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Structural, Spectroscopic, and Mechanistic Insights Into the Three Phases of Nitrification Metallobiochemistry

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Structural, Spectroscopic, and Mechanistic Insights Into the Three Phases of Nitrification Metallobiochemistry STRUCTURAL, SPECTROSCOPIC, AND MECHANISTIC INSIGHTS INTO THE THREE PHASES OF NITRIFICATION METALLOBIOCHEMISTRY A Dissertation Presented to the Faculty of the Graduate School of Cornell University In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy | Chemistry and Chemical Biology By Meghan Anne Smith August 2018 © 2018 Meghan Anne Smith STRUCTURAL, SPECTROSCOPIC, AND MECHANISTIC INSIGHTS INTO THE THREE PHASES OF NITRIFICATION METALLOBIOCHEMISTRY Meghan Anne Smith, Ph. D. Cornell University 2018 Biological ammonia (NH3) oxidation, referred to as nitrification, is a critical part of the biogeochemical nitrogen cycle. Nitrification is mediated by both bacteria and – archaea to ultimately oxidize NH3 to nitrite (NO 2 ), though there are also complete NH3-oxidizing (comammox) bacteria capable of oxidizing NH3 completely to nitrate – (NO3 ). In addition to these products, nitrification is also a major source of the by- products and environmental pollutants nitric oxide (NO), nitrous oxide (N2O) and nitrogen dioxide (NO2). Many steps of biological nitrification, including those leading to the production of these harmful products, are not currently clear; however, the work presented in this dissertation describes recent efforts and discoveries towards a complete understanding of the nitrification pathway. This process begins in both bacteria and archaea with the enzyme ammonia monooxygenase (AMO), which oxidizes NH3 to hydroxylamine (NH2OH). There exist two metal-binding sites in AMO of interest as these are highly conserved in AMOs and related enzymes. The true active site of this enzyme remains in debate, but here we show that both sites must remain intact for effective catalysis. In bacteria, the formed NH2OH is further oxidized to NO by the enzyme NH2OH oxidoreductase (HAO), though prior convention stated – that HAO was able to oxidize NH2OH fully to NO2 . There exists another enzyme in NH3–oxidizing bacteria (AOB) known as cytochrome (cyt) P460 that can oxidize NH2OH to NO and N2O. Here we present structural and mechanistic studies that describe how the unusual P460 cofactor and surrounding amino acids allow for this – catalysis. The recent discovery that the true product of HAO is NO and not NO2 presents a challenge to find the enzyme in AOB which can complete this oxidation to – the final product NO2 . Here we present a potential candidate, nitrosocyanin (NC), and describe preliminary experiments on its interaction with NO. BIOGRAPHICAL SKETCH Meghan A. Smith attended Creighton University in Omaha, NE and graduated cum laude with honors in 2013. She completed a B.S. in Chemistry on the Biochemistry track with a minor in Cognitive and Behavioral Neuroscience. During her time at Creighton, she received a fellowship from the Institutional Development Award Program (IDeA) Networks of Biomedical Research Excellence (INBRE) program in Nebraska. As part of this fellowship she completed a summer of research in the lab of Prof. James R. Alfano at the University of Nebraska, Lincoln and three years of research in the lab of Prof. Karin van Dijk at Creighton University. This work was focused on effector protein interactions of the type three secretion system (TTSS) that lead to virulence in the bacteria Pseudomonas syringae pv. tomato. After graduating from Creighton, Meghan pursued her PhD at Cornell University where she worked for Prof. Kyle M. Lancaster on the enzymology supporting ammonia (NH3) oxidation in both bacteria and archaea. The following describes her work completed at Cornell University under the guidance of Prof. Lancaster. v ACKNOWLEDGMENTS I gratefully acknowledge all current and former members of the Lancaster Research Group for their support, advice, and, importantly, friendship. Specifically, I would like to recognize Dr. Jonathan D. Caranto for his help in designing and performing experiments on projects concerning AMO, cyt P460, and nitrosocyanin. As well, I would like the thank him for providing general guidance and always making sure we were on track and had set goals in mind. I would also like to acknowledge Dr. Avery C. Vilbert for her support and help in setting up various cyt P460 experiments. I’d like to acknowledge Dr. Sudipta Chatterjee, Ida M. DiMucci, and Sean H. Majer for help in experimental setup and techniques. I’d also like to thank Rachael Coleman, Ben Looker, and Dr. Richard Walroth for their support. In addition, I wish luck to Sean, Rachael, and Ben for their continued work on these projects! I’d finally like to acknowledge Prof. Kyle M. Lancaster for his continued guidance, advice, support, and music tastes. I gratefully acknowledge Dr. Michael Fenwick for assistance with X-ray data collection