate Hydroxylamine to Ammonia and Nitrite, Despite a Strongly Favorable Driving Force Matthew Youngblut University of Wisconsin - Milwaukee Daniel J. Pauly University of Wisconsin - Milwaukee Natalia Stein University of Wisconsin - Milwaukee Daniel Walters University of Wisconsin - Milwaukee John A. Conrad University of Wisconsin - Milwaukee See next page for additional authors Follow this and additional works at: https://epublications.marquette.edu/physics_fac Part of the Physics Commons Recommended Citation Youngblut, Matthew; Pauly, Daniel J.; Stein, Natalia; Walters, Daniel; Conrad, John A.; Moran, Graham R.; Bennett, Brian; and Pacheco, A. Andrew, "Shewanella oneidensis Cytochrome c Nitrite Reductase (ccNiR) Does Not Disproportionate Hydroxylamine to Ammonia and Nitrite, Despite a Strongly Favorable Driving Force" (2014). Physics Faculty Research and Publications. 7. https://epublications.marquette.edu/physics_fac/7 Authors Matthew Youngblut, Daniel J. Pauly, Natalia Stein, Daniel Walters, John A. Conrad, Graham R. Moran, Brian Bennett, and A. Andrew Pacheco This article is available at e-Publications@Marquette: https://epublications.marquette.edu/physics_fac/7 Marquette University e-Publications@Marquette Physics Faculty Research and Publications/College of Arts and Sciences This paper is NOT THE PUBLISHED VERSION; but the author’s final, peer-reviewed manuscript. The published version may be accessed by following the link in the citation below. Biochemistry, Vol. 53, No. 13 (8 April 2014): 2136–2144. DOI. This article is © American Chemical Society Publications and permission has been granted for this version to appear in e- Publications@Marquette. American Chemical Society Publications does not grant permission for this article to be further copied/distributed or hosted elsewhere without the express permission from American Chemical Society Publications.
    [Show full text]
  • The Noncanonical Pathway for in Vivo Nitric Oxide Generation: the Nitrate-Nitrite-Nitric Oxide Pathway
    1521-0081/72/3/692–766$35.00 https://doi.org/10.1124/pr.120.019240 PHARMACOLOGICAL REVIEWS Pharmacol Rev 72:692–766, July 2020 Copyright © 2020 by The Author(s) This is an open access article distributed under the CC BY-NC Attribution 4.0 International license. ASSOCIATE EDITOR: CHRISTOPHER J. GARLAND The Noncanonical Pathway for In Vivo Nitric Oxide Generation: The Nitrate-Nitrite-Nitric Oxide Pathway V. Kapil, R. S. Khambata, D. A. Jones, K. Rathod, C. Primus, G. Massimo, J. M. Fukuto, and A. Ahluwalia William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University London, London, United Kingdom (V.K., R.S.K., D.A.J., K.R., C.P., G.M., A.A.) and Department of Chemistry, Sonoma State University, Rohnert Park, California (J.M.F.) Abstract ...................................................................................694 Significance Statement. ..................................................................694 I. Introduction . ..............................................................................694 A. Chemistry of ·NO and Its Metabolism to Nitrite and Nitrate . .........................695 II. Inorganic Nitrite and Nitrate . ............................................................697 A. Historical Uses of Inorganic Nitrite.....................................................697 B. Historical Uses of Inorganic Nitrate ....................................................698 III. Sources and Pharmacokinetics of Nitrate . ................................................698
    [Show full text]
  • Protein Disulfide Isomerase May Facilitate the Efflux
    Redox Biology 1 (2013) 373–380 Contents lists available at SciVerse ScienceDirect Redox Biology journal homepage: www.elsevier.com/locate/redox Protein disulfide isomerase may facilitate the efflux of nitrite derived S-nitrosothiols from red blood cells$ Vasantha Madhuri Kallakunta a, Anny Slama-Schwok b, Bulent Mutus a,n a Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada N9B 3P4 b INRA UR892, Domaine de Vilvert, 78352 Jouy-en-Josas, France article info abstract Article history: Protein disulfide isomerase (PDI) is an abundant protein primarily found in the endoplasmic reticulum Received 20 June 2013 and also secreted into the blood by a variety of vascular cells. The evidence obtained here, suggests that Received in revised form PDI could directly participate in the efflux of NO+ from red blood cells (RBC). PDI was detected both in 8 July 2013 RBC membranes and in the cytosol. PDI was S-nitrosylated when RBCs were exposed to nitrite under Accepted 9 July 2013 ∼50% oxygen saturation but not under ∼100% oxygen saturation. Furthermore, it was observed that hemoglobin (Hb) could promote PDI S-nitrosylation in the presence of ∼600 nM nitrite. In addition, three Keywords: lines of evidence were obtained for PDI–Hb interactions: (1) Hb co-immunoprecipitated with PDI; (2) Hb Nitrite reductase quenched the intrinsic PDI fluorescence in a saturable manner; and (3) Hb–Fe(II)–NO absorption S-nitrosohemoglobin spectrum decreased in a [PDI]-dependent manner. Finally, PDI was detected on the surface RBC under Hypoxic vasodilation ∼100% oxygen saturation and released as soluble under ∼50% oxygen saturation.
