INVESTIGATION OF NITROUS OXIDE BIOSYNTHESIS BY A BACTERIAL NITRIC OXIDE REDUCTASE (NOR) AND AN ENGINEERED NOR MIMIC USING STABLE ISOTOPE RATIO MASS SPECTROMETRY By Clarisse Marie Finders A THESIS Submitted to Mic higan State University in partial fulfillment of the requirements for the degree of Biochemistry and Molecular Biology Master of Science 2018 ABSTRACT INVESTIGATION OF NITROUS OXIDE BIOSYNTHESIS BY A BACTERIAL NITRIC OXIDE REDUCTASE (NOR) AND AN ENGINEERED NOR MIMIC USING STABLE ISOTOPE RATIO MASS SPECTROMETRY By Clarisse Marie Finders While carbon dioxide (CO 2 ) is the most prevalent greenhouse gas, nitrous oxide (N 2 O) is far more potent , with a global warming potential ~ 265 times greater than that of carbon dioxide over a 100 - year period. 1 Additionally, N 2 O is capable of destroying ozone, making it doubly concerning as a greenhouse gas. Approximately half of the N 2 O produced yearly is from anthropogenic sources. The largest contributor to anthropogenic N 2 O is the over - fertilization of agricultural soils, which fuels a host of microbial nitrogen cycling processes that produce N 2 O. One of these processes is denitrification , and N 2 O is known to be an obligate intermediate in this process . In d e nitrification , N 2 O is sy nthesized by an enzyme known as nitric oxide reductase (NOR). A thorough understanding of the enzymatic mechanisms by which N 2 O is produce d is essential to mitigating anthropogenic N 2 O emissions . To this end, this thesis contains the examination of N 2 O produced by a bacterial cytochrome c NOR (cNOR) from Paracoccus dentrificans and a cNOR mimic, I107EFe B Mb, using stable isotope ratio mass spectrometry (IRMS). The first chapter provides the reader with an introduction to the nitrogen cycle, the known NO Rs, and the basics of isotope theory. The studies on the native cNOR and I107EFe B Mb are contained in the second and third chapters, respectively. Conclusions and future directions are presented in the final chapter. iii This thesis is d edicated to Eric Hegg, who saw my potential as a scientist, welcomed me into his lab, and gave me guidance along the way ; to Joshua Haslun, who challenged me to be come what Eric could see ; to Aaron Mebane, Kathleen Thomps on, Stephen, Mary , John, Victoria, and James Finders, who encouraged me and listened to me ; and finally to Elise Rivett, who put up and t rials of daily life lab with me . iv ACKNOWLEDGEMENTS I would like to acknowledge Hasand Ghandi for his assistance with samples and insight i nto experiments and isotopic data, and Peggy and Nathaniel Ostrom for their insight s and allowing me to use their instruments and Nathaniel Ostrom and Bob Hausinger for agreeing to be my committee members . I would also like to acknowledge Yi Lu , Julian Reed, and Pia Ä de lroth for generously providing purified enzyme, as well as Nicolai Le h nert, Jen nifer Dubois , and Judith Klinman for their hel pful discussions and insights. v TABLE OF CONTENTS LIST OF TABLES ................................ ................................ ................................ ........................ vii LIST OF FIGURES ................................ ................................ ................................ ..................... viii KEY TO SYM BOLS AND ABBREVIATIONS ................................ ................................ .......... ix CHAPTER 1: Nitrogen, NORs, and Navigating Stable Isotopes ................................ ................... 1 Introduction to the nitrogen cycle ................................ ................................ ............................... 1 Isotope notation ................................ ................................ ................................ ........................... 2 From N 2 to NH 3 : Nitrogen fixation ................................ ................................ ............................. 4 NH 3 to NO 3 - /N 2 : Nitrification, Comammox, and Anammox ................................ ...................... 6 NO 3 - to NH 4 + /N 2 O/N 2 : DNRA, Denitrification, Nitrifier - denitrification, and Co - denitrification ................................ ................................ ................................ ................................ ..................... 8 The Importance of N 2 O ................................ ................................ ................................ ............. 11 Nitric Oxide Reductases: Detoxifying NORs ................................ ................................ ........... 12 Respiratory NORs ................................ ................................ ................................ ..................... 13 Stable Isoto pes as a mechanistic probe ................................ ................................ ..................... 20 Zero - Point Energy and the ZPE Approximation ................................ ................................ ....... 22 Other contributors to the KIE ................................ ................................ ................................ .... 25 Summary ................................ ................................ ................................ ................................ ... 27 CHAPTER 2: Not so elementary: Isotopic fractionation and kinetic isotope effects of a pu rified bacterial nitric oxide reductase (NOR) ................................ ................................ ......................... 29 ABSTRACT ................................ ................................ ................................ .............................. 29 INTRODUCTION ................................ ................................ ................................ ..................... 30 MATERIALS AND METHODS ................................ ................................ .............................. 33 Materials ................................ ................................ ................................ ................................ 33 Enzymatic Assays: ................................ ................................ ................................ ................. 34 Isotope nomenclature ................................ ................................ ................................ ............. 35 ................................ ................................ ................................ ............................... 36 Statistics and Figures ................................ ................................ ................................ ............. 37 RESULTS ................................ ................................ ................................ ................................ .. 38 Enzymatic N 2 O Production ................................ ................................ ................................ ... 38 vi 18 15 N of N 2 O Produced by P. denitrificans cNOR .......................... 39 15 N 15 N , and S P Values for N 2 O Produced by P. denitrificans cNOR ........................... 43 DISCUSSION ................................ ................................ ................................ ........................... 45 Considerations of Observed KIEs ................................ ................................ ......................... 45 Insights from Site Preference ................................ ................................ ................................ 46 Mechanisms and Mechanistic insights ................................ ................................ .................. 48 Contributions to KIE ................................ ................................ ................................ ............. 50 CONCLUSIONS ................................ ................................ ................................ ....................... 51 APPENDIX ................................ ................................ ................................ ............................... 53 CHAPTER 3: New Dog, Old Tricks: Isotopic Studies of a Cytochrome c Nitric Oxide Reductase (cNOR) Mimic ................................ ................................ ................................ .............................. 60 ABSTRACT ................................ ................................ ................................ .............................. 60 INTRODUCTION ................................ ................................ ................................ ..................... 61 MATERIALS AND ME THODS ................................ ................................ .............................. 64 NO Solution Preparation ................................ ................................ ................................ ....... 64 Enzymatic Assays: ................................ ................................ ................................ ................. 64 RESULTS AND DISCUSSION ................................ ................................ ............................... 67 Enzymatic N 2 O Production ................................ ................................ ................................ ... 68 15 18 15 N 15 N of N 2 O produced by I107EFe B Mb .................. 69 CHAPTER 4:Knowledge Gained and Next Steps ................................ ................................ ........ 72 REFERENCES ................................ ................................ ................................ .......................... 74 vii LIST OF TABLES p - values, R 2 values, and kinetic isotope effects (KIEs) of P. denitrificans cNOR. ................................ ................................ ................................ ............. 42 - values, R 2 , and kinetic isotope effects of (KIEs) of P. denitrificans cNOR performed to test for 18 O exchange. ................................ ............................. 59 viii LIST OF FIGURES Figure 1. Diagram of the nitrogen cycle. ................................ ................................ ........................ 4 Figure 2. The ato mic structure of nitrous oxide. ................................ ................................ ........... 11 Figure 3. Crystal structure of cNOR from Pseudomonas aeruginosa . ................................ ......... 15 Figure 4. Three distinct propo sed mechanisms of cNOR, adapted from Moënne - Loccoz. 50 ....... 15 Figure 5. The proposed mechanism of P450nor.. ................................ ................................ ......... 19 Figure 6. A diagram illustrating a bond as a harmonic quantum oscillator. ................................ . 22 Figure 7. A diagram of a normal and inverse isotope effect. ................................ ........................ 24 Figure 8. Three potential transition states during N 2 O production by cNOR ............................... 32 Figure 9. Production of N 2 O by P. denitrificans cNOR ................................ ............................... 38 18 15 N (B) of N 2 O produced by cNOR. ................................ .................... 41 18 N 15 N (B) of N 2 O produced by cNOR. ................................ ................. 43 Figure 12. S P ( S P 15 N - 15 N ) for cNOR over the course of the enzymatic reaction. .............. 45 Figure 13. Three distinct proposed mechanisms of cNOR, adapted from Moënne - Loccoz. 35 ..... 48 Figure 14. Production of N 2 O by P. denitrificans cNOR with data points past 240 min ............. 54 Figure 15. Production of N 2 O by P. denitrificans cNOR with 18 O enriched water. ..................... 55 18 15 N (B) of N 2 O produced by three biological replicates ....................... 56 15 N 15 N (B) of N 2 O produced by three biological replicates. .................. 57 Figure 18. S P ( S P 15 N - 15 N ) for cNOR ................................ ................................ ............... 58 Figure 19. Production of N 2 O from NO controls and I107EFe B Mb reactions. ............................ 67 Figure 20. Isotopic fractionation of bulk nitrogen (A), oxygen (B), N (middle nitrogen, C), and N (terminal nitrogen, D). ................................ ................................ ................................ ............. 69 ix KEY TO SYMBOLS AND ABBREVIATIONS Fractionation factor delta Isotopic enrichment factor Net isotope effect Frequency Anammox Anaerobic ammonia oxidation AOA/B Ammonia Oxidizing Archaea/Bacteria BNC Binuclear center BNF Biological Nitrogen Fixation CO 2 Carbon dioxide Comammox Complete ammonia oxidation DFT Density Functional Theory DNRA Dissimilatory Nitrate Reduction to Ammonia ECD Electron Capture Detector EPR Electron Paramagnetic Resonance EXC Excited states FeMo - co Iron - molybdenum cofactor FMN Flavin mononucleotide FNOR/FIRd Flavorubredo x in HAO Hydroxylamine oxidoreductase HCO Heme Copper Oxidases Hcp Hybrid cluster protein HNO 3 Nitric acid KIE Kinetic Isotope Effect KIE int intrinsic KIE x KIE obs observed KIE LSR Least Squares Regression Mb myoglobin MMI Mass Moment of Inertia N 2 Dinitrogen N 2 O Nitrous oxide Nar Nitrate reductase NH 3 Ammonia NH 2 OH Hydroxylamine Nir Nitrite reductase NO 2 - Nitrate NO 3 - Nitrate NO x Nitric oxides NOB Nitrate Oxidizing Bacteria NOR Nitrous Oxide Reductase Nos Nitrous oxide reductase N r Reactive Nitrogen Species NXR Nitrate Oxidoreductase qNOR quinol Nitric Oxide Reductase RLM Robust Linea r Modeling S P Site Preference TG - IRMS Trace G as Isotope Ratio Mass Spectrometry TS Transition State TSDF Transition State Decomposition Frequency UV - Vis Ultra violet - visible ZnPP Zinc protoporphyrin IX ZPE Zero Point Energy 1 C HAPTER 1: N itrogen, NORs, and N avigating Stable Isotopes Introduction to the nitrogen cycle Nitrogen is an essential element to life due to its presence in amino acids and nucleic acids. The nitrogen cycle, as the name implies, is the circulation of nitrogen species in the atmosphere, landmasses, and bodies of water. All nitrogen species fall into one of two categories: largely inert di nitrogen (N 2 ) or reactive nitrogen species (N r ). N 2 an d N r are linked together in a triangle with the three vertices as N 2 , nitrate (NO 3 - ) as the most oxidized N r , and ammonia (NH 3 ) as the most reduced N r . These three nitrogen species are interconverted through a host of bio chemical processes that produce oth er N r intermediates including hydroxylamine (NH 2 OH), nitric acid (HNO 3 ), nitrite (NO 2 - ), nitrogen oxides (NO x ), and nitrous oxide (N 2 O) ( Figure 1). 