and Prof. Brian Crane for advice about structural refinement. Further, Boris Dzikhovsky for his help collecting EPR spectra. In addition, Dr. Nicholas Coleman for supplying the pMycoFos vector and Prof. Harry B. Gray for providing the pEC86 plasmid containing the cyt c maturation genes ccmABCDEFGH. Finally, I’d like to acknowledge all my friends and family, without whom none of this would have been possible! vi TABLE OF CONTENTS Biographical Sketch v Acknowledgements vi Table of Contents vii List of Figures xi List of Tables xii List of Schemes xiii Chapter 1: Introduction 1 A: Nitrification 2 B. Ammonia Monooxygenase 3 C. Hydroxylamine Oxidoreductase and Cytochrome P460 11 D. Nitric Oxide Oxidation 15 E. Addressed in this Dissertation 17 F. References 17 Chapter 2: Recombinant Expression of Active Archaeal Ammonia Monooxygenase Variants in Heterotrophic Mycobacterium smegmatis 29 A: Materials and Methods 32 B. Results 37 C. Discussion 42 D. Conclusion 45 E. References 46 Chapter 3: The Eponymous Cofactors in Cytochrome P460s from Ammonia- vii Oxidizing Bacteria are Iron Porphyrinoids whose Macrocycles are Dibasic 51 A: Materials and Methods 55 B. Results 59 C. Discussion 71 D. Conclusion 74 E. References 75 Chapter 4: Outer-Sphere Gating of Substrate Oxidation and N–N Bond Formation in Cytochrome P460 82 A: Materials and Methods 84 B. Results 88 C. Discussion 99 D. Conclusion 105 E. References 105 Chapter 5: Interaction of the Red Copper Protein Nitrosocyanin with Nitric Oxide 110 A: Materials and Methods 114 B. Results 118 C. Discussion 122 D. Conclusion 124 E. References 125 Appendix A: Molecular Biology A.A.1 A: pMycoFos Sequences A.A.2 B. pMycoFos–CXAB Insert Sequence A.A.7 C. CXAB amoB Mutants A.A.9 D. CXAB amoC Mutants A.A.9 E. Nitrosomonas sp. AL212 cyt P460 Sequence A.A.10 viii F. Nitrosomonas sp. AL212 cyt P460 Mutants A.A.10 G. Nitrosomonas europaea cyt P460 Sequence A.A.11 H. Nitrosomonas europaea cyt P460 Glu97Ala Sequence A.A.11 I. Nitrosocyanin Gene Sequence A.A.12 J. Nitrosocyanin Mass Spectrometry Analysis A.A.12 Appendix B: Crystallographic Data A.B.1 A: Crystallographic Data Table A.B.2 Appendix C: ORCA Input Files and Final Geometry Optimized Structures A.C.1 ix A: Geometry Optimization ORCA Input File A.C.2 B. TDDFT ORCA Input File A.C.3 C. Cyt P460 Isoporphyrin Final Geometry Optimized Structure Coordinates A.C.4 D. Cyt P460 Porphyrin, Meso C–N Final Geometry Optimized Structure Coordinates A.C.7 E. Cyt P460 Phlorin Final Geometry Optimized Structure Coordinates A.C.10 F. Metmyoglobin Final Geometry Optimized Structure Coordinates A.C.13 G. Cyt P460 Porphyrin, Meso C–C Final Geometry Optimized Structure Coordinates A.C.16 H. Cyt P460 Porphyrin, Meso C–N Final Geometry Optimized Structure Coordinates A.C.19 I. Heme Restraint Files A.C.22 x LIST OF FIGURES Figure Description Page 1.1 Summarized current understanding of biological nitrification 4 1.2 Structural homology in CuMMOs 7 1.3 Sequence homology between aAMO, bAMO, and pMMO 9-10 1.4 Heme P460 cofactors in cyt P460 and HAO 13 1.5 Proposed cyt P460 catalytic cycle 14 2.1 AMO gene cluster 31 2.2 Structural homology model of aAMO 31 2.3 M. smegmatis colony PCR 38 2.4 Detection of AMO in M. smegmatis cells 39 2.5 O2 Consumption by M. smegmatis cells 40 2.6 Effect of allyltiourea on O2-consumption 40 2.7 NH4Cl-induced changes in O2-consumption 41 3.1 Isoporphyrin, porphyrin, phlorin configurations 54 3.2 SDS-PAGE gel of Nitrosomoas sp. AL212 cyt P460 57 3.3 UV-vis of cyt P460s from different bacterial species 60 3.4 Spectroscopic characterization of cyt P460 species 61 3.5 Crystal structure of N. sp. AL212 cyt P460 62 3.6 P460 Cofactor binding pocket 65 3.7 Calculated heme difference density 66 3.8 2F0–Fc simulated annealing omit map 68 3.9 TD-DFT and experimental UV-vis 68 3.10 TD-DFT and experimental UV-vis 69 3.11 Four-orbital porphyrin model for cyt P460 70 3.12 Energy diagram for cyt P460 71 4.1 N. europaea and N. sp. AL212 active sites 84 4.2 N. sp. AL212 cyt P460 NH2OH and NO Kds 89 4.3 Spectrochemical potentiometric titration 90 4.4 UV-vis characteristics of cyt P460 variants 92 4.5 Ala131Glu mutation allows for NH2OH oxidation 94 4.6 Steady-state NH2OH oxidase activity plot 94 4.7 EPR Spectra of cyt P460 variants 96 4.8 WT AL212, Ala131Glu, Ala131Gln crystal structures 97 4.9 Active site configuration of various cyt P460 species 97 4.10 2F0–Fc simulated annealing composite omit map 98 4.11 Sequence homology of various cyt P460 genes 103 5.1 UV-vis Absorption of CuII or CuI NC with NO 113 5.2 EPR Spectra of NC 119 5.3 NO concentration in solution with CuII or CuI NC 119 5.4 CuIINC NO oxidase assay trials 121 5.5 CuINC NO oxidase assay trials 121 I – 5.6 Cu NC interaction with NO2 122 xi LIST OF TABLES Table Description Page 2.1 AMO cloning primers 33 3.1 X-ray data collection and refinement statistics 63 3.2 Geometric parameters of P460 cofactor 65 3.3 Normal-coordinate structural decomposition analysis 67 4.1 N.
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