    [Show full text]
  • Nitrate- and Nitrite-Reductase Activities in Mycobacterium
    NITRATE- AND NITRITE-REDUCTASE ACTIVITIES IN MYCOBACTERIUM AVIUM A5 By Nitin S. Butala Thesis submitted to the Faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in BIOLOGICAL SCIENCES Joseph O. Falkinham, III, Chairman Eugene M. Gregory Biswarup Mukhopadhyay June, 2006 Blacksburg, Virginia Keywords: Mycobacterium avium , nitrate- and nitrite- reductases, and nitrate reduction test Copyright 2006, Nitin Butala Nitrate- and Nitrite-reductase Activities in Mycobacterium avium A5 By Nitin S. Butala Committee Chairman: Joseph O. Falkinham, III Biology (ABSTRACT) Mycobacterium avium is human and animal opportunistic pathogen responsible for disseminated disease in immunocompromised patients. Mycobacteria have a capacity to adapt to the environmental conditions by inducing enzyme activities and altering their metabolism. M. avium A5 cells were grown in a defined minimal medium (Nitrogen Test Medium) with glutamine, nitrite, nitrate, or ammonia as sole nitrogen source at a concentration of 2 mM at 37 0C aerobically. The strain grew well on all the nitrogen sources except nitrite. It grew slowly on nitrite with a generation time of 6 days and cultures were not viable after 4 weeks of storage. These data confirm that M. avium can utilize a single nitrogen source in a defined minimal medium as documented by McCarthy (1987). M. avium genome has been sequenced and contains genes sharing sequence similarities to respiratory nitrate reductase and dissimilatory nitrite reductases. Because, M. avium can use nitrate or nitrite as sole nitrogen source for growth (McCarthy, 1987), it must have assimilatory nitrate- and nitrite-reductases. Nitrate- and nitrite-reductase activities of M.
    [Show full text]
  • Protein Disulfide Isomerase May Facilitate the Efflux Of
    Protein disulfide isomerase may facilitate the efflux of nitrite derived S-nitrosothiols from red blood cells Vasantha Madhuri Kallakunta, Anny Slama-Schwok, Bulent Mutus To cite this version: Vasantha Madhuri Kallakunta, Anny Slama-Schwok, Bulent Mutus. Protein disulfide isomerase may facilitate the efflux of nitrite derived S-nitrosothiols from red blood cells. Redox Biology, Elsevier, 2013, 1 (1), pp.373-380. 10.1016/j.redox.2013.07.002. hal-02647054 HAL Id: hal-02647054 https://hal.inrae.fr/hal-02647054 Submitted on 29 May 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Redox Biology 1 (2013) 373–380 Contents lists available at SciVerse ScienceDirect Redox Biology journal homepage: www.elsevier.com/locate/redox Protein disulfide isomerase may facilitate the efflux of nitrite derived S-nitrosothiols from red blood cells Vasantha Madhuri Kallakunta a, Anny Slama-Schwok b, Bulent Mutus a,n a Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada N9B 3P4 b INRA UR892, Domaine de Vilvert, 78352 Jouy-en-Josas, France article info abstract Article history: Protein disulfide isomerase (PDI) is an abundant protein primarily found in the endoplasmic reticulum Received 20 June 2013 and also secreted into the blood by a variety of vascular cells.