2 One techniqu e that has proven to be highly useful in elucidating transformations within the nitrogen cycle is stable isotope tracing. 3 Stable isotopes are atoms of the same element that differ in mass only and are not radioactive. By examining the rate of the reaction for either 14 N (more common isotope) or 15 N (less common isotope), it is possible to assign isotopic identifiers (called isotopic enrichment factors, discussed later) to a process. This is particularly helpful in field studies where multiple nitrogen cycling processes occur simultaneously. As the marine and atmospheric nitrogen cycles are reviewed elsewh ere, 2 , 4 the scope of this chapter is limited to the terrestrial nitrogen cycle. Basic information needed to understand isotopic measurements is presented first. The known processes in the terrestrial nitrogen cycle 2 are discussed, as well as a brief mention of any relevant stable isotope studies. Particular attention is given to N 2 O as a greenhouse gas and the current state of know ledge of the enzymes that produce it. Finally, the theory of using stable isotopes to gain enzymatic mechanistic insights is also presented. Isotope notation Isotopes are traditionally measured as ratios with the isotopic ratio R defined as the abundance of the heavy isotope over the abundance of the light isotope: (1) To enable standardization and comparison of isotopic measurements, R is converted to the delta (2) where R sample is the isotope ratio of the sample and R standard is the isotope ratio of the international 15 N is atmospheric N 2 . 5 Many reactions in the nitrogen cycle react with the two N isotopes at different rates. 3 The ratio of (3) w here k 2 is the reaction rate for the heavy isotope and k 1 is the reaction ra te for the light isotope. 6 3 (4) Rayleigh equation: 7 (5) where f is the fraction of the substrate remaining in the reaction. Eqn. 5 is most accurate for a unidirectional single step reaction in a closed system. This encompasses some enzymatic assays, but not experiments that measure an entire process involving multiple reactions, such as pure net isotope effect is the isotope effect of a multistep process that contains observable intermediates. For example, a net isotope effect would be observed for a monoculture study on nitrification as NH 3 is oxidized to NO 3 - with NH 2 OH as an intermediate. In this manuscript, a in the product indicates preferential incorporation of the heavy isotope. 4 Figure 1 . Diagram of the nitrogen cycle. Nitrogen fixation occurs when N 2 is turned into NH 3 . Anammox is the simultaneous oxidation of ammonia and reduction of nitrite to produce N 2 and water. Nitrification (red boxes) occurs in two stages: ammonia oxidizing archaea/bacteria (AOA/B) transform NH 3 to NH 2 OH to NO to NO 2 - , and nitrite oxidizing bacteria (NOB) catalyze the formation NO 3 - from NO 2 - . Comammox (green box) is the same process as nitrification but performed entirely in one organism. Dissimilatory nitrate reduction to ammonia (DNRA) is the reverse process of nitrif ication and comammox. The three denitrification processes (blue box) enable conversion of oxidized N r to N 2 . Denitrification is the reduction of NO 3 - to N 2 via NO 2 - , NO, and N 2 O. Nitrifier - denitrification is the same process, but performed by nitrifying ba cteria and fungi. Co - denitrification is the utilization of co - substrates during denitrification, using an oxidized N r (NO or N 2 O) and a reduced N r to form N 2 O or N 2 . From N 2 to NH 3 : Nitrogen fixation The largest bioavailable nitrogen reservoir is the atmo sphere, the vast majority of which is N 2 . As N r are continually being depleted through assimilation, sequestration, and conversion back to N 2 , nitrogen fixation must be carried out constantly to sustain life. 8 Reduction of N 2 to N r is difficult, requiring catalysis either by a specialized enzyme known as nitrogenase or industrially 5 by the Haber - Bosch process. 4 Biological nitrogen fixation (BNF) is performed by select microbes that synthesize the enzyme nitrogenase. Thus far, three different types of nitrogenases have been discovered, and are named for the metal found in the active site. These are molybd enum nitrogenase, vanadium nitrogenase, and iron nitrogenase. 9 As molybdenum nitrogenase is the most common nitrogenase, it is the only o ne discussed here. The molybdenum nitrogenase complex is a dimer of trimers, with one half of the enzyme containing an Fe protein clusters are found in nitrogena se: the F cluster in the Fe protein, which is a [ 4Fe - 4S ] cluster, the [ 8Fe - 7S cluster ] ) and the iron - molybdenum cofactor (FeMo - co) - co is an [ Mo - 7Fe - 9S - C - ho mocitrate ] cluster. This takes the form of two [ 4Fe - 4S ] clusters bound together in which the sulfur connecting the two clusters is replaced by a carbon atom and a terminal iron is replaced with Mo. Three sulfurs bridge the two clusters and the homocitrate coordinates the Mo. 8 , 10 Sto i chiometrically, nitrogenase catalyzes the reaction: N 2 + 8 e - + 16 ATP + 8 H + 3 + H 2 + 16 ADP + 16 P i As evidenced by the large number of ATPs used, nitrogen fixation is a highly energy intensive process for t he cell. 8 Anthropogenic nitrogen fixation is also energetically expensive , with more than 1% of the energy produced in the world used for the Haber - Bosch process. 11 Developed between 1909 and 1913, the Haber - Bosch process converts N 2 and H 2 to NH 3 through the use of a catalyst , high heat , and pressure. While the conversion of N 2 and H 2 to NH 3 is an exothermic reaction, the natural rate at which this reduction occurs is not practical economically. Heat can be applied to 6 increase the rate of the reaction ; because heat drives the equilibrium away from ammonia production, however, obtain the desired product. The Haber - Bosch process accounts for approximately two thirds of all terrestrial nitrogen fixation while BNF accoun ts for the remaining third. 4 There have been some stable isotope studies performed on BNF. 12 , 13 Zhang et al. used mutant strains that could express only one nitrogenase and observed that there was a slight discrimination against the heavier isotope ( - 6 to - 14 Taking all of the available data together indicates a slight - 3 NH 3 to NO 3 - /N 2 : Nitrification, Comammox, and Anammox Once N 2 is fixed as NH 3 , another set of nitrogen t ransformations can begin, finishing with either NO 3 - or N 2 as the final product. These processes are known as nitrification, complete ammonia oxidation (comammox), and anaerobic ammonia oxidation (anammox). Nitrification is the oxidation of ammonia or ammonium to nitrite or nitrate under aerobic conditions , and both chemotrophic nitrification and heterotrophic nitrification have been discovered. 15 Nitrification can be carried out by two different sets of microbes, ammonia oxidizing archaea/bacteria (AOA/B) and nitrate oxidizing bacteria (NOB). 16 The reactions in the pathway are shown below: 17 , 18 NH 3 + O 2 + 2 e - + 2 H + 2 OH + H 2 O NH 2 OH + H 2 - + 3 H + + H 2 O NO + 2 OH - 2 - + e - NO 2 - + H 2 3 - + 3 e - + 2 H + 7 AOA/B catalyze the first set of reactions while NOB carry out the last step of converting nitrite to nitrate. The first reaction is catalyzed by an enzyme known as ammonia monooxygenase (AMO) while the conversion of NH 2 OH to NO is performed by hydroxylamine oxidoreducta se (HAO). Until recently it was thought that HAO oxidized hydroxylamine directly to nitrate. Caranto et al. definitively demonstrated that under both aerobic and anaerobic conditions, HAO produces NO as its final product rather than NO 2 - . 17 Other side products that can be released during nitrification are NO 3 - , and N 2 O. 17 Catalysis of NO to NO 2 - occurs by O 2 oxidation in aerobic conditions 17 while an as yet unknown enzyme performs the conversion under anaerobic conditions. Nitrite oxidoreductase (NXR) performs the last step of nitrification. 18 A host of stable isotope experiments have been performed in pure culture studies for nitrifying bacteria. In general, discrimination against the heavy isotope was seen when examining NH 4 + oxidation to NO 2 - and conversion of NH 2 OH to N 2 - - 56.6 ± , respectively) . 3 Oxidation of NH 4 + to N 2 O also showed preferential incorporation of the - 3 In contrast, NO 2 - transformation to NO 3 - revealed a preference for the heavy isotope = - 3 Comammox combines the processes of AOA/B and NOB, enabling NH 3 /NH 4 + oxidation to NO 3 - to occur in a single organism. Discovered in 2015, relatively little is known about this process. Thus far, two bacterial species in the genus Nitrospira have been identified as comammox organisms, and it is known that the A subunit of the AMO in the coma mmox organisms is distinct from that of AOA/B AMO. The distinct AMO has enabled researchers to identify Nitrospira in forest soils, rice paddy soils, rice rhizospheres, and brackish lake water. Metagenomic studies suggest comammox organisms are even more w idespread, with putative 8 Nitrospira species found in wastewater treatment plants, drinking water treatment systems, agricultural soils, and freshwater habitats. 19 Finally, ammonia and ammonium can be processed by what is known as anammox. Rather than converting ammonia to NO 2 - or NO 3 - , the end produc t of anammox is N 2 : 20 NH 4 + + NO 2 - 2 + H 2 O Most of the research performed on anammox rev olves around wastewater treatment, but anammox has been seen in natural settings as well , including freshwater environments, marine environments, and terrestrial environments. 21 Thus far, all bacteria capable of anammox are in the planctomycete phylum which contains aquatic bacteria. Planctomycetes are characterized by pits in the cell surface, no peptidoglycan in the cell walls, budding reproduction, and internal cell compartmen talization . As HAO has only been found inside a cytoplasmic membrane bound 21 Stable isotope tracing for anam mox was reported by Brunner et al. who determined that the 14 N is preferentially incorporated into the product. 22 No stable isotope tracing for comammox has not yet been reported. NO 3 - to NH 4 + /N 2 O/N 2 : DNRA, Denitrification, Nitrifier - denitrification, and Co - denitrification NO 3 - in the soil has one of three ultimate fates: assimilatory nitrate reduction (plant uptake), conver sion into ammonium by what is known as the dissimilatory nitrate reduction to ammonium (DRNA) pathway , or conve rsion into N 2 O or N 2 in one of the many denitrification processes. In a bio logical context, DRNA and denitrification are simply different methods of utilizing nitrate for anaerobic respiration. Each process has its own advantage: the DRNA 9 pathway transfers more electrons than denitrification (eight as opposed to five per initial nitrate substrate ) , but denitrification is ubiquitous. 23 , 24 DNRA is an anaerobic process in which microbes convert NO 3 - to NO 2 - to NH 4 + . Two types of DNRA are known, one paired to sulfur oxidation and the other coupled to fermentation. 23 Several different species of bacteria are capable of fermentative DNRA including Pseudomonas spp. , Clostridia, Vibrio spp . , and Desulfovibrio spp . Environments that favor DNRA over denitrification are still being elucidated , but there is some evidence that D N RA prevails in highly reducing conditions with low nitrate availability and high organic matter . 25 , 26 No stable isotope studies have been performed at the time of writing this chapter. The process that closes the nitrogen cycle and reduces NO 3 - /NO 2 - to N 2 is known as denitrification , and this process is performed under anaerobic conditions by denitrifying organisms. Some traditional nitrifiers have been found to be capable of denitrification as well, and to differentiate de nitrification performed by nitrifiers and denitrifiers, denitrification by nitrifying microbes is termed nitrifier - denitrification. 27 Both pa thways use the same four enzymes to transform nitrate to N 2 , and these enzymes catalyze the following stepwise reactions: NO 3 - + 2 e - + 2 H + 2 - + H 2 O NO 2 - + e - + 2 H + 2 O 2 NO + 2 e - + 2 H + 2 O + H 2 O N 2 O + 2 e - + 2 H + 2 + H 2 O These reactions are carried out sequentially by nitrate reductase (Nar), nitrite reductase (Nir), nitric oxide reductase (Nor), and lastly, nitrous oxide reductase (Nos). 28 Archaea, yeast, fungi, 10 and bacteria are all capable of denitrification, though studies have mostly focused on fungal a nd bacterial denitrification. 27 Fungi are the predominant denitrifiers in grassland and woodland soils, while bacteria dominate in agricultur al soils. 