The Pennsylvania State University The Graduate School Eberly College of Science EXPLORING THE FUNCTIONAL AND MECHANISTIC DIVERSITY OF DIIRON OXIDASES AND OXYGENASES A Dissertation in Biochemistry, Microbiology, and Molecular Biology by Lauren J. Rajakovich 2017 Lauren J. Rajakovich Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2017 ii The dissertation of Lauren J. Rajakovich was reviewed and approved* by the following: J. Martin Bollinger, Jr. Professor of Chemistry Professor of Biochemistry and Molecular Biology Dissertation Co-Advisor Committee Co-Chair Carsten Krebs Professor of Chemistry Professor of Biochemistry and Molecular Biology Dissertation Co-Advisor Committee Co-Chair Squire J. Booker Howard Hughes Medical Investigator Professor of Chemistry Professor of Biochemistry and Molecular Biology Amie K. Boal Professor of Chemistry Professor of Biochemistry and Molecular Biology Christopher House Professor of Geosciences Scott Selleck Professor of Biochemistry and Molecular Biology Head of the Department of Biochemistry, Microbiology, and Molecular Biology *Signatures are on file in the Graduate School iii ABSTRACT Approximately half of all enzymes in Nature utilize a metal to perform their biological function. Many of the metalloenzymes that harbor transition metals activate dioxygen to catalyze a diverse array of oxidation reactions to functionalize unreactive sites in biomolecules. These enzymatic transformations are often inaccessible to synthetic chemists, and consequently, understanding the naturally-evolved mechanisms by which these enzymes enact such challenging reactions will enable the development of new biocatalysts for industrial and therapeutic applications. One common strategy employed in Nature is the coupling of two transition metals, typically iron, to carry out oxidation and oxygenation reactions. Decades of research on this class of non-heme diiron enzymes has focused on three founding members, which has revealed unifying mechanistic features that enable them to enact one- and two-electron oxidation reactions. Chapter 1 summarizes the principles for dioxygen activation employing this bioinorganic scaffold that emerged from this foundational work. Chapter 1 also introduces more recent discoveries of novel non-heme diiron enzymes, facilitated by advancements in genome sequencing and bioinformatics, that have expanded the scope of chemical transformations and mechanistic strategies possible within this extensive metalloenzyme class. My dissertation research focused on three of these newly discovered diiron enzymes that invoke alternative mechanistic strategies to carry out non- canonical transformations. Chapter 2 covers the hydrocarbon-producing cyanobacterial diiron enzyme, ADO, which catalyzes a C-C bond cleavage reaction to effectively remove the chemical functional group from its fatty aldehyde substrate, producing linear hydrocarbons. My work demonstrates that ADO operates by a free-radical mechanism to enact this redox-neutral transformation, and that a cyanobacterial ferredoxin (PetF)/ferredoxin reductase/NADPH reducing system can act as an efficient reducing partner, a requisite for ADO catalysis. These mechanistic studies enabled the identification of inherent vulnerabilities that limit enzymatic iv efficacy, thereby highlighting direct targets for bioengineering and optimization of biofuel processes deploying this catalyst. Chapter 3 describes a project designed to biochemically characterize a diiron enzyme belonging to a new functional class. This work culminated in the discovery of a novel microbial phosphonate degradation pathway, consisting of two iron- dependent oxygenases with previously misannotated functional assignments. Finally, Chapter 4 describes progress on studies of another potential biofuel catalyst, an iron-dependent enzyme, UndA. This work provides evidence that UndA employs a diiron cofactor to convert fatty acids into terminal alkenes. This finding corrects its original cofactor assignment, rationalizes its ability to perform this transformation, and suggests a feasible catalytic mechanism. Collectively, these mechanistic studies contribute unique insight into the emerging reactivities of diiron enzymes that diversify their potential biotechnological applications. v TABLE OF CONTENTS List of Figures .......................................................................................................................... vii List of Tables ........................................................................................................................... xviii Acknowledgements .................................................................................................................. xix Chapter 1 Non-heme diiron oxygenases and oxidases ............................................................. 1 1.1 Introduction ................................................................................................................ 2 1.2 Canonical ferritin-like diiron-carboxylate oxidases and oxygenases (FDCOOs) ...... 4 1.3 Novel outcomes and variations on the FDCOO mechanistic theme .......................... 12 1.3.1 Electrophilic diferric-peroxide intermediates as oxidants. .............................. 13 1.3.2 A nucleophilic dioxygen moiety in aldehyde deformylating oxygenase. ....... 24 1.3.3 Use of transition metals other than iron by ferritin-like proteins. ................... 29 1.4 O2-activating diiron cofactors within non-ferrtin-like protein architectures .............. 39 1.4.1 Integral membrane diiron oxidases/oxygenases. ............................................. 40 1.4.2 Deoxyhypusine hydroxylase. .......................................................................... 44 1.4.3 Diiron β-hydroxylases. .................................................................................... 47 1.4.4 HD-domain mixed-valent diiron oxygenases. ................................................. 50 1.5 Outlook....................................................................................................................... 56 1.6 References .................................................................................................................. 57 Chapter 2 A cyanobacterial hydrocarbon production pathway employing a non-heme diiron oxygenase .............................................................................................................. 75 2.1 Exploring physiological reducing partners for ADO catalysis .................................. 82 2.1.1 Selection of the cyanobacterial [2Fe-2S] ferredoxin, PetF, as a physiologically-relevant reducing partner ........................................................ 84 2.1.2 Employment of PetF as a reducing system to support ADO catalysis under single turnover conditions ................................................................................ 87 III/III 2.1.3 Reduction of the Fe2 ADO by PetF, monitored by stopped-flow absorption spectroscopy (SF-Abs) ................................................................... 88 III/III 2.1.4 Rapid reduction of the ADO Fe2 -PHA intermediate by PetF, monitored by SF-Abs ....................................................................................... 90 2.1.5 Rapid reduction of the ADO Fe2(III/III)-PHA intermediate by PetF, monitored by rapid freeze-quench (RFQ) Mössbauer spectroscopy ................ 94 2.1.6 Sequential delivery of two electrons from PetF to ADO in a tightly coupled fashion ................................................................................................. 98 2.2 Interrogating the postulated free-radical mechanism of ADO catalysis .................... 101 2.2.1 Accumulation of radicals upon reduction of the ADO Fe2(III/III)-PHA intermediate ...................................................................................................... 102 2.2.2 Association of the transient EPR signal with a substrate-derived alkylperoxyl radical .......................................................................................... 106 2.2.3 Dependence of peroxyl radical accumulation on the concentration of O2 ...... 110 2.2.4 Diminished alkane yields due to unproductive alkyl radical quenching by O2 ...................................................................................................................... 111 vi 2.2.5 Detection of a protein-based sulfinyl radical using short-chain aldehyde substrates .......................................................................................................... 114 2.2.6 Exploring potential hydrogen atom donors for quenching of the alkyl radical intermediate .......................................................................................... 122 2.3 Materials and Methods ............................................................................................... 125 2.4 Acknowledgements .................................................................................................... 136 2.5 References .................................................................................................................. 136 Chapter 3 A new microbial pathway for organophosphonate degradation catalyzed by two previously misannotated non-heme-iron enzymes .................................................... 142 3.1 Introduction ................................................................................................................ 143 3.2 Discovery of the TMAEP hydroxylation activity