    [Show full text]
  • Effect of Nitrate, Nitrite, Ammonium, Glutamate, Glutamine and 2
    Physiol. Mol. Biol. Plants (2007), 13(1) : 17-25. Research Article Effect of Nitrate, Nitrite, Ammonium, Glutamate, Glutamine and 2-oxoglutarate on the RNA Levels and Enzyme Activities of Nitrate Reductase and Nitrite Reductase in Rice Ahmad Ali1, S. Sivakami1 and Nandula Raghuram1,2★ 1Department of Life Sciences, University of Mumbai, Vidyanagari, Santacruz (E), Mumbai 400 098, India 2School of Biotechnology, Guru Gobind Singh Indraprastha University, Kashmiri Gate, Delhi 110 006, India In this study the induction and regulation of NR and NiR by various N metabolites in excised leaves of rice (Oryza sativa ssp. indica var. Panvel I) seedlings grown hydroponically (nutrient starved) for 10-12 days and adapted for 2 days in darkness was examined. Nitrate induced the activity of both the enzymes reaching an optimum at 40 mM in 6 hrs. Nitrite and ammonium inhibited NR in light in a concentration-dependent manner. Glutamine, which had little effect of its own on NR in light and no effect on NiR in both light and dark, strongly inhibited Nitrate-induced NR in the dark. When the activities of these enzymes were measured from leaves treated with glutamate and 2-oxoglutarate, a similar pattern of induction was observed for NR and NiR. The transcript levels of NR and NiR increased to a similar extent in the presence of nitrate. However light did not cause any significant change in transcript levels. These results indicate that both the enzymes are under tight regulation by nitrogen metabolites and light and are co-regulated under certain conditions. INTRODUCTION carbon and nitrogen metabolites, growth regulators, light, temperature and carbon dioxide concentration The assimilatory nitrate reduction pathway is a vital (Aslam et al., 1997).
    [Show full text]
  • Enhancing Nitrite Reductase Activity of Modified Hemoglobin: Bis-Tetramers and Their Pegylated Derivatives† Francine E
    11912 Biochemistry 2009, 48, 11912–11919 DOI: 10.1021/bi9014105 Enhancing Nitrite Reductase Activity of Modified Hemoglobin: Bis-tetramers and Their PEGylated Derivatives† Francine E. Lui and Ronald Kluger* Davenport Chemical Research Laboratories, Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario, Canada M5S 3H6 Received August 12, 2009; Revised Manuscript Received November 2, 2009 ABSTRACT: The clinical evaluation of stabilized tetrameric hemoglobin as alternatives to red cells revealed that the materials caused significant increases in blood pressure and related problems and this was attributed to the scavenging of nitric oxide and extravasation. The search for materials with reduced vasoactivity led to the report that conjugates of hemoglobin tetramers and polyethylene glycol (PEG) chains did not elicit these pressor effects. However, this material does not deliver oxygen efficiently due to its lack of cooperativity and high oxygen affinity, making it unsuitable as an oxygen carrier. It has been recently reported that PEG- conjugated hemoglobin converts nitrite to nitric oxide at a faster rate than does the native protein, which may compensate for the scavenging of nitric oxide. It is therefore important to alter hemoglobin in order to enhance nitrite reductase activity while retaining its ability to deliver oxygen. If the beneficial effect of PEG is associated with the increased size reducing extravasation, this can also be achieved by coupling cross-linked tetramers to one another, giving materials with appropriate oxygen affinity and cooperativity for use as circulating oxygen carriers. In the present study it is shown that cross-linked bis-tetramers with good oxygen -1 -1 delivery potential have enhanced nitrite reductase activity with kobs = 0.70 M s (24 °C), compared to -1 -1 -1 -1 native protein and cross-linked tetramers, kobs = 0.25 M s and kobs = 0.52 M s , respectively, but are -1 -1 less active in reduction of nitrite than Hb-PEG5K2 (kobs = 2.5 M s ).
