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Machovina-Enzymatic-2018.Pdf ENZYMATIC STRATEGIES FOR CONTROLLING AND HARNESSING THE OXIDATIVE POWER OF O2 by Melodie M. Machovina A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry MONTANA STATE UNIVERSITY Bozeman, Montana November 2018 ©COPYRIGHT by Melodie Marie Machovina 2018 All Rights Reserved ii DEDICATION I would like to dedicate this work to my parents, Alberto and Gina, whose outstanding dedication to excellence and hard-work has encouraged me to persevere through all that life has given. This dissertation is also dedicated to my godfather, Murray Oxman, a true friend and inspiration. Your wisdom has guided me along this journey and continues to guide me! iii ACKNOWLEDGMENTS My first and foremost thanks go to my advisor, Prof. Jennifer DuBois; your passion and dedication towards excellent science is an inspiration. Thank you for your guidance along this significant chapter in my life! I would also like to acknowledge my thesis committee members, Profs. Joan Broderick, Valerie Copie, and Brian Bothner, and Dr. Gregg Beckham for all that you’ve taught me along the way. I am grateful to my collaborators for providing excellent data and being great to work with. A special thanks to Gregg Beckham for taking me under his wing at NREL and always being so excited and positive. I would also like to acknowledge John McGeehan and Sam Mallinson in the UK; it’s been a pleasure working with you on the amazing GcoA stories. Thank you for making our collaboration so enjoyable and productive! Doreen Brown, thank you for helping make grad school a smooth and enjoyable experience. Members of the DuBois lab, I’m so glad we get to interact on a daily basis – it makes coming into work a pleasure. Arianna, I’m overjoyed that we got to share this adventure together! To Bryan, thank you for helping me navigate through these years and always being there for me; your strength is inspiring. My utmost gratitude to my family; you always remind me of what is important in life. iv TABLE OF CONTENTS SECTION I. AN INTRODUCTION TO THE IMPORTANT ROLE OF O2-UTILIZING ENZYMES 1. STRATEGIES OF O2-DEPENDENT ENZYMES .........................................................1 The Paradox of O2 Utilization .........................................................................................1 Enzymatic Strategies for Activating O2 ..........................................................................2 ABM Enzymes Activate O2 Differently ..........................................................................7 New P450 Class Degrades Plant Matter to Make High-value Products .........................9 Understanding How Different Enzymes Use O2 for Variable Reactions ......................11 References .....................................................................................................................11 SECTION II. UNDERSTANDING HOW A FAMILY OF SMALL, RELATIVELY SIMPLE BACTERIAL ENZYMES USES O2 IN THE PRESENCE AND ABSENCE OF A COFACTOR 2. TIME-RESOLVED STUDIES OF ISDG IDENTIFY MOLECULAR SIGNPOSTS ALONG THE NON-CANONICAL HEME OXYGENASE PATHWAY....................................................................................................................16 Contribution of Authors and Co-Authors ......................................................................16 Manuscript Information Page ........................................................................................18 Abstract .........................................................................................................................19 Introduction ...................................................................................................................20 Methods and Materials ..................................................................................................22 IsdG purification, expression and reconstitution with heme .................................22 IsdG-heme reaction with O2, and reducing equivalents monitored by UV/visible (UV/vis) spectroscopy .........................................................................24 IsdG-heme reaction with O2/ascorbate monitored by time resolved in proteo mass spectrometry .................................................................................................24 IsdG-heme reaction with O2/ascorbate monitored by UV/vis stopped flow spectroscopy ..........................................................................................................25 Shunt reactions with H2O2 and peracetic acid ......................................................26 Extraction of tetrapyrrole products .......................................................................26 Tetrapyrrole product analysis by liquid chromatography/tandem mass spectroscopy (LC-MS/MS) ................................................................................... 27 Quantification of C1 products ................................................................................27 Results and Discussion ..................................................................................................28 Conclusions ..................................................................................................................36 Tables and Figures........................................................................................………….38 Associated Content .......................................................................................................44 Acknowledgements ...............................................................................................44 Abbreviations ........................................................................................................ 44 v TABLE OF CONTENTS CONTINUED Footnote ....................................................................................................................... 45 References ....................................................................................................................45 3. MONOXYGENASE SUBSTRATES MIMIC FLAVIN TO CATALYZE COFACTORLESS OXYGENATIONS .......................................................................49 Contribution of Authors and Co-Authors .....................................................................49 Manuscript Information Page ........................................................................................50 Abstract ........................................................................................................................51 Introduction ...................................................................................................................52 Methods and Materials .................................................................................................54 Expression and purification of NMO ....................................................................54 Generation of site-directed mutants ......................................................................54 Dithranol stocks and reaction media .....................................................................55 NMO/substrate binding monitored by fluorometric quenching…………….........55 pH titration of dithranol ........................................................................................56 Analysis of reactants and products by high-performance liquid chromatography (HPLC) .......................................................................................56 Monitoring uncatalyzed reactions over time .........................................................57 Monitoring the NMO-catalyzed reaction over time in the steady state ................57 Effects of pH on the steady state reaction .............................................................58 Transient kinetics of the reaction between the NMO-substrate complex and O2 ...........................................................................................................................59 Analysis of reaction products………………………………………………….…59 - Effects of superoxide (O2 ) trapping on product distribution…………………….60 Detection and quantification of superoxide via the CM-radical by continuous wave electron paramagnetic resonance (EPR) spectroscopy ................................60 Sequence analysis .................................................................................................61 Results ...........................................................................................................................61 Characterization of the enzyme, substrate, and products ......................................61 Uncatalyzed oxidation of dithranol in air ...............................................................62 Enzymatic oxidation of dithranol in air ..................................................................64 pH dependence of the steady state reaction ............................................................65 Dependence of substrate affinity of pH ..................................................................67 Dependence of the steady state enzymatic reaction on O2 concentration ..............67 Transient kinetics of the reaction between NMO-dithranol complex and O2 ........68 Radical pair formation and recombination .............................................................69 Analysis of sequence and structure ........................................................................71 Discussion .....................................................................................................................72 Schemes, Tables,
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