Heme Axial Base Effects on Heme-Peroxo- Copper Adduct Formation, Properties, and Reactivity by Patrick J. Rogler Dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, MD October, 2018 Abstract Over the course of the next century, a fundamental understanding of the critical factors that control the efficiency, and selectivity of the four proton, four electron reduction of O2 to H2O will likely continue to grow in importance for inorganic and materials chemists. This may be driven in large part by the necessity and promise of sustainable and economically feasible alternative energy technologies which will undoubtedly rely on fundamental chemical insights into the discrete metal-oxy species formed in the course of reductive O—O bond activation and cleavage in both homogeneous and heterogeneous media. Bio-inorganic chemists seek to address these important problems by looking to natural systems, wherein lie excellent examples of efficient, selective O2 reduction provided by Heme-Copper Oxidases (HCOs). This superfamily of integral membrane proteins serve as the terminal electron acceptors of the mitochondrial electron transport chain where they bind and reduce O2 to H2O at a hetero-binuclear active site consisting of a tris-histidyl ligated CuB ion, and a heme iron cofactor. The discrete steps of this reductive O—O bond cleavage reaction are coupled to transmembrane proton pumping and oxidative phosphorylation. A long standing interest of our group has been to utilize heme-peroxo-copper adducts as model systems to understand in detail the factors which result in O2 reduction to water by drawing upon structure function relationships in the binuclear active site. The III 2- II metal bridging peroxide formulation (heme)-Fe -(O2 )-Cu has never been observed during turnover, however it has been explored computationally and is an informative starting point as a model. ii In the chapters below we present a design approach wherein the heme axial base moiety attached to the porphyrin periphery is changed from a covalently attached pyridyl base, to an imidazole base, and we describe the effects of these axial base functionalities on heme-peroxo-copper adduct formation, and stability. Finally, we report the synthesis, and characterization of a small library of mononuclear heme-ferric peroxides which serve to sharpen relevant questions related to heme-peroxo-copper adduct reactivity towards exogenous reductant. Advisor: Prof. Kenneth D. Karlin Thesis Committee: Dr. David P. Goldberg Dr. John P. Toscano iii Acknowledgements I would first and foremost like to thank my thesis advisor, Prof. Kenneth D. Karlin for allowing me the opportunity to conduct my thesis research in hi laboratory. His vast knowledge and experience within not only our field, but also on a broad range of topics in chemistry and biology has helped me to grow great deal in the course of my graduate career, and I most certainly would not have been able to complete this work without his help and guidance. I would also like to thank the members of my thesis committee, Prof. David P. Goldberg, and Prof. John P. Toscano for their insights, as well as their patience and kindness. During my time as PhD candidate, especially early on, senior group members have helped me to grow a great deal. In particular I would like to thank Dr. Savita K. Sharma, Dr. Isaac Garcia-Bosch, Dr. Ryan L. Peterson, and Dr. Sunghee Kim all of whom have helped me either through their superb teaching skills, or their mentorship. I would not have grown as much as a person, or a scientist during my time in graduate school were it not for their influence. Several lab group members have also been particularly impactful as well. Daniel, Mayukh Jeff, Hyun, Haley, Austin, and Diego are all wonderful people, and I have been privileged to be in their company both in a scientific, and personal sense, and I will look back on our time together in lab with great fondness. I would also like to thank the NIH for funding, and the Chemistry Department for financial support. Also, several staff members including Boris, Phil, Cathy, Joel, Rosalie Dennis and Lauren have been very helpful throughout my time here. iv Lastly, and most importantly of all, I would like to thank my parents, Charles and Leslie Rogler, my brother Chris, my sister Kimberley and Valerie. Without their love and support I would never have made it through, and I will never forget how important they are to me. In addition to my family, many of my friends have been highly supportive, and great to talk to, and I have very many fond memories of my time with Zaid, Darius, Ivan, Jordan, James, and Eddie. Sincerely, Patrick J. Rogler v Table of Contents CHAPTER 1. UNDERSTANDING BIOLOGICAL O2 REDUCTION BY HEME-COPPER OXIDASES: SMALL MOLECULE SCALE HEME-CU MODELS CAN PROVIDE ENZYME INSIGHTS ..................................................................................................................................................... 1 1.1. DIOXYGEN REDUCTION AND ACTIVATION: GENERAL CONSIDERATIONS ........................................ 2 1.2. DIOXYGEN ACTIVATION AND REDUCTION BY METALLOENZYMES ................................................. 4 1.3. EFFICIENT, SELECTIVE O2 REDUCTION BY HEME-COPPER-OXIDASES ............................................. 6 1.4. KEY STRUCTURAL FEATURES OF THE HCO SUPERFAMILY, AND ESSENTIAL REDOX COFACTORS ... 9 1.5. CATALYTIC PHASES OF O2 REDUCTION BY BOVINE HEART CYTOCHROME C OXIDASE ................. 10 1.5.1. Catalytic mechanism of cytochrome c oxidase ...................................................................... 11 1.6. UNDERSTANDING O2 REDUCTION BY HCOS USING A SYNTHETIC MODELING APPROACH ............ 14 1.7. DIOXYGEN DERIVED HEME-PEROXO-COPPER ASSEMBLIES AS HCO SYNTHETIC MODELS: GENERATION, STRUCTURES AND SPECTROSCOPIC PROPERTIES .................................................................. 16 REFERENCES: ............................................................................................................................................ 28 CHAPTER 2. ISOCYANIDE OR NITROSYL COMPLEXATION TO HEMES WITH VARYING TETHERED AXIAL BASE LIGAND DONORS: SYNTHESIS AND CHARACTERIZATION ....... 39 2.1. ABSTRACT .................................................................................................................................. 40 2.2. INTRODUCTION ........................................................................................................................... 41 2.3. EXPERIMENTAL ........................................................................................................................... 44 2.3.1. Materials and Methods .......................................................................................................... 44 2.3.2. Synthesis of DIMPI, and NO bound ferrous heme porphyrinates ......................................... 46 2.3.3. X-Ray structure determination .............................................................................................. 48 2.4. RESULTS AND DISCUSSION .......................................................................................................... 50 2.4.1. Stable heme–isocyanide complex formation: ........................................................................ 50 2.4.2. Crystal structure of isocyanide complex, [(PIm)FeII-(DIMPI)] (3)-DIMPI ........................... 53 2.4.3. Stable heme–Fe–Nitrosyl formation ...................................................................................... 61 2.5. CONCLUSIONS ............................................................................................................................. 65 REFERENCES: ............................................................................................................................................ 67 APPENDIX A ........................................................................................................................................... 72 CHAPTER 3. REACTIONS OF A HEME-SUPEROXO COMPLEX TOWARD A CUPROUS CHELATE AND •NO(G): CCO AND NOD CHEMISTRY .................................................................... 76 3.1. ABSTRACT .................................................................................................................................. 77 3.2. INTRODUCTION ........................................................................................................................... 77 3.3. EXPERIMENTAL ........................................................................................................................... 79 3.3.1. Materials and Methods .......................................................................................................... 79 3.3.2. Synthesis ................................................................................................................................ 80 2 3.3.3. Preparation of H NMR and EPR samples ............................................................................ 81 3.3.4. Procedure for Nitrate/Nitrite test .......................................................................................... 82 3.3.5. Nitration of the 2,4-Di-tert-butylphenol (DTBP) ................................................................... 82 3.4. RESULTS AND DISCUSSION .......................................................................................................... 83 I 3.4.1. Reactivity of the iron(III)-superoxo complex (2) towards [Cu (AN)][B(C6F5)4]