    [Show full text]
  • Intact Tissue Assay for Nitrite Reductase in Barley Aleurone Layers1
    Plant Physiol. (1971) 47, 790-794 Intact Tissue Assay for Nitrite Reductase in Barley Aleurone Layers1 Received for publication October 14, 1970 THOMAS E. FERRARI2 AND JOSEPH E. VARNER Department of Horticulture and Michigan State University-Atomic Energy Commission Plant Research Labo- ratory, Michigan State University, East Lansing, Michigan 48823 ABSTRACT activity be measured in the intact tissue under conditions similar to those in which nitrite release occurs. This report A method has been devised for the detection and measure- describes a method for the detection and measurement of ment of nitrite reductase activity in intact barley (Hordeum nitrite reductase activity in intact barley aleurone layers, and vulgare L. cv. Himalaya) aleurone layers. The technique in- the effects of conditions favoring nitrite release in the activity volves feeding aleurone layers nitrite and measuring nitrite of the enzyme. The technique involves administering nitrite disappearance after a given time period. The method also to aleurone layers and measuring nitrite disappearance with allows simultaneous determination of nitrite uptake by the time. The method also tissue. Quantitative recovery of nitrite is obtained by rapid allows simultaneous determination of heating of tissue in the presence of dimethyl sulfoxide. nitrite uptake by the tissue. Using the procedure described, nitrite reductase activity in intact barley aleurone layers was determined. Enzyme activity MATERIALS AND METHODS was increased by prior incubation of the tissue with nitrate, Tlsse Preparation. Aleurone layers were isolated from but considerable activity was present in tissue incubated barley (Hordeum vulgare L. cv. Himalaya, 1965 Harvest) seeds without nitrate. Nitrate-induced activity was inhibited by as described previously (4).
    [Show full text]
  • Allosteric Control of Internal Electron Transfer in Cytochrome Cd1 Nitrite Reductase Ole Farver*†, Peter M
    Allosteric control of internal electron transfer in cytochrome cd1 nitrite reductase Ole Farver*†, Peter M. H. Kroneck‡, Walter G. Zumft§, and Israel Pecht¶ *Department of Analytical Chemistry, Danish University of Pharmaceutical Sciences, DK-2100 Copenhagen, Denmark; ‡Fachbereich Biologie, Universita¨t Konstanz, D-78457 Konstanz, Germany; §Lehrstuhl fu¨r Mikrobiologie, Universita¨t Fridericiana, D-76128 Karlsruhe, Germany; and ¶Department of Immunology, Weizmann Institute of Science, 76100 Rehovot, Israel Communicated by Harry B. Gray, California Institute of Technology, Pasadena, CA, May 6, 2003 (received for review February 10, 2003) Cytochrome cd1 nitrite reductase is a bifunctional multiheme en- (5). The N-terminal tail of Ps-cd1 NIR differs markedly from zyme catalyzing the one-electron reduction of nitrite to nitric oxide those of the other two enzymes suggesting a different mode of and the four-electron reduction of dioxygen to water. Kinetics and interaction. Therefore, we have now studied both the thermo- thermodynamics of the internal electron transfer process in the dynamics and kinetics of internal ET in the Ps enzyme by pulse Pseudomonas stutzeri enzyme have been studied and found to be radiolytically produced N-methylnicotinamide radicals. dominated by pronounced interactions between the c and the d1 hemes. The interactions are expressed both in dramatic changes in Materials and Methods the internal electron-transfer rates between these sites and in Cytochrome cd1 from P. stutzeri strain ZoBell (ATCC 14405) marked cooperativity in their electron affinity. The results consti- was purified, and its biochemical and spectroscopic parameters tute a prime example of intraprotein control of the electron- were characterized as described (9).
    [Show full text]
  • The Nitrite Reductase from Pseudomonas Aeruginosa: Essential Role of Two Active-Site Histidines in the Catalytic and Structural Properties
    The nitrite reductase from Pseudomonas aeruginosa: Essential role of two active-site histidines in the catalytic and structural properties Francesca Cutruzzola` *, Kieron Brown†, Emma K. Wilson*, Andrea Bellelli*, Marzia Arese*, Mariella Tegoni†, Christian Cambillau†, and Maurizio Brunori*‡ *Dipartimento di Scienze Biochimiche ‘‘A. Rossi Fanelli’’ and Centro di Biologia Molecolare del Consiglio Nazionale delle Ricerche, Universita`di Roma ‘‘La Sapienza,’’ 00185 Rome, Italy; and †Architecture et Fonction des Macromole´cules Biologiques, Unite´Mixte de Recherche 6098, Centre National de la Recherche Scientifique and Universite´s de Marseille I and II, 13402 Marseille Cedex 20, France Edited by Harry B. Gray, California Institute of Technology, Pasadena, CA, and approved December 18, 2000 (received for review August 3, 2000) Cd1 nitrite reductase catalyzes the conversion of nitrite to NO in essential for NIR activity (NIRV) but have no effect on the oxygen denitrifying bacteria. Reduction of the substrate occurs at the d1- reductase activity. The 3D structures of both mutants shows that (i) heme site, which faces on the distal side some residues thought to be Ala replaces His in the distal d1-heme pocket of both mutants; (ii) essential for substrate binding and catalysis. We report the results Tyr-10 slips away together with the N-terminal arm; and (iii) the obtained by mutating to Ala the two invariant active site histidines, c-heme domain experiences a large topological change relative to His-327 and His-369, of the enzyme from Pseudomonas aeruginosa. the d1-heme domain, which is unmodified. Our results allow us to Both mutants have lost nitrite reductase activity but maintain the propose a mechanism for catalysis of nitrite reduction, based largely ability to reduce O2 to water.