29 , 30 All known denitrifying fungi lack N 2 O reductase, thus releasing N 2 O as their final product. 30 A process termed co - denitrification can occur simultaneously with denitrification. In co - denitrification, the co - substrates of denitrification are used to nitrosate an amino com pound, resulting in N 2 O or N 2 formation. 31 , 32 This is known to occur during fungal denitrification and particularly during catalysis of NO to N 2 O by the fungal nitric oxide reductase, P450nor (discussed later). 31 Although the biological significance of this process is not fully understood, it has been proposed that co - denitrification acts as a nitrogen immobilizing process as the inorganic nitrogen is bound to th e organic N compounds. 32 Denitrification has been particularly well studied , with stable isotopes and isotopic enrichment factors known for each step in the process, albeit with more or less precision. NO 3 - to NO 2 - reduction has been examined via soil incubations, water samples, and both fungal and bacterial pure culture studies. The general trend across all these experiments for both bacteria and fungi is = - 3 Pure culture studies of reduction of NO 2 - to NO show a similar though weaker trend as nitrate reduction (average - - 3 Soil inc ubation and pure culture studies on the reduction of N 2 O to N 2 saw preferential incorporation of the light isotope as well, - 3 The only study that examined the isotopic 2 O catalysis was a fungal enzymatic study , and they observed an overall discrimination against the light isotope ( al though the - 12.6 ± 3.0). 33 11 The Importance of N 2 O One of the common intermediates in the nitrogen cycle is N 2 O. N 2 O is a potent greenhouse gas with a 100 - year warming potential approximately 265 times greater than that of carbon dioxide. 1 Additionally, N 2 O is currently the chief anthropogenic contributor to ozone depletion in the atmosphere. Approximately t wo thirds of the processes in the known terre strial nitrogen cycle (nitrification, comammox, denitrification, nitrifier - denitrification, and co - denitrification) produce N 2 O as either an obligate intermediate or side product. Moreover, the nitrogen fluxes through processes that can produce N 2 O are high. One study examining nitrogen loss in large northeastern United States water sheds estimated that half of all N r applied to agricultural soils in these watersheds was denitrified. 34 It is therefore crucial to understand the environmental conditions in which the different nitrogen cycling processes dominate. Figure 2 . The atomic structure of nitrous oxide. The nitrogen atoms are in blue and the atom s are labeled in the figure. Here again, stable isotope studies have proven invaluable. The two nitrogen atoms in the asymmetric N 2 O molecule are distinct , and ) , Figure 2). The difference in 15 N between N and N is known as site preference ( S P ). S P has been shown to be a robust indicator of the processes m aking N 2 15 N measurements, S P has been shown to be nearly 12 constant in pure bacterial cultures as a function of substrate concentration . 35 , 36 , 37 Moreover, S P is different for different processes. The S P for fungal denitrification is ~ - studies, which is similar to the S P obtained from pure culture bacterial nitrification studies ( S P = 35 , 36 However, pure culture studies examining bacterial denitrification reveal an S P value of approximately - 10 to denitrification. 35 Haslun et al. recently examined the effect of varied carbon source c oncentrations of two bacterial dentirifiers and showed that S P remained consistent while growth rate and bulk 15 N varied markedly . 37 While identifying the microbial source of N 2 O is a vital step to ward mitigation, it is equally important to understand the enzymes involved in N 2 O production. Approximately 80% of all anthropogenic N 2 O produced is the result of agricultural fertilization practices , and both bacterial and fungal denitrification take place in agricultural fields. 29 , 38 , 39 Because both processes utilize NORs to produce N 2 O, understanding the types and biochemical mechanisms of the different NORs will further our ability to identify N 2 O production pathways as a means for reduc ing the emission of anthropogenically produced N 2 O. Nitric Oxide Reductases: Detoxifying NORs A variety of NORs has been discovered with their physiological functions encompassing two categories: NO detoxification and/or anaerobic respiration. 31 , 40 , 41 , 42 Two known detoxifying NORs are FNOR (flavorubredoxin) and Hcp (hybrid cluster protein). A fairly recent discovery, flavorubredoxin ( known as both FNOR and FIRd) belongs to the A - type flavoprotein family. Crystal structures show that FIRd consists of an N - terminal metallo - - lactamase - like domain and a C - terminal flavodoxin domain. The diiron catalytic site is contained in the N - terminus while a 13 flavin mononuc leotide (FMN) resides in the C - terminus. 43 Initial characterization revealed that FIRd is reduced by NADH:oxidoreducta se (FIRd - red), which is encoded in the same gene locus as FIRd. Activity assays indicate that FIRd has a high specificity for NO, with turnover numbers similar to that of the more well - known cytochrome c NOR (cNOR, discussed later). 41 Studies to elucida te its mechanism are currently underway. 44 Hybrid cluster prote ins (Hcp) are soluble proteins known to contain a unique iron - sulfur - oxygen cluster ( [ 4Fe - 2O - 2S ] ). 42 First proposed to be involved in nitrate/nitrite respiration, it has recently been demonstrated that, in Escherichia coli at least, Hcp is an NO detoxifying NOR. A combination of microbial gene deletion studies and purified enzyme studies demonstrated that Hcp is a high - affinity, low - capacity NOR that is expressed under ambient physiological conditions, while other NORs are expressed unde r high NO stress conditions. It is further known that Hcp is reduced by an NADH - dependent reductase, Hcr, which also serves to protect Hcp from inactivation by nitrosative damage during high NO concentrations. 42 As the function of Hcp has just been elucidated, a mechanism has not yet been proposed. Resp iratory NORs Three types of respiratory bacterial NORs are known as well as one fungal respiratory NOR. The most well studied bacterial NOR is cNOR, which was first purified in 1989 from the bacterium Pseudomonas stuzeri. 45 cNOR is an integral membrane protein that consists of two subunits named NorC and NorB. NorC (17 kDa) is the smaller of the two, and contain s a globular region with a low spin heme c that faces the periplasmic side of the membrane. NorC also has a single transmembrane helix which anchors it to the membrane. Functionally, NorC serves as the electron shuttle betwee n cytochrome c 550 and NorB, which is the catalytic unit. NorB (56 kDa) is 14 a membrane protein, containing 12 helices . A low - spin heme b in NorB passes electrons from NorC to the active site in NorB, which is a binuclear center (BNC) made of a high - spin heme b , called heme b 3 , and a non - heme iron, termed Fe B, which are antiferromagnitically coupled to each other in the oxidized state. 46 , 47 cNOR is a member of the heme - copper oxidase (HCO) superfamily and structurally homologous to the cbb 3 - type oxidases. All six ligand coordinating hist idines found in HCOs are strictly conserved in cNOR , with the main differences between HCO and cNOR being the exchange of the Cu for an Fe at the active site and no proton translocation channels in cNOR. Due to the homology of the cNOR to cytochrome c oxid ases, the cytochrome c oxidase nomenclature was adopted for NOR. 47 cNOR was successfully crystalized from Pseudomonas aeruginosa in 2012 (Figure 3 ). New i - oxo - bridge between the BNC in the resting state and a calcium binding site in NorB. The calcium is located between the propionates of heme b and b 3 , and it is thought to possibly control electron transfer b etween heme b and heme c or to stabilize the interaction of NorC and NorB. 48 15 Figure 3 . Crystal structure of cNOR from Pseudomonas aeruginosa . NorC is shown in grey with the heme c in pink. NorB, the catalytic subunit, is shown in rainbow with heme b (cyan heme), heme b 3 (purple heme), Fe B (brown sphere) and the calcium ion ( grey sphere). The figure was prepared with PyMol using PDB:3O0R. 49 Figure 4 . Three distinct proposed mechanisms of cNOR, adapted from Moënne - Loccoz. 50 16 At present, three different mechanisms of N 2 O production are proposed for cNOR ( Figure 4 ). The first proposed mechanism is termed the cis - heme b 3 mechanism. This mechanism proposes that all catalysis occurs on the heme Fe, and the Fe B helps to coordinate the NO and donate electrons. There is some experimental evidence for this mechanism in the form of membrane potential studies and optical spectroscopy, 51 but the greatest support comes from computational studies, which shows this mechanism contains the lowest energy barriers. 52 The second mechanism is known as the cis - Fe B mechanism, which is similar to the cis - heme b 3 mechanism but catalysis occurs on the Fe B . Currently there are some kinetic studies and cyclic voltammetry data that are consistent with this mechanism. 53 , 54 While a mechanism involving a dinitrosyl iron comp lex on the Fe B site has been ruled out, 55 it could be possible to have a mechanism in which the first NO molecule binds to the Fe B and is then attacked by the second NO before forming the N - N b ond (as shown in Figure 4 ). The third mechanism is known as the trans mechanism, in which one NO molecule binds to each Fe in the BNC before N - N bond formation occurs. Structural studies, steady state kinetics, electron paramagnetic resonance ( EPR ) , and r e sonance Raman studies support this mechanism. 48 , 56 , 57 , 58 This is also the mechanism proposed for a cNOR mimic, which has been well studied . 59 , 60 , 61 A second type of bacterial respiratory NOR is known as qNOR, and this enzyme uses quinol as its biological reductant. Structurally, it is quite similar to cNOR, the major differences between the two be ing an extended N - terminus on qNOR and a water channel to the active site that is not 17 present in cNOR. 40 , 62 Due to its high similarity to cNOR, qNOR will not be discussed in extensive detail in this chapter. The only NOR known to generate an electrochemical gradient was isolated from Bacillus azotoformans and is known as Cu A NOR . 63 Though no crystal stru ctures have been solved yet, homology modeling to cytochrome ba 3 from Thermus thermophil u s predicts that Cu A NOR consists of 12 transmembrane helices and has a di - copper site, one heme b , and a binuclear center that contains a heme b 3 and a non - heme iron. It is known that Cu A NOR uses cytochrome c 551 as a reductant , and is used in anaerobic respiration. 64 No mechanism has yet been proposed for this NOR, but given the high structural similarity of Cu A NOR to cNOR, it is likely that the mechanism of the two NORs are similar. In denitrifying fungi, one type of NOR has b een discovered. Known as P450nor due to the P450 heme that serves as the catalytic site, P450nor catalyzes the reduction of NO to N 2 O via: NAD(P)H + H + + + H 2 O + N 2 O P450nor (~46 kDa) is a soluble protein consisting of a single subunit . It is encoded by a single gene (CYP55) and is found in both the cytoplasm and mitochondria. 31 , 65 Early research indicated that P450nor received electrons from NAD(P)H, but it was not until a crystal structure was solved that it was suggested that NAD(P )H directly reduced P450nor. The authors noted that the distal pocket of the heme was much more open to the solvent than P450 mono o xygenases and could potentially be an NAD(P)H binding site. 65 Kinetic studies and additional crystal structures with NADH analogs confirmed direct reduction of P450nor by NAD(P)H. This is quite surprising given that all other P450s must be reduced one electron at a time. 31 18 As the known fungal denitrifiers do not contain a Nos, they cannot catalyze the final step i n denitrification and convert N 2 O to N 2 . 31 This is somewhat overcome by the process of co - denitrification in which nitrite and a second nitrogen donor (reduced relative to N 2 ) are combined by P450nor to form either N 2 O or N 2 . The final product is dependent on the redox state of the second nitrogen atom, with more oxidized species yielding N 2 O and more reduced species yielding N 2 . 19 Figure 5 . The proposed mechanism of P450nor . NO binds to the ferric heme, which is then reduced directly by NAD(P)H. The reduced intermediate is doubly protonated before the second NO attacks, forming what is proposed to be a cyclic inter mediate. In the last step, the cyclic intermediate breaks apart to yield H 2 O and N 2 O. The mechanism of P450nor (Figure 5 ) has been studied extensively, and while the general mechanism is known, details about some of the intermediates are still being elucidated. In brief, the general mechanism of P450nor consists of two phases. First, one molecule of NO binds to the ferric heme , and this complex is reduced directly by NAD(P)H. After reduction, both the N and O are protonated before the second NO molecu le binds and N 2 O and H 2 O are formed. 66 D ensity functional theory (D FT ) calculations have shed light on the redox states of the various intermediates. For example, the (FeNO) species that forms after the first NO binds to the heme is best characterized as Fe(II) - NO + , while after reduct ion it resembles Fe(II) - NO - . 