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Spectroscopic Study of the Formation and Degradation of Metalated Tetrapyrroles by the Enzymes Cfba, Isdg, and Mhud Ariel E

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Spectroscopic Study of the Formation and Degradation of Metalated Tetrapyrroles by the Enzymes Cfba, Isdg, and Mhud Ariel E University of Vermont ScholarWorks @ UVM Graduate College Dissertations and Theses Dissertations and Theses 2019 Spectroscopic Study of the Formation and Degradation of Metalated Tetrapyrroles by the Enzymes CfbA, IsdG, and MhuD Ariel E. Schuelke-Sanchez University of Vermont Follow this and additional works at: https://scholarworks.uvm.edu/graddis Part of the Inorganic Chemistry Commons Recommended Citation Schuelke-Sanchez, Ariel E., "Spectroscopic Study of the Formation and Degradation of Metalated Tetrapyrroles by the Enzymes CfbA, IsdG, and MhuD" (2019). Graduate College Dissertations and Theses. 1155. https://scholarworks.uvm.edu/graddis/1155 This Dissertation is brought to you for free and open access by the Dissertations and Theses at ScholarWorks @ UVM. It has been accepted for inclusion in Graduate College Dissertations and Theses by an authorized administrator of ScholarWorks @ UVM. For more information, please contact [email protected]. SPECTROSCOPIC STUDY OF THE FORMATION AND DEGRADATION OF METALATED TETRAPYRROLES BY THE ENZYMES CFBA, ISDG, AND MHUD A Dissertation Presented by Ariel E. Schuelke-Sanchez to The Faculty of the Graduate College of The University of Vermont In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Specializing in Chemistry October, 2019 Defense Date: August 21, 2019 Dissertation Examination Committee: Matthew D. Liptak, Ph. D., Advisor Scott W. Morrical, Ph. D., Chairperson Rory Waterman, Ph. D. José S. Madalengoitia, Ph. D. Cynthia J. Forehand, Ph. D., Dean of the Graduate College ABSTRACT Metal tetrapyrroles represent a large class of earth-abundant catalysts but are limited to naturally-occurring combinations. Chelatase enzymes are responsible for the catalyzed metal insertion into a specific tetrapyrrole. CfbA is a class II chelatase from Archaeaglobis fulgidus that catalyzes the insertion of nickel into sirohydrochlorin to give rise to Coenzyme F430 required for methanogensis. This archaeal chelatase was used to study the substrate scope of divalent metals and tetrapyrroles to probe various metal tetrapyrrole combinations. A spectroscopic study established that the CfbA is capable of binding to cobalt and nickel, in addition to various tetrapyrroles. Magnetic circular dichroism (MCD) established that the metal binding site of CfbA contains a labile high spin 6-coordinate cobalt species that is ligated to nitrogen- or oxygen- atoms. The two residues involved in metal binding is likely His10 and His74. Tetrapyrrole binding resulted in a shift in energy. Computational studies have shown that resultant red-shift in energy is due to slight ruffling upon binding to CfbA. The enzymatic capabilities of CfbA was probed with various metal and tetrapyrrole combinations. The rate of insertion was significantly impacted by identity of the metal and the position of the propionate and acetate side chains on the rings of the tetrapyrrole as compared to sirohydrochlorin. Modifications to these side chains resulted in changes in ruffling. An increase in the ruffling resulted in a decrease in the rate of the reaction. These results have shown a significant expansion of the tetrapyrrole substrate scope. Additionally, detailed insights into the proposed chelatase mechanism have been established. IsdG serves as the primary enzyme involved in iron acquisition from heme in Staphylococcus aureus. The active site contains a tryptophan residue at 67 that is expected to be involved in heme ruffling. Trp67 was substituted with a smaller amino acid, phenylalanine to determine the role it plays in heme ruffling and degradation. The optical spectroscopic characterization of W67F IsdG resulted in changes to the geometric and electronic structure. The absorbance spectrum of W67F blue-shifted in the Q- and Soret bands indicating a change in the heme ruffling. MCD, VTVH and 1H NMR spectroscopy 2 have shown that the electronic ground state is indicative of a Eg state, consistent with reduced heme ruffling. The degradation of heme by W67F IsdG resulted in the formation of biliverdin, a product seen in canonical HOs. These data suggest that Trp67 significantly influences heme ruffling and degradation. Additionally, W67F IsdG follows a unique reaction mechanism compared to IsdG. These data provide information on the development of a selective inhibitor of IsdG to prevent pathogenesis. ACKNOWLEDGEMENTS I would like to thank my advisor, professor Matthew Liptak, for his role in completing my Ph.D. I would also like to thank the members of my committee, Drs. Rory Waterman, José Madalengoitia, and Scott Morrical. Each of you has expressed support in my progress throughout my time here. I am forever grateful of having the support of my group members and dear friends. Thank you for all the fun adventures. Finally, I would like to thank my family and my partner. To my parents, I would have never made it this far without your unwavering, constant support. You have pushed me to be the best person I can possibly be. To my partner, thank you for your constant love and encouragement. I truly could not have done this without you by my side. ii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ................................................................................................ ii LIST OF ABBREVIATIONS ........................................................................................... vii LIST OF TABLES ............................................................................................................. ix LIST OF FIGURES ........................................................................................................... xi CHAPTER 1: FORMATION AND DEGRADATION OF METALATED TETRAPYRROLES ............................................................................................................1 1.1 Tetrapyrroles ..................................................................................................................2 1.2 Synthetic Pathway of Tetrapyrroles ...............................................................................3 1.3 Biosynthetic Pathway and Degradation of Tetrapyrroles ..............................................6 1.4 Experimental and Theoretical Approach to Study Tetrapyrroles ................................11 1.5 Chapter 1 References ..................................................................................................15 CHAPTER 2: CFBA CATALYZES INSERTION OF COBALT INTO RUFFLED TETRAPYRROLES ..........................................................................................................21 2.1 Introduction ..................................................................................................................22 2.2 Experimental Methods .................................................................................................24 2.3 Results ..........................................................................................................................33 2.4 Discussion ...................................................................................................................48 iii 2.5 Conclusions ..................................................................................................................53 2.6 Chapter 2 References ...................................................................................................54 CHAPTER 3: SPECTROSCOPIC STUDY OF NICKEL ENZYMATIC ACTIVITY BY CFBA...........................................................................................................................61 3.1 Introduction ..................................................................................................................62 3.2 Experimental Methods .................................................................................................65 3.3 Results ..........................................................................................................................69 3.4 Discussion ...................................................................................................................76 3.5 Conclusions ..................................................................................................................79 3.6 Chapter 3 References ...................................................................................................79 CHAPTER 4: MODELLING THE HEME TRANSMEMBRANE PROTEINS MMPL3-E1 AND MMPL11-E1 USING MHUD .............................................................84 4.1 Introduction ..................................................................................................................85 4.2 Experimental Methods .................................................................................................89 4.3 Results ..........................................................................................................................92 4.4 Discussion ...................................................................................................................96 4.5 Conclusions ..................................................................................................................97 4.6 Chapter 4 References ...................................................................................................98 iv CHAPTER 5: IDENTIFYING THE ROLE OF TRP67 IN HEME RUFFLING AND DEGRADATION BY THE HEME-DEGRADING ENZYME ISDG ............................101 5.1 Introduction ................................................................................................................102
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