    [Show full text]
  • In Vivo Assay of Nitrate Reductase in Cotton Leaf Discs
    Plant Physiol. (1973) 51, 332-336 In Vivo Assay of Nitrate Reductase in Cotton Leaf Discs EFFECT OF OXYGEN AND AMMONIUM' Received for publication August 11, 1972 J. W. RADIN Western Cotton Research Laboratory, United States Department ofAgriculture, 4135 East Broadway, Phoenix, Arizona 85040 ABSTRACT was quite uncritical between 0.3 and 2%; within this range of concentrations, there was no difference in the response to Factors affecting nitrate reduction by leaf discs of cotton nitrate. In some studies disodium arsenate or other salts were (Gossypium hirsutum L.) were investigated. When incubated in added to the medium as experimental treatments. Generally 30 mM nitrate, discs reduced nitrate much more slowly under air there were 3 discs per tube, with three replicates per treat- or 02 than under N2. Inhibition by 02 did not occur at nitrate levels of 100 mM or greater. Treatment with arsenate had little ment. For time course studies, 20 ml of assay medium and 20 effect under Na but stimulated nitrate reduction under air. discs were placed in 50-ml flasks, with two replicates per treat- Similarly, ammonium inhibited nitrate reduction, with the in- ment. Tubes or flasks were connected to a manifold and evac- hibition being partially relieved by arsenate. Uptake of nitrate uated with a vacuum pump to a pressure of less than 5 mm was unaffected by ammonium. The NAD/NADH ratio increased Hg. Anaerobiosis was maintained while releasing the vacuum in response to both oxygen and amnmonium. The effects of these by introducing nitrogen gas from a cylinder connected to the treatments on nitrate reduction can be explained by competition line.
    [Show full text]
  • Reciprocal Regulation of Sulfite Oxidation and Nitrite Reduction By
    Nitric Oxide 89 (2019) 22–31 Contents lists available at ScienceDirect Nitric Oxide journal homepage: www.elsevier.com/locate/yniox Reciprocal regulation of sulfite oxidation and nitrite reduction by mitochondrial sulfite oxidase T ∗ Alexander T. Kaczmareka,b, Marc J.F. Strampraadc, Peter-Leon Hagedoornc, Guenter Schwarza,b, a Institute of Biochemistry, Department of Chemistry, University of Cologne, Cologne, 50674, Germany b Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, 50931, Germany c Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ, Delft, the Netherlands ARTICLE INFO ABSTRACT Keywords: The oxygen-independent nitrate-nitrite-nitric oxide (NO) pathway is considered as a substantial source of NO in Sulfite oxidase mammals. Dietary nitrate/nitrite are distributed throughout the body and reduced to NO by the action of various Molybdenum cofactor enzymes. The intermembrane spaced (IMS), molybdenum cofactor-dependent sulfite oxidase (SO) was shown to Nitrate-nitrite-NO pathway catalyze such a nitrite reduction. In this study we asked whether the primary function of SO – sulfite oxidation – Nitrite reduction and its novel function – nitrite reduction – impact each other. First, we utilized benzyl viologen as artificial Nitric oxide −1 electron donor to investigate steady state NO synthesis by SO and found fast (kcat =14s ) nitrite reduction of SO full-length and its isolated molybdenum domain at pH 6.5. Next, we determined the impact of nitrite on pre- steady state kinetics in SO catalysis and identified nitrite as a pH-dependent inhibitor of SO reductive and oxidative half reaction. Finally, we report on the time-dependent formation of the paramagnetic Mo(V) species following nitrite reduction and demonstrate that sulfite inhibits nitrite reduction.
    [Show full text]