66 , 67 The double protonation event promotes binding of the second NO molecule and formation of N 2 O and H 2 O, 20 though exact details of this last step are lacking. It has been proposed that a cyclic intermediate forms when the second NO binds, however experimental evidence for this complex is lacking. Stable Isotopes as a mechanistic probe In comparison with the P450nor, the mechanistic knowledge about cNOR is relatively limited, as evidenced by the three proposed mechanisms. As an integral membrane protein, cNOR is difficult to isolate and purify. Even when enough protein is purified, the signals from the thr ee hemes make gathering and interpreting spectroscopic data a challenge, at times providing results that could support multiple mechanisms. 51 Stable isotopes are uniquely suited to p robe enzymatic mechanisms and have been used for decades in such studies across multiple disciplines. 68 , 69 Stable isotopes have also been used to examine the mechanism of enzymes in the nitrogen cycle. Most relevant to this chapter is the study by Yang et al. , who used natural abundance isotopes of NO to examine the catalysis of NO to N 2 O via purified P450nor from the fung us Histoplasma capsulatum . 33 This study revealed that the first N in the final N 2 binding. Yang et al . reached this conclusion by analyzing the kinetic isotope effect s (KIE s ) for the reaction . A KIE is the reaction rate of the light isotope ( k L ) over the reaction rate of the heavy isotope ( k H ). (6) (7) 21 In this definition, the KIE can be greater than one, one, or less than one. If the KIE is greater than one, this is referred to as a normal isotope effect, in which the light isotope reacts more quickly than the heavy isotope. When a KIE less than one is obtained, the reverse is true, and the heavy isotope reacts more quickly than the light isotope. If the KIE i s equal to one, both isotopes react at the same rate. Several factors determine the KIE: (8) These include the zero - point energy (ZPE), excited vibrational states (EXC), the mass moment of inertia (MMI), and the tra nsition state decomposition frequency (TSDF). Each of these terms is discussed in more detail below. 22 Zero - Point Energy and the ZPE Approximation Figure 6 . A diagram illustrating a bond as a harmonic quantum oscillator. As the atoms move from extremely close together (far left ) to the point where the bond break s (dashed line), the energy forms a well. In this well is the lowest energy state of the nucleus, term ed the zero - point energy (ZPE). The ZPE for the lighter isotope (ZPE L , blue line) is different than that of the heavier isotope (ZPE H , red line) due to the differences in mass between the two isotopes. In a simplified form, a bond between two atoms in th e ground state can be thought of as a harmonic quantum oscillator. If energy is plotted as a function of internuclear distance, it can be seen that the energy decreases as the nuclei move further apart. After a certain point however, the energy begins incr easing and eventually the bond connecting the nuclei is broken, resulting in a plateau (Figure 6 ) In the well that occurs in between these two extremes is a point where the nucleus is at its lowest energy level (i.e., the ground state). This lowest energy level is designated the zero - point energy. Due to quantum physics, it is possible to relate the zero - point energy to mass through the following equations: (9) Energy Internuclear Distance ZP E L ZPE H 23 (10) (11) w here h c is the speed of light, k is 1 and m 2 are the masses of the atoms in the bond of interest. It can be seen that energy is proportional to the frequency of the bond, which is inversely proportional to the square root of the reduced mass of the atoms in the bond. Thus, if the mass changes (i.e., when one isotope is replaced with another), the energy will also change, yielding different ZPE A similar phenomenon is observed in the transition state 24 Figure 7 . A diagram of a normal and inverse isotope effect. Energy is plotted against an arbitrary reaction coordinate. A normal isotope effect (in which the light isotope reacts more quickly than the heavier isotope) is shown on the left while an inverse isotope effect (where the heavier isotope reacts more quick ly) is on the right. The red lines in the energy wells designate the ZPEs of the heavy isotope and the blue lines represent the light isotope ZPE. The difference in ZPE between the two isotopes is shown by the green arrow in the ground state and the purple reactant/TS ) . E L * and E H * stand for the activation energies of the light isotope and heavy isotope, respectively. The wells in F igure 7 are Born - Oppenheimer electronic energy surfaces, which can be thought of as force fields. These force fields vary in steepness, which has implications for the nuclei of interest. If the well is shallow, then the force holding the two nuclei together is we ak and the , then the force holding the nuclei together is E L * reactant E L E L E H E H E L * E H * TS reactant Energy Reaction Coordinate E L E L E H E H E H * TS Normal Isotope Effect (KIE>1) Normal Isotope Effect= reactant TS Inverse Isotope Effect (KIE< 1) Inverse Isotope Effect= reactant < TS 25 determining the type of isotope effect that is obs erved and making mechanistic conclusions. For example, if the bond between the atoms of interest is looser in the TS than the ground state ( Fig. 7, left panel) the light an d heavy isotopes are close together in the TS. Because the lighter isotope has a higher ZPE in the ground state than the heavier isotope, the activation energy for the light isotope is lower than that of the heavy isotope. Thus, the light isotope reacts mo re quickly than the heavier isotope. Conversely, if the bond of interest is tighter in the TS than the ground state ( Fig. 7, right panel) , an inverse isotope effect occurs , and the heavy isotope reacts more quickly than the light isotope. This is because i are farther apart , which translates to a lower energy of activation for the heavier isotope. 70 Over the course of many hydrogen/deuterium studies, it was noted that the ZPE was the largest single contributor to the observed KIE. As a result, the ZPE approximation was coined, which states that for all practical purposes, KIEs occur due to the differences in ZPE between the two isotopes of interest. While the ZPE approximation is valid for many H/D studies, it can begin to break down in certain circumstances, such as when examining heavier atom isotopes (like 14 N/ 15 N). In this case, the other three terms in Equation 8 begin to contribute more substantially to the observed KIE. Other contributors to the KIE Excited vibrational states (EXC) accounts for the ability of different isotopes to populate the excited state levels of a bond. These levels are any of the energy levels above the ZPE. The 26 energy levels in a harmonic quantum oscillator are evenly spaced apart at h times the frequency). Because bond frequency is directly related to bond strength, the excited st ate levels of bonds with higher frequencies are further apart. Above a certain vibrational frequency, however, the ambient energy provided by temperature is not sufficient to allow substantial occupation of these excited state levels. It has been demonstra ted that EXC only 1500 cm - 1 . Solving EXC mathematically for frequencies greater than 1500 cm - 1 results in an answer very close to unity, thus highlighting that EXC does not greatly influence the final KIE in these instances. 71 The mass moment of inertia (MMI) is the contri bution to the isotope effect from the energy from translation and rotation of the molecule of interest. Previous experiments have shown that MMI begins to compete with ZPE as the prevailing contributor to the KIE under certain conditions. One case is when the molecules of interest are small enough that isotopic substitution could substantially change the mass and moment of inertia. 70 The ZPE and EXC encompass the vibrational contributions to the KIE while the MMI is the contribution from the rotation and translation of the molecule. While the vibrational, rotational, and translational terms can be separated into different components, they are still ultimately relat ed to one another. It is unsurprising , therefore , that changes in MMI result in changes in ZPE and EXC. For example, a study involving model calculations of secondary isotope effects (isotope effects occurring at a site other than the bond of interest) rev ealed that in some cases, changes in MMI were compensated by corresponding changes in ZPE and EXC. 70 In model studies of heavy atom KIEs , however, the compensation of EXC and ZPE was smaller, and MMI was the single largest contributor to the KIE. When MMI is the predominant driver of the KIE, 27 the final KIE can still be interpreted as a normal or inverse isotope effect, just as when the ZPE approximation holds. However, one can no longer use the ZPE approximation to explain the isotope effect. Additionally, calculating theoretical KIEs for reactions becomes more difficult due to the complicated interaction between MMI , ZPE , and EXC. 70 The final contributor to the KIE is known as the TSDF. As the name implies, the TSDF is the tendency of the transition state complex to decompose into products. In theory, the TSDF contribution to the KIE can be calculated by hand if the correct parameters are known or can be approximated, as was demonstrated for hydrogen and deuterium in the theoretical reaction of a hydrogen transfer between two simple molecules. 72 With heavy atom isotopes , however, the calculations quickly become unwieldly and require advanced computational power to determine the TSDF. 70 Summary In this introduction, I have introduced the nitrogen cycle and reactions contained within the terrestrial portion of the nitrogen cycle. The different types of nitric oxide reductases were discussed with particular focus given to the two most well - studied NORs and the need for more mechanistic studi es of cNOR. Uses of s table isotope s use s as both a tracer in the nitrogen cycle and a mechanistic probe w ere presented , and the quantum physical components that contribute to a KIE were discussed. The remainder of this thesis will cover stable isotope mechanistic studies performed on purified cNOR from Paracoccus denitrificans (Chapter 2) and a cNOR mimic, I107EFe B Mb (Chapter 3). The isotopic studies on the native cNOR in C hapter 2 revealed that the enzyme did not discriminate between 14 N and 15 2 O molecule and discriminated 28 B Mb in C hapter 3, N 2 O production was observed , but the limited extent of reactio n that was captured did not allow for KIE determination. Conclusions and future directions are presented in Chapter 4. 29 C HAPTER 2: Not so elementary: Isotopic fractionation and kinetic isotope effects of a purified bacterial nitric oxide reductase (NOR) Cl arisse M. Finders, Joshua A. Haslun, Hasand Gandhi, Peggy H. Ostrom, Nathaniel E. Ostrom, Eric L. Hegg Joshua Haslun and Hasand Gandhi performed isotope ratio analysis of the N 2 O molecules. Peggy Ostrom helped with data analysis and Nathaniel Ostrom and Eric Hegg helped with data analysis and guided the research. ABSTRACT Nitrous oxide (N 2 O) is a serious concern due to its role in global warming and ozone destruction with a 100 - year time horizon global warming potential approximately 265 times greater than that of carbon dioxide. Agricultural practices account for ~80% of all anthropogenic N 2 O produced, due in large part to the stimulation of microbial denitrification as a result of over fertilization. To effectively mitigate N 2 O emissions, it is vital to understand the microbial processes and enzymes responsible for N 2 O production. Stable isotopes are uniquely suited to examine both microbial N 2 O sources and the mechanism of N 2 O biosynthesis through the use of site preference ( S P ) and kinetic isotope effects (KIEs), respectively. Using trace gas isotope ratio mass spectrometry (TG - IRMS), we determined the 15 15 N 15 N 18 O for purified cytochrome c nitric oxide reductase (cNOR) from the model denitrifier Paracoccus denitrificans . We also calculated the 30 S P ( S P 15 N 15 N ). Normal isotope effects were observed for bulk 15 N, 15 N , and 18 O wit h average KIE s of: 0.0079 ± 0.0009 - 7.8 ± ± 0.0052 (Avg. = - 12.2 ± ± 0.0008 (average = - 17.7 ± 15 N was not statistically different from one (Avg. KIE=0 .9993 ± ± S P changed over the . We present implications for the mechanism of N 2 O production from cNOR based on our data as well as the main contributors to the observed KIE. INTRODUCTION Nitrogen is a key element to all life and exists as either reactive nitrogen species or dinitrogen gas (N 2 ). These two categories are connected to each other through an intricate cycle of pathways that includes nitrification, denitrification, anammox, comammox, dissimilatory nitrate reduction to ammonia (DNRA), and potentially others. 2 , 18 Nitrous oxide (N 2 O) is one of the key intermediates in the nitrogen cycle. Unfortunately, N 2 O is also a potent greenhouse gas, with a 100 - year global warming potential approximately 265 times greater than that of carbon dioxide (CO 2 ), and is the largest anthropogenic source of ozone depletion. 73 Since the invention of the Haber - Bosch process, which fixes N 2 to ammonia, humans have had an enormous e ffect on both the amount and flux of bioavailable nitrogen, including N 2 O. Currently, anthropogenic N 2 O accounts for ~40% of all N 2 O generated, with ~80% of this produced by microbial activity in response to agricultural practices. 38 With atmospheric N 2 O concentrations increasing ~0.26% every year, 73 fully understanding the enzymatic processes that directly contribute to anthropogenic agricultural N 2 O production is especially important. 31 Soil microbial processes that produce N 2 O include nitrification, denitrification, and nitrifier - denitrification. Nitrification is the conversion of ammonium and ammonia (NH 4 + and NH 3 ) to nitrate (NO 3 - ) via hydroxylamine (NH 2 OH) under aerobic conditions. During the oxidation of NH 2 OH, N 2 O is released as a side product. Denitrification and nitrifier - denitrification are processes that under anaerobic conditions convert NO 3 - to N 2 through a series of intermediates including nitric oxide (NO) and N 2 O. Expanding our knowledge of enzymatic mechanisms of N 2 O production is crucial to reducing anthropogenically produced N 2 O. Ni tric oxide reductase (NOR), which produces N 2 O from NO, is one of the most important enzymes in soil microbial N 2 O production. 18 NOR activity can be found in both bacteria and fungi. 18 , 31 In its most generic form, NOR catalyzes the following reaction: 2 NO +2 e - + 2 H + 2 O + H 2 O A single NOR occurs in fungi, consisting of one subunit (46 kDa). A P450 - type heme serves as the catalytic center of the enzyme, giving P450nor it s name. 65 Catalysis b egins when an NO molecule binds to the ferric P450 heme, producing a heme - NO complex. The heme - NO complex is then reduced by NAD(P)H and doubly protonated, producing an intermediate that is best described as (Fe(III) - NHOH). 66 The final steps of the reaction that include binding of the second NO molecule followed by N 2 O formation are less well understood. 31 , 67 T hus far, four distinct bacterial NORs have been discovered, and several crystal structures have been documented. 43 , 42 , 62 , 74 Structural and sequence evidence show that quinol - dependent (qNOR) and cytochrome c - dependent (cNOR) NORs are evolutionarily related to the heme - copper oxidase family. 40 The m ost well studied bacterial NOR is cNOR, which is composed of two subunits, NorC (17 kDa) and NorB (56 kDa). NorC consists of one transmembrane helix, a 32 cytochrome c binding site, and a heme c which transfers electrons from cytochrome c to NorB. NorB is the catalytic unit and is an integral membrane protein with twelve transmembrane helices. Contained in the helices are two heme b molecules, termed heme b and heme b 3 , analogous to the cytochrome c oxidase nomenclature . 75 Heme b receives electrons from NorC and passes them to heme b 3 , which makes up half the catalytic site. The other half is a non - heme iron, termed Fe B , coordinated to three histidines and a glutamate. 75 Compared to our understanding of P450nor, our knowledge of the bacterial NOR mechanism is relatively limited. Three classes of mechanisms are currently proposed for cNOR, ( Figure 8 ) mainly differing in where and how the NO molecules bind to th e catalytic center. Two of the mechanisms propose that all of the chemistry occurs on one of the two Fe centers, with the first NO molecule binding to either the Fe B iron ( cis - Fe B mechanism) or the heme ( cis - heme b 3 mechanism) before being attacked by the second NO molecule.). 52 , 53 The third mechanism, known as the trans mechanism, suggests that one NO molecule binds to each iron. 58 Figure 8 . Three potential transition states during N 2 O production by cNOR, adapted from Blomberg. 52 The catalytic site is a binuclear center that consists of the heme b 3 and a non - heme iron, Fe B. 33 Stable isotopes have long been used to study enzymatic mechanisms through measuring kinetic isotope effects (KIEs). A KIE is defined as the rate constant of the light isotope divided by the rate constant of the heavy isotope. KIEs are useful in determining mechanisms and transition state structures because they provide a snapshot of the v ibrational environment of the bonds of interest. 76 The two nitrogen atoms in the N 2 O molecule are not equivalent, and therefore the making stable isotopes particularly well - suited to investigate N 2 O biosynthesis. In this context, we previously used stable isotopes to examine the production of N 2 O by purified P450nor from Histoplasma capsulatum . 33 In this manuscript, we present the enrichment factors and KIEs for 15 15 N , 15 N 18 O of purified cNOR from the bacterium Paracoccus denitrificans. We also discuss energetic contributors to the KIE and mechanistic implications of our data. MATERIALS AND METHODS Materials NO was purified by attaching a stainless steel trap (20 cm x 0.6 cm) and a 160 mL side - arm flask to a cylinder of 99.5% NO (Airgas) in a chemical fume hood. The molecular sieves (5A, 8 x 12 mesh; Arcos Organics) in both the trap and the flask were activated by heating the system to 180 °C under vacuum (~10 - 2 Torr) for 24 h. After cooling to room temperature, NO was allowed to enter the evacuated flask after passing through the molecular sieve trap. All reagents were purchased from Sigma - Aldrich unless otherwise noted and used without further purification. P urified P. denitrificans cNOR was generously provided by Professor Pia Ädelroth. 77 34 Enzymatic Assays: The cNOR activity assay (12.8 mL) was prepared by mixing 50 mM anaerobic 4 - (2 - hydroxyethyl) - 1 - piperazineethanesulfonic acid ( HEPES) buffer (pH 7.5), 50 mM KCl, 0.5 mM - tetramethyl - p - phenylenediamine (TMPD, Arcos Organics), 3 mM c, and 0.04% n - dodecyl - - D - maltoside (DDM, An atrace) in a 160 mL glass serum bottle in an anaerobic chamber (Coy). After the serum bottle was capped with a butyl stopper (Geo - Microbial Technologies, Inc.) and crimped, NO gas (10.3 using a gas - tight syringe (Hamilton) and allowed to equilibrate for 15 min while shaking at 100 rpm at room temperature on a benchtop shaker (Thermo Scientific). To avoid potential diffusion of N 2 O out of the reaction vial , the serum bottle was inverted be fore the cNOR (20 µL, 78 nM final concentration) was injected to initiate the reaction. The bottle was continuously shaken at 100 rpm during the course of the reaction. Prior to sampling, a volume of N 2 gas equivalent to the intended sample was injected to maintain a constant pressure upon sampling. The gas sample was then injected into a 30 mL sealed serum bottle that had been sparged with Ultra High Purity N 2 . To ensure the enzyme was producing N 2 O at the expected rate, one replicate was analyzed via gas chromatography (GC) on a Shimadzu Greenhouse Gas Analyzer, model GC - 2014 (Columbia, MD), equipped with a Haysep N separation column and an electron capture detector (ECD). The conditions used were: 5% C H 4 /95% Ar make up gas (Airgas) with a flowrate = 2.5 mL/min; N 2 carrier gas with a flowrate = 25 mL/min; 100°C GC oven temperature; 350°C ECD temperature. To obtain the isotopic composition of the N 2 O produced, samples were analyzed on an IsoPrime 100 stab le isotope ratio mass spectrometer (IRMS) interfaced to a TraceGas inlet system (Elementar; Mt. Laurel, NJ) as previously described. 33 Samp le reproducibility (1 standard deviation) based on N 2 15 N, 35 18 15 N 15 N , and S P 18 15 15 N 15 N values of the laboratory pure N 2 O standard were 40.15, - 0.72, 10.19, and - Two different sets of experiments were performed. The first set was performed to determine the isotopic enrichment factor for N 2 O production by cNOR. Three replicates were sampled every forty min until enzyma tic N 2 O production began to plateau (~240 min). To determine if isotopic exchange between the water and NO was occurring, a second set of experiments was performed. Three replicates that spanned at least 1 0 % of reaction completion were assayed, two of whic h were spiked with 18 control to test for the abiotic production of N 2 O was prepared that included all reagents except the cNOR enzyme. N 2 O above background was not detected via GC - E CD analysis under our reaction conditions, indicating that the N 2 O observed in the first set of experiments was enzymatically produced. Isotope nomenclature The isotopic ratio R is defined as the abundance of the rare isotope over the abundance of the mor e abundant isotope: (1) (2) 36 18 15 N are Vienna Standard Mean Ocean Water and atmospheric N 2 , respectively. 5 Mass overlap corrections (e.g., due to the presence of N 2 17 O) were performed following the procedure of Toyoda and Yoshida. 78 For a closed system with an irreversible reaction, which describes our reaction conditions for the reduction of NO to N 2 the Rayleigh equation: 7 (5) where f obtained, the kinetic isotope effect (KIE), the ratio of the reaction rate of the light over the heavy isotopically substituted compounds, can be calculated from the following equations: (6) (7) (8) k L and k H are the rate constants for the light and heavy isotopes, respectively. If the Rayleigh equation (Eq. 5) is plotted with the y - axis as the isotopic composition of N 2 O at any point in time and the x - axis as f *ln f / ( 1 - f ), the slope is equivalent to isotopic enrichment factor ( ). In this relationship, the reaction begins at 1 on the right side of the graph (all substrate) and ends at 0 (no substrate remaining) on the left. To make 37 the data presentation more intuitive, all graphs presented in this manuscript use a close approximation of f *ln f /(1 - f ) for the x - axis, (1 - f ), in which reaction progress proceeds from left to right. All calculations of fitting, however, employ the full Rayleigh equation (Eqn. 5) as described above. Stati stics and Figures outliers based on the sample mean. Data obtained prior to a 1 - f value of 0.1 were not included in the analysis to grant suffi cient time for the reaction mixture to equilibrate and reach steady state. Least squares regression (LSR) was initially applied. However, because LSR is sensitive to influence by outliers, we applied Robust Linear Modeling (RLM) to determine the underlying trends in the data. 79 Results between the two approaches were compared and RLM decreased the standard er ror of the model without affecting the estimated parameters. All statistics were performed in the statistical software package R (R Foundation, version 3.4.1). 38 RESULTS Figure 9 . Production of N 2 O by P. denitrificans cNOR. Each replicate contained 78 of cNOR 2 O that could be produced was Enzymatic N 2 O Production The production of N 2 O by cNOR was monitored via GC - ECD over the course of 240 minutes. The rate of N 2 O production averaged18 ± 7 nmol/min over the 180 min reaction (Fig. 9 ). Sufficient N 2 O was produced over this time period to allow analysis by isotope ratio mass spectrometry . 39 18 15 N of N 2 O Produced by P. denitrificans cNOR 15 15 N 15 N 18 O, and S P ) for the reduction of NO to N 2 O by cNOR were measured over a range of ~20% - 74% substrate conversion to product. Enzymatic activity began 2 O produced (62 - 74% conversion, Figure S1). It is important to note that all KIEs presented in the manuscript are observed KIEs (KIE obs ) as opposed to intrinsic KIEs (KIE int ), which are defined as the inherent isotope effect for a specific step in a reaction. 80 Because the KIE int ca n be partially masked by other steps in the reaction sequence, the observed KIEs should therefore be considered the lower limits of the intrinsic KIEs. 81 18 O was observed and application of th e Rayleigh model (Eq. 5) yielded an - 17.7 ± 0.7 for the three replicates (Figure 10 A , p - 2 The positive slopes indicate a normal isotope effect in which the 16 O isotope reacts more rapidly than the 18 O isotope. This leads to an enrichment of 18 O in the residual substrate pool because 16 O is incorporated into the product more quickly. As the reaction proceeds, both substrate and product become more enriched in the heavy isotope over time, yielding a pos 1 (Table 1). The average KIE across all three samples was 1.0180 ± 0.0008, An experiment was also performed with water enriched in 18 O to examine the possibility of isotopic exchange of oxygen between water and NO ( Fig . 14 - 18 ). If exch ange occurred, there would be a difference in the slopes of the isotopically enriched and unenriched replicates, with the slope of the 18 O enriched sample tending toward zero. As evident in Fig. S3A, there does not appear to be a difference between the 18 O spiked and non - spiked replicates, suggesting that if any isotopic exchange of oxygen occurred under our reaction conditions, it was below our detection 40 limit and could therefore be ignored. Although the f range of this control experiment was too small to 2 18 O as the reaction progressed is further indication of a normal isotope effect for 18 O during N 2 O production. 15 N of N 2 - 15 the measured reaction (Figure 10 B, p - 2 15 N KIE of the three samples was 1.0079 ± 0.0009, indicating that the reaction rate for 14 NO is faster than 15 NO. 41 Figure 10 . 18 O 15 N (B) of N 2 O produced by cNOR as a function of the fraction of substrate (NO) reduced (1 - f ) with reactions progressing from left to right. Three biological replicates were assayed. The discontinuity between the 0.2 and 0.6 range of Replicate 1 resulted from a plugged needle during sampling. 42 Table 1 . - values, R 2 values, and kinetic isotope effects (KIEs) of P. denitrificans cNOR. Measurement Replicate p - value R 2 KIE 15 N 1 - 7.6 0.0014 0.971 1.0077 2 - 8.8 0.0366 0.993 1.0089 3 - 7.1 0.0242 0.809 1.0072 Avg ± SD - 7.8 ± 0.8 1.0079 ± 0.0009 15 N 1 3.0 0.4298 a - 0.045 a 0.9971 2 0.8 0.8327 a - 0.865 a 0.9992 3 - 1.6 0.3987 a - 0.009 a 1.0016 Avg ± SD 0.7 ± 2.3 0.9993 ± 0.0023 15 N 1 - 17.8 0.0172 0.847 1.0181 2 - 10.6 0.0013 1.000 1.0107 3 - 8.1 0.0697 0.625 1.0082 Avg ± SD - 12.2 ± 5.0 1.0123 ± 0.0052 18 O 1 - 16.8 0.0027 0.954 1.0171 2 - 18.2 0.0015 1.000 1.0186 3 - 18.00 0.0014 0.971 1.0183 Avg ± SD - 17.7 ± 0.7 1.0180 ± 0.0008 S P 1 - 20.7 0.0563 0.672 0.9797 2 - 11.4 0.1572 0.881 0.9887 3 - 6.4 0.0345 0.750 0.9936 Avg ± SD - 12.9 ± 7.3 0.9873 ± 0.0071 a The p - values and R 2 coefficients for N are largely meaningless because the N isotopic 43 Figure 11 . 18 N 15 N (B) of N 2 O produced by cNOR as a function of the fraction of substrate (NO) reduced (1 - f ) with reactions progressing from left to right. Three biological replicates were assayed. 15 N 15 N , and S P Values for N 2 O Produced by P. denitrificans cNOR 15 N values remained essentially constant over the course of the reaction (Fig. 3A, p = 0.39 to 0.83), yielding a KIE of 0.9993 ± 0.0023, indicating that there is no significant discrimination 44 against 15 Table 1) may indicate a real change, the standard deviation of 2.3 does not allow us to conclude that the slope is different than zero. 15 N 15 N is driven by 15 N - 12.2 ± 5.0 (Figure 11 B). A normal KIE of 1.0123 ± 0.0052 was calculated using all three replicates (Table 1), indicating that 14 sition more rapidly than 15 N (p = 0.0013 to 0.0697). The control experiment with H 2 18 15 N and 15 N as discussed above (Figures 16 A and 16 B). From in vivo studies of N 2 O production, S P has been found to be nearly constant over time, making it a valuable tool for distinguishing N 2 O derived from nitrification versus denitrification. 6 , 82 In the current study, we have a closed system with a finite pool of substrate. We would therefore expect S P to change over the course of the reaction as substrate is depleted if the and positions in N 2 the replicates differ, (Table 1) an overall trend of decreasing S P during the course of the reaction can be seen (Figure 12 ). 45 Figure 12 . S P ( S P 15 N - 15 N ) for cNOR over the course of the enzymatic reaction. S P is plotted against the fraction of substrate (NO) reduced with the reaction progressing from left to right. Three biological replicates were assayed. DISCUSSION Considerations of Observed KIEs In this manuscript, we measured the observed KIEs for the N , N , and 18 O to gain mechanistic insights into cNOR from P. denitrificans. As previously mentioned, these are observed KIEs and should be taken as the lower limit of the intrinsic KIEs for the isotopically sensitive steps in the reaction. Any slight varia tion in enzymatic assay conditions could affect the rate of individual steps in the reaction sequence. This could, in turn, alter the extent to which the intrinsic KIEs are expressed. 80 46 It is also important to note that other reactions could influence the isotope ratio of the e nzymatically - produced N 2 O. One such reaction is the exchange of oxygen between water and NO. Results from monoculture experiments led Kool et al . to propose that exchange of oxygen occurs during either the reduction of nitrite (NO 2 - ) to NO or the conversion of NO to N 2 O. 83 The lack of oxygen exchange observed in our experiments implies t hat any exchange must occur before conversion of NO to N 2 O. Furthermore, as the reduction of NO 2 - smallest energy gain of the entire denitrification process, it can be argued that this is the most likely step to be reversed and therefore al lows for a greater chance for oxygen exchange. 83 Other reactions to consider are any proces ses that consume or produce NO or N 2 O. There is no indication in the current literature that cNOR can reverse N 2 O formation (i.e., oxidize N 2 O to 2 O at pH 7 is + 1.18 V, indicating that this reactio n is highly exergonic and unlikely to be easily reversed. 84 In addition, there is currently no evidence that cNOR can further reduce N 2 O to N 2 . Another way N 2 O could be produced is via co - denitrification, a process in which inorganic nitrogen (NO 3 - or NO 2 - ) reacts with an organic source, such as an amino acid. 85 Due to the reducing conditions of our reaction and the sources of nitrogen available (NO, TMPD, and the buffer), it is highly unlikely that co - denitrification is taking place. Finally, control experiments in the absence of cNOR did not produce any detectable N 2 O. Insights from Site Preference Previous bacterial culture studies examining N 2 O production during denitrification demonstrated that S P values remained essentially constant . 35 , 37 One explanation for this observation could be that 2 O are the same. If this were the case, the difference 47 between N and N would remain constant during the course of the reaction, resulting in an unvarying S P S P - 6.4 to - is consistent with our previous in vitro study of P450nor. 33 As a result, S P fractionates over the course of the enzymatic reaction, and it has the potential to do so within cells as well. An alternative explanation for the consistent S P observed during bacterial denitrification is that f is being held constant in the cell, 33 , 37 which could occur if the rate of NO production is approximately equal to its reduction to N 2 O. The lack of expression of the enzymatic fractionation within bacterial cultures implies that NO is effectively held at steady - state levels within cells. It should be noted that an S P P. denitrificans cNOR has been reported by Yamazaki et al. 86 This measurement is the result of data taken at a single time point in the reaction rather than over the course o S P value and the S P data presented here for cNOR cannot be directly compared. 48 Mechanisms and Mechanistic insights Figure 13 . Three distinct proposed mechanisms of cNOR, adapted from Moënne - Loccoz. 35 Currently there are three distinct mechanisms proposed for cNOR ( Figure 13 ), all with varying levels of evidence. One mechanism is the cis - heme b 3 mechanism, in which the majority of the chemistry is proposed to occur at the heme iron. 87 In this mechanism, the first NO enters the catalytic site and binds to the reduced ferrous heme. The second NO then directly attacks the first NO to form the N - N bond. From here, it is possible that a cyclic intermediate forms which breaks apart to yield N 2 O. Optical spectroscopy and membrane potential studies by Hendriks et al. 88 support this mechanism. This mechanism was also calculated to be the most energetically favorable by Blom berg, who compared all three different mechanisms. 52 A second mechanism is 49 the cis - Fe B mechanism, which is identical to the cis - heme b 3 mechanism except that the reaction occurs on the non - heme iron. Steady - state kinetic studies 53 and cyclic voltammetry 54 data are consistent with this mechanism. In the trans mechanism, one NO binds to each iron, and the NO molecules then combine to form the N - N bond. This mechanism is supported by a large amount of experimental data, including steady state kinetics, EPR studies, and structural studies. 56 , 58 , 75 Additional evidence comes from a cNOR mimic, engineered by the Lu lab. 89 Lin et al. not only confirmed N 2 O production by this engineered enzyme, 59 they also provided evidence that catalysis proceeded through a trans mechanism. 61 It is worth remembering, however, that this mimi c could utilize a different mechanism than the natural enzyme. For several decades, KIEs have been used as a probe for enzymatic mechanisms. 90 , 69 To draw mechanistic information from KIEs, hypothetical bond order and bond distances in the transition states must be determined. An example may be seen in o ur previous work on the P450nor from H. capsulatum, in which the first NO binding event to the enzyme was examined . 33 When considering the mechanism of cNOR, it is important to realize that there are at least two different steps that could affect the observed KIE; one when the first NO binds to an Fe, and the second when the N - N bond forms. At present, there is very little data available for either bond length or order in the transition states of these two states. Blomberg calculated 2.07 Å for the N - N bond distance during the second transition state in the cis - heme b 3 mechanism. 52 As this is a very long and weak bond, one would not expect to observe an isotope effect for this step because the bond order for NO has essentially not changed. This could be co nsistent with our observed N data . Berto et al. combined spectroscopy and DFT calculations to study a model of the Fe B site. 91 Adding NO to the reduced Fe(II) form of the models resulted in the formation of an Fe(III) - NO - complex, with an electron moving to a largely NO antibonding orbital and a concomitant 50 lengthening of the NO bond. Additionally, changing the coordinating ligand on the model resulted in a range of N - O stretching frequencies, indicating that it is possible to tune the properties of the {F - bonding between the Fe and NO. 91 If cNOR behaves similarly to these models, a normal isotope effect would be expected for either cis mechanism, with the magnitude - bonding between the NO and the Fe. The small normal isotope effect observed for N could be consistent with this sort of binding event. Contributions to KIE The zero - point energy (ZPE) approximation has been used to explain the cause of KIEs on a physical level. The ZPE is the lowest energy level occupied by a nucleus, and the ZPE approximation states that isotope effects are due to differences in ZPE between bonds comprised of the lighter and heavier iso topes in both the reactant state and transition state (TS). In addition to ZPE, however, three other energetic components exist that also contribute to the observed KIE. These components are: (1) excited vibrational states (EXC), which account for the abil ity of the different isotopes of interest to populate the excited vibrational states, (2) mass - moments of inertia (MMI), which is the contribution to the isotope effect from the energy from translation and rotation, and (3) the transition state decompositi on frequency (TSDF), the probability of the transition state complex to decompose into products rather than reverting to reactants. Thus, in mathematical form, a KIE as defined by Bigeleisen and Wolfsberg is: (9) The ZPE approx imation has been used to explain KIEs in many H/D studies where the difference in ZPE between the two isotopes is great. In heavy - atom isotope effects, however, the energy 51 difference between the light and heavy atom is relatively minor and the ZPE approxim ation can break down. In those instances, EXC, MMI, and the TSDF may contribute substantially to the observed KIE. While the complexity of TSDF requires calculations of theoretical KIEs to determine the contribution of the TSDF, 70 EXC only significantly contributes to the KIE when - 1 . 71 Previous work has shown that the NO bond in reduced hemoglobin and myoglobin has a frequency greater than 1500 cm - 1 , 92 while models of the Fe B site revealed NO bond freq uencies between 1720 - 1790 cm - 1 . 91 Thus, it is likely that EXC is not a substantial contributor to the KIEs observed in cNOR. MMI can be the prevailing contributor to the KIE under certain circumstances. Such a case occurs when the molecules of interest are small enough that isotopic substitution could substantially change the mass and moment of inertia. 70 While the small size of NO and modest magnitude of our observed KIE might suggest that MMI could be a major contributor to the KIEs, some KIE calculations have observed MMI does not significantly impact the overall KIE in reactions with early transition states (as proposed in the cis - heme b 3 mechanism). 93 , 94 Thus, calculation of all transition states and theoretical KIEs are needed to understand which component is dominant in our system. CONCLUSIONS In this manuscript we have presented the isotopic enrichment factors and KIEs of purified cNOR from P. denitrificans . A small normal isotope effect was observed for the 18 O, 15 N, and 15 N , while no significant isotope effect was observed for the 15 N . By comparing our enzymatic S P with the S P from pure culture studies, we have provided further evidence that bacteria, like fungi, 52 hold NO at approximately steady - state levels resulting in a consistent S P over time. While it is tempting to apply the ZPE a pproximation to our data to infer an enzymatic mechanism, the relatively small KIE values require a thorough computational analysis before more definitive mechanistic conclusions can be made. ACKNOWLEDGMENT We thank Nicolai Lenhert (University of Michigan ) and Jennifer Dubois (Montana State University) for their helpful discussions. 53 APPENDIX 54 APPENDIX Figure 14 . Production of N 2 O by P. denitrificans cNOR with data points past 240 min included. All data points before individual replicates plateaued (Rep.1 before 280 min., Rep 2 before 160 min. and Rep 3. before 240 min.) were used in data analysis. 55 Figure 15 . Production of N 2 O by P. denitrificans cNOR with 18 O enriched water. Two replicates (red squares and dark red triangles) contained 18 NO ( g) and 78 nM cNOR. The maximum potential N 2 O 56 Figure 16 18 O (A) and 15 N (B) of N 2 O produced by three biological replicate s as a function of the fraction of substrate (NO) reduced (1 - f) with 18 O enriched water (red squares and dark red triangles) and unenriched water (blue circles). 57 Figure 17 . 15 N (A) 15 N (B) of N 2 O produced by three biological replicates as a function of the fraction of substrate (NO) reduced (1 - f) with 18 O enriched water (red squares and dark red triangles) and unenriched water (blue circles). 58 Figure 18 . S P ( S P 15 N - 15 N ) for cNOR over the course of the enzymatic reaction) with 18 O enriched water (red squares and dark red triangles) and non - enriched water (blue circles). S P is plotted against the fraction of substrate (NO) reduced with the reaction progressing from left to right. Three biological replicates were assayed. 59 Table 2 . - values, R 2 , and kinetic isotope effects of (KIEs) of P. denitrificans cNOR performed to test for 18 O e xchange . Measurement Replicate a p - value R 2 15 N No H 2 18 O Added - 18.3 0.0447 0.7163 H 2 18 O Enriched Rep. 1 - 5.4 0.8716 0.4753 H 2 18 O Enriched Rep. 2 - 11.9 0.0291 0.914 15 N No H 2 18 O Added - 3.6 0.5431 - 0.1535 H 2 18 O Enriched Rep. 1 16.1 0.6943 - 0.3599 H 2 18 O Enriched Rep. 2 30.1 0.3021 0.2306 15 N No H 2 18 O Added - 32.9 0.0165 0.8503 H 2 18 O Enriched Rep. 1 12.9 0.7716 - 0.4218 H 2 18 O Enriched Rep. 2 - 42.4 0.2252 0.4005 18 O No H 2 18 O Added - 54.3 0.0013 0.9715 H 2 18 O Enriched Rep. 1 - 40.3 0.1776 0.5144 H 2 18 O Enriched Rep. 2 - 39.2 0.034 0.8997 SP No H 2 18 O Added 29.3 0.0109 0.8862 H 2 18 O Enriched Rep. 1 3.2 0.6666 - 0.3332 H 2 18 O Enriched Rep. 2 72.5 0.2561 0.3301 a While p - values, and R 2 coefficients are provided , due to the small f cannot be taken as an accurate measurement of the entire reaction. . 60 CHAPTER 3 : N ew D og , O ld T ricks : I sotopic S tudies of a Cytochrome c N itric O xide R eductase (cNOR) M imic Clarisse M. Finders , Joshua A. Haslun , Hasand Gandhi , Peggy H. Ostrom , Nathaniel E. Ostrom , * , Eric L. Hegg , * Joshua Haslun and Hasand Gandhi helped with experiment design and isotope ratio analysis of the N 2 O molecules. Peggy Ostrom helped with analyzing the data. Nathaniel Ostrom and Eric Hegg helped with data analysis and directed the research . ABSTRACT Unlike many other nitrogen species, nitrous oxide (N 2 O) is a powerful greenhouse gas (100 - year global warming potential ~265 times that of carbon dioxide) and is the major pathway of stratospheric ozone destruction . Because approximately half of all N 2 O formed in a given ye ar is the result of various human activities, it is necessary to identify and mitigate anthropogenic sources of N 2 O. The single largest source of anthropogenic N 2 O is due to fertilization practices, which exacerbates the microbial process of denitrificatio n. In denitrification, NO 3 - and NO 2 - are reduced to N 2 , with N 2 O produced as an obligate intermediate. N 2 O is directly produced by the enzyme nitric oxide reductase (NOR), which catalyzes the conversion of NO to N 2 O. The most well - known bacterial NOR is th e cytochrome c NOR (cNOR), which contains multiple metal centers and is an integral membrane protein, making it challenging to study. In an effort to gain 61 more insight into the mechanism of cNOR, the Yi Lu lab at the University of Illinois Urbana - Champaign mutated sperm whale myoglobin (Mb) to generate a mimic of cNOR. The mimic, called I107EFe B Mb, has been shown to be a structural and functional model of the NOR active site and is capable of producing N 2 O. To f urther establish the accuracy of I107EFe B Mb as a effects (KIEs) of I107EFe B Mb to compare them to the values we previously obtained for cNOR. While I107EFe B Mb di d produce N 2 O, the limited extent of reaction we observed prevent ed us values or the KIEs of I107EFe B Mb. The isotopic data that was gathered are presented along with a discussion o f future possibilities to extend the f values and KIEs. INTRODUCTION Nitrous oxide (N 2 O) is an obligate intermediate in denitrification , a process in which NO 3 - and NO 2 - are reduced to N 2 . N 2 O is also a potent greenhouse gas, having a 100 - year global warming potential ~265 times greater than that of carbon dioxide. 1 Approximately half of all N 2 O emitted per year is anthropogen ically produced and , of the anthropogenic N 2 O, ~80% is the result of agricultural practices. 95 , 38 Excess fertilization stimulates a range of microbial nitrogen cycling processes that can produce N 2 O. One of these is bacterial denitrification which has been cited as the predominant source of N 2 O emissions in agricultural soils. 29 Mitigation strategies therefore should not only examine the microbes that produce N 2 O, but also the enzymes that directly catalyze N 2 O formation. 62 The enzyme responsible for reducing NO to N 2 O in bacterial denitrification is termed nitric oxide reductase. Currently the most well studied bacterial NOR is cNOR, which is named for its natural reductant cytochrome c. cNOR is an i ntegral membrane protein that consists of two subunits, NorC and NorB. NorC contains a single heme c that serves to pass electrons between cytochrome c and NorB, where catalysis occurs. NorB is comprised of 12 transmembrane helices, a calcium ion, two heme b molecules, and a non - heme Fe, termed Fe B . Once the heme c in NorC receives an electron, the electron is passed to one of the b - type hemes in NorB. The first heme b then passes electrons to the second heme b in NorB. This second heme b, called heme b 3 , s erves as half of the catalytic site, with Fe B as the other half. 75 While there has been great interest in the cNOR mechanism for many years, a single mechanism has not yet been agreed upon. 52 , 51 This is due in part to the difficulties of isolating and purifying membrane proteins and the spectroscopic challenges associated with multiple metal centers in one protein. Recognizing the need for a simplified cNOR mimic that could be produced in large quantities, Yeung et al. engineered a cNOR mimic from sperm whale myoglobin (Mb) in 2009. 89 The mimic, called Fe B Mb, was produced by engineering an iron binding site into Mb by mutating three residues to histidines and a glutamate (L29H, F43H, V68E). U ltra violet - v is ible (UV - Vis) spectroscopy confirmed iron binding to the engineered site and EPR spectroscopy revealed spin - coupling between the heme iron and non - heme iron, as is observed in cNOR. More impressively, Fe B Mb was able to reduce NO to N 2 O, demonstrating that it is both an active site structural model and a function al mimic of cNOR. 89 Further improvement of the model was made when a second glutamate was in troduced into Fe B Mb, resulting in a new enzyme termed I107EFe B 2 O conversion from ~10% to ~24% N 2 O production , revealing the importance of the second 63 conserved glutamate in cNOR. It is hypothesized that this second Glu e nhances proton uptake in I107EFe B Mb, as it was seen that I107E interacts with a water molecule in the crystal structure. Rapid - freeze - quench Raman spectroscopy studies further highlighted the importance of the second glutamate as it was found that this glu tamate prevents the formation of a dead - end complex seen in the Fe B Mb enzymes. 96 By replacing Fe with Cu or Zn in the Fe B site, it was shown that the metal in the Fe B site influences the reduction potential of the heme. 59 In the Fe B Mb enzymes, exposure to excess NO leads to a five coordinate heme - nitrosyl complex that does not yield N 2 O formation. It was determined that this dead - end complex was the result of a second NO molecule displacing the proximal histidine to the heme. It was suggested that this five coordinate intermediate is avoided in I107EFe B Mb due to the second glutamate proto nating the NO bound on the distal side of the heme. 96 To further elucidate the mechanism by which I107EFe B Mb produces NO, any chemistry occurring at Fe B site can be examined by replacing the native heme in Mb with other protoporphyrins. Chakraborty et al. replaced the native heme with Zn protoporphyrin IX (ZnPP) and examined the chemistry occurring at the Fe B site. M ö ssbauer and EPR spectroscopy of ZnPPFe B Mb1 with NO showed the formation of a radical bound to the Fe in the Fe B site. These data , taken together with the previous studies that reveal NO binding to the heme in I107EFe B Mb , lend support to the trans mechanism proposed to cNOR. 97 Given the evidence that I107EFe B Mb is a good mimic of cNOR, we performed experiments with B Mb to gain mechanistic insights into the react ion catalyzed by this mimic. A second goal was to examine the mechanistic similarity of I107EFe B Mb and the native cNOR by comparing the KIEs obtained from these two enzymes. Because the enzyme is only capable of a single turnover, however, the 64 extent of re action was not sufficient values or the KIEs. The N 2 O production and isotopic data that w ere collected is presented and discussed in the following sections. MATERIALS AND METHODS NO Solution Preparation An NO solution was prepared by attaching a 160 mL side arm flask containing degassed water and two stainless steel traps (20 cm x 0.6 cm) to a cylinder of 99.5% NO (Airgas). The molecular sieve (5A, 8 x 12 mesh; Arcos Organics) and Ascarite II (20 - 30 mesh, Thomas Scientific) contained in the traps were heated overnight under vacuum to degas and precondition that trap material (180 °C and ~10 - 2 Torr, respectively). After cooling the traps to room temperature, NO was allowed to flow through the traps and ente r the degassed water in the side arm flask. The concentration of the NO solution was determined according to the assay by Hogg and Kalyanaraman. 98 The concentration of the NO solution was found to be 0.33 mM. All glassware was washed in both acid and base baths prior to experiments. I107EFe B Mb was generously provided by Professor Lu (University of Illinois, Urbana - Champaign) . Enzymatic Assays: I107EFe B Mb (25 nmol) was reduced with 1. 77 equivalents of sodium dithionite in an anaerobic chamber (Coy). Excess dithionite was removed using a desalting column (PD MiniTrap TM G - 25, GE Healthcare) that had been equilibrated with 100 mL of degassed 50 mM Bis - Tris propane buffer (pH 7.1, Sigma - Aldrich, 10 mL of 0.25 mM dithionite spiked buffer and 90 mL unspiked buffer). Two equivalents of FeSO 4 7 H 2 O (Mallinckrodt) w ere added to the collected enzyme fraction, and the solution was transferred to a 13 mL serum vial. 65 After the serum vial was capped with a butyl stopper (Geo - Microbial Technologies, Inc.) and crimped, the reaction was initiated by injecting of NO solution (80 n mol) into the serum vial using a gastight syringe (Hamilton). Controls were performed in duplicate to determine if abiotic N 2 O production was occurring. Because one equivalent of both dithionite and Fe 2+ is required for enzyme activity, the controls conta ined only the excess of these reagents. 0.77 equivalents of sodium dithionite was run through a column and collected. One equivalent of FeSO 4 7 H 2 O was added to the collected fraction and the solution was transferred to a 13 mL serum vial. The vial was cap ped, crimped, and NO solution was added (80 nmol, of solution) to initiate the reaction. Both the controls and enzymatic reactions were constantly shaken at 100 rpm at 30 ° C during the course of the reactions. To determine the amount and isotopic composition of the N 2 O produced, the samples and controls were analyzed on an isotope ratio mass spectrometer (IRMS) as previously described in the same way presen ted in Chapter 2, and the delta ( ) values for our laboratory pure N 2 O 18 15 15 N 15 N are the same as in the previous chapter. In the Rayleigh model, the two variables that are plotted against each other are for the product (y - axis) and f *ln f /(1 - f ), (x axis) with f being the fraction of substrate remaining The range in f is synonymous with the extent of the reaction. Therefore , ( the slope of the line ) and the KIE (derived from ) , will be most accurate when the largest extent of reaction is captured. Because the enzyme mimic is presently capable of only a single turnover, we simulated an f range by making individual reactions at different time points (18, 26, 33, 43, and 53 hours) , with 66 each time point assayed in triplicate. Because it was only possible to perform two assays due to limited enzyme, statistic al tests were not performed on the obtained data. 67 RESULTS AND DISCUSSION Figure 19 . Production of N 2 O from NO controls and I107EFe B Mb reactions. (A) N 2 O observed in controls at 18, 26, 33, and 53 hours. No controls were made for the 43 - hour time point. (B) N 2 O produced by I107EFe B Mb experiments after subtracting out the average background N 2 O from the controls. 68 Enzymatic N 2 O Production The production of N 2 O by both the controls and I107EFe B Mb was obtained from the samples and analyzed on the IRMS. As can be seen in Figure 19 (A), the amount of N 2 O in the controls remained fairly constant over time. Because there is no clear increase in N 2 O production, we believe that this N 2 O is most likely simply background in the NO stock solution and not the result of abiotic reactions. To be consistent in our analysis, the average amount of N 2 O in all of t he controls was subtracted from the N 2 O observed in the I107EFe B Mb assays, yielding a normalized N 2 O amount (Figure 19 (B)). A marked increase in N 2 O can be seen in both samples up to ~43 hours at which point N 2 O production plateaus. There appears to be a difference in production rates between the two batches of I107EFe B Mb analyzed. While Batch 1 beg an producing N 2 O later than Batch 2, Batch 1 produce d slightly more product. One explanation for this is that these are different sample preparations and therefore not exactly the same. Overall, I107EFe B Mb is capable of catalyzing NO reduction, albeit at an extremely slow rate compared to the native enzyme . 69 Figure 20 . Isotopic fractionation of bulk nitrogen (A), oxygen (B), N (middle nitrogen, C), and N (terminal nitrogen, D). All isotope ratios are plotted against the fraction of substrate (NO) remaining progressing from right to left. Two batches of I107EFe B Mb were assayed . 15 18 15 N 15 N of N 2 O produced by I107EFe B Mb 15 18 15 N 15 N ) were measured for the reduction of NO to N 2 O by I107EFe B Mb during 5 different time points. All results only span a range of ~5% conversion of substrate to product, greatly limiting the conclusions that can be drawn and making it impractical to determine S P a 70 trend in the data. For example, if the data for the bulk nitrogen (Figure 2 0 A) are taken all together, a positive trend may be present . In the oxygen data on the other hand (Figure 2 0 B), a downward trend (ignoring one point) seems to be present . There does not seem to be a trend emerging in the N (Figure 2 0 C), while the N (Figure 2 0 D) may have a general negative trend, similar to the bulk nitrogen. It must be reemphasized, however, that the very small f range does the dangers of drawing co nclusions from an f range this small. There are a few ways a larger f range could be accomplished. One strategy is to purge any background N 2 O so as to be certain that all N 2 O observed in t he I107EFe B Mb reactions was produced by the prote i n . While it might be tempting to rais e the NO concentration to increase the probability of NO binding to I107EFe B Mb , f is defined as the fraction of substrate remaining . Therefore increasing the NO would only yield an even smaller f range. One of the greatest barriers to a large f range is the single turnover limitation of I107EFe B Mb . It may be possible to achieve multiple turnovers by supplying excess reductant. Because dithionite produces N 2 O abiotically (data not shown), however, it is not possible to add excess dithionite to the reactions. Cysteine, NADH, ascorbate, glyceraldehyde, and hydroquinone have all been used as reductants for metmyoglobin in the past. 99 If one of these molecules could reduce I107EFe B Mb and does not produce N 2 O on its own, then it may be possible to achieve multiple turnovers for I107EFe B Mb without si mply increasing the abiotic reduction of NO . Further work needs to be done to examine this possibility. In this chapter, we have examined the N 2 O production and isotopic fractionation of a cNOR mimic, I107EFe B Mb. While I107EFe B is capable of reducing NO to N 2 O, the limited extent of the reaction we observed does not allow us to make any definitive conclusions about either the 71 isotopic fractionation or the mechanism of I107EFe B Mb. Future studies examining different reductants and improving the turnover numbe r of the enzyme will need to be accomplished before accurate isotopic information can be gathered. ACKNOWLEDGMENT We thank Julian Reed (University of Illinois Urbana - Champaign) for his help with the I107EFe B Mb assays and providing protein . 72 C HAPTER 4:Knowledge Gained and Next Steps In this thesis I have presented two studies examining N 2 O biosynthesis and isotopic fractionation from two different systems : a natural cytochrome c nitric oxide reductase or cNOR (Chapter 2) and an engineered cNOR mimic, I107EFe B Mb (Chapter 3) using isotope ratio mass spectrometry. Our results revealed that the native cNOR from Paracoccus deni t rificans discriminates against the heavy isotope for the oxygen, N , and the bulk nitrogen while no discrimination between the two isotopes is observed for the N . Three important conclusions were made from these results. The first is that while our data are consistent with either of the two proposed cis mechanisms, we are not able to definitively rule out the proposed trans mechanism due to the small magnitude of our isotope effects. The magnitude of our observed KIEs leads to the second conclusion, which is that the zero - point energy difference between the two isotopes may not be the dominant contributor to the final KIE. Ot her factors, such as the mass moment of inertia (MMI) or transition state decomposition frequency (TSDF) may be contributing significantly to the observed KIE. KIE calculations of the transition states for all of the proposed mechanisms will be necessary to pinpoint the predominant contributor. Other future experiments include examining the isotopic composition of N 2 O produced from purified quinol NOR (qNOR). As qNOR and cNOR have a high degree of sequence similarity and presumably follow an identical reac tion mechanism 62 , the isotopic fractionations would be expected to be similar. If the KIEs from qNOR displayed similar trends as cNOR but were larger in magnitude, more refined mechanistic conclusions could be made. Finally, in our study of cNOR we demonstrated that under our reaction conditions, oxygen exchange between water and NO does not occur. This 73 implies that any oxygen exchange that does occur during denitrification takes place before the formation of N 2 O. In our experiments with I107EFe B Mb, measurable N 2 O production was detected. However, the extremely short extent of reaction that was captured did not allow for accurate dete rmination of isotopic enrichment factors and KIEs. The isotopic data that were obtained could indicate the beginnings of trends , but without further experiments it is impossible to say for certain. Future experiments with I107EFe B Mb will focus on improving N 2 O production. This could be accomplished through tuning the binding of I107EFe B Mb to the substrate/Fe B so that more of the enzyme mimic turned over under single turnover conditions. N 2 O production could also be improved by changing the assay conditions to promote multiple turnovers so that this sample becomes a true en zyme. 74 REFERENCES 75 REFERENCES (1) Myher, G., Shindell, D., Breon, F. M., Collins, W., Fuglestvedt, J., Huang, D., Koch, J., Lamarque, F., Lee, D., Mendoza, B., Nakajima, T., Robock, A., Stephens, G., Takemura, T., and Zhang, H. (2013) Anthropogenic and Natural Radiative Forcing, in Cli mate Change 2013: The Phycsical Science Basis. Contribution of Working Group I to the Fifth Assescment Report of the Intergovernmental Panel on Climate Change (Stocker, T. F., Qin, D., Plattner, G. K., Tignor, M., Allen, S. K., Boschung, J., Nauels, A., Be x, V., and Midgley, P. M., Eds.), pp 73 79. Cambridge University Press, Cambridge, UK. (2) Galloway, J. N., Aber, J. D., Erisman, J. W., Seitzinger, S. P., Howarth, R. W., Cowling, E. B., and Cosby, B. J. (2003) The Nitrogen Cascade. Bioscience 53 , 341 35 6. (3) Denk, T. R. A., Mohn, J., Decock, C., Lewicka - Szczebak, D., Harris, E., Butterbach - Bahl, K., Kiese, R., and Wolf, B. (2017) The nitrogen cycle: A review of isotope effects and isotope modeling approaches. Soil Biol. Biochem. 105 , 121 137. (4) Fowler , D., Coyle, M., Skiba, U., Sutton, M. A., Cape, J. N., Reis, S., Sheppard, L. J., Jenkins, A., Grizzetti, B., Galloway, N., Vitousek, P., Leach, A., Bouwman, A. F., Butterbach - bahl, K., Dentener, F., Stevenson, D., Amann, M., Voss, M., and Fowler, D. (201 3) The global nitrogen cycle in the twenty - first century. Philisophical Trans. R. Soc. B . (5) Bond, A. L., and Hobson, K. A. (2012) Reporting Stable - Isotope Ratios in Ecology: Recommended Terminology, Guidelines and Best Practices. Waterbirds 35 , 324 331. (6) Ostrom, N. E., and Ostrom, P. H. (2012) The Isotopomers of Nitrous Oxide: Analytical Considerations and Application to Resolution of Microbial Production Pathways, in Handbook of Environmental Isotope Geochemistry , pp 453 476. (7) Mariotti, A., Germon , J. C., Hubert, P., Kaiser, P., Letolle, R., Tardieux, A., and Tardieux, P. (1981) Experimental determination of nitrogen kinetic isotope fractionation: Some principles; illustration for the denitrification and nitrification processes. Plant Soil 62 , 413 430. (8) Hoffman, B. M., Lukoyanov, D., Yang, Z. Y., Dean, D. R., and Seefeldt, L. C. (2014) Mechanism of nitrogen fixation by nitrogenase: The next stage. Chem. Rev. 114 , 4041 4062. (9) Eady, R. R. (1996) Structure - function relationships of alternative nitrogenases. Chem. Rev. 96 , 3013 3030. (10) Hu, Y., and Ribbe, M. W. (2011) Biosynthesis of nitrogenase FeMoco. Coord. Chem. Rev. 255 , 1218 1224. (11) Nørskov, J., Chen, J., Miranda, R ., Fitzsimmons, T., and Stack, R. (2016) Sustainable Ammonia Synthesis Exploring the scientific challenges associated with discovering alternative, sustainable processes for ammonia production. 76 (12) Macko, S. A., Fogel, M. L., Hare, P. E., and Hoering, T . C. (1987) Isotopic fractionation of nitrogen and carbon in the synthesis of amino acids by microorganisms. Chem. Geol. Isot. Geosci. Sect. 65 , 79 92. (13) Bauersachs, T., Schouten, S., Compaore, J., Wollenzien, U., Stal, L. J., and Sinninghe Damste, J. S . (2009) Nitrogen isotopic fractionation associated with growth on dinitrogen gas and nitrate by cyanobacteria. Limnol. Oceanogr. 54 , 1403 1411. (14) Zhang, X., Sigman, D. M., Morel, F. M. M., and Kraepiel, A. M. L. (2014) Nitrogen isotope fractionation by alternative nitrogenases and past ocean anoxia. Proc. Natl. Acad. Sci. 111 , 4782 4787. (15) Focht, D. D., and Verstraete, W. (1977) Biochemical Ecology of Nitrification and Denitrification, in Advances in Microbial Ecology (Alexander, M., Rosswall, T., Sh ilo, M., and Veldkamp, H., Eds.), pp 135 214. Plenum Press, New York. (16) Cai, M., Ng, S. - K., Lim, C. K., Lu, H., Jia, Y., and Lee, P. K. H. (2018) Physiological and Metagenomic Characterizations of the Synergistic Relationships between Ammonia - and Nitri te - Oxidizing Bacteria in Freshwater Nitrification. Front. Microbiol. 9 , 1 13. (17) Caranto, J. D., and Lancaster, K. M. (2017) Nitric oxide is an obligate bacterial nitrification intermediate produced by hydroxylamine oxidoreductase. Proc. Natl. Acad. Sci. 114 , 8317 8222. (18) Kuypers, M. M. M., Marchant, H. K., and Kartal, B. (2018) The microbial nitrogen - cycling network. Nat. Rev. Microbiol. (19) Hu, H. W., and He, J. Z. (2017) Comammox a newly discovered nitrification process in the terrestrial nitroge n cycle. J. Soils Sediments 17 , 2709 2717. (20) Jetten, M. S. M., Wagner, M., Fuerst, J., Van Loosdrecht, M., Kuenen, G., and Strous, M. process. Curr. Opin. Biotechnol. 12 , 283 288. (21) Jin, R. C., Yang, G. F., Yu, J. J., and Zheng, P. (2012) The inhibition of the Anammox process: A review. Chem. Eng. J. 197 , 67 79. (22) Brunner, B., Contreras, S., Lehmann, M. F., Matantseva, O., Rollog, M., Kalvelage, T., Klockgether, G., Lavik, G., Jetten, M. S. M., Kartal, B., and Kuypers, M. M. M. (2013) Nitrogen isotope effects induced by anammox bacteria. Proc. Natl. Acad. Sci. 110 , 18994 18999. (23) Burgin, A. J., and Hamilton, S. K. (2007) Have we overemphasized the role of denitrifi Front. Ecol. Environ. 5 , 89 96. (24) Tiedje, J. M., Sexstone, A. J., Myrold, D. D., and Robinson, J. A. (1983) Denitrification: ecological niches, competition and survival. Antonie Van Le euwenhoek 48 , 569 583. 77 (25) Rütting, T., Boeckx, P., Müller, C., and Klemedtsson, L. (2011) Assessment of the importance of dissimilatory nitrate reduction to ammonium for the terrestrial nitrogen cycle. Biogeosciences 8 , 1779 1791. (26) Silver, W. L., Her man, D. J., Firestone, M. K., and Sep, N. (2012) Dissimilatory Nitrate Reduction to Ammonium in Upland Tropical Forest Soils. Ecology 82 , 2410 2416. (27) Wrage, N., Velthof, G. L., Van Beusichem, M. L., and Oenema, O. (2001) Role of nitrifier denitrificati on in the production of nitrous oxide. Soil Biol. Biochem. 33 , 1723 1732. (28) Tavares, P., Pereira, A. S., Moura, J. J. G., and Moura, I. (2006) Metalloenzymes of the denitrification pathway. J. Inorg. Biochem. 100 , 2087 2100. (29) Zumft, W. G. (1997) Cel l Biology and Molecular Basis of Denitrification. Microbiol. Mol. Biol. Rev. 61 , 533 616. (30) Baggs, E. M. (2011) Soil microbial sources of nitrous oxide: Recent advances in knowledge, emerging challenges and future direction. Curr. Opin. Environ. Sustain . 3 , 321 327. (31) Shoun, H., Fushinobu, S., Jiang, L., Kim, S. - W., and Wakagi, T. (2012) Fungal denitrification and nitric oxide reductase cytochrome P450nor. Philos. Trans. R. Soc. B Biol. Sci. 367 , 1186 1194. (32) Spott, O., Russow, R., and Stange, C. F. (2011) Formation of hybrid N 2 O and hybrid N 2 due to codenitrification: First review of a barely considered process of microbially mediated N - nitrosation. Soil Biol. Biochem. 43 , 1995 2011. (33) Yang, H., Gandhi, H., Ostrom, N. E., and Hegg, E. L. (2014) Isotopic Fractionation by a Fungal P450 Nitric Oxide Reductase during the Production of N 2 O. Environ. Sci. Technol. 48 , 10707 10715. (34) Van Breemen, N., Boyer, E. W., Goodale, C. L., Jaworski, N. A., Paustian, K., Seitzinger, S. P., Lajtha, K., Mayer, B ., Van Dam, D., Howarth, R. W., Nadelhoffer, K. J., Eve, M., and Billen, G. (2002) Where did all the nitrogen go? Fate of nitrogen inputs to large watersheds in the northeastern U.S.A. Biogeochemistry 57 58 , 267 293. (35) Sutka, R. L., Ostrom, N. E., Ostro m, P. H., Breznak, J. A., Gandhi, H., Pitt, A. J., and Li, F. (2006) Distinguishing Nitrous Oxide Production from Nitrification and Denitrification on the Basis of Isotopomer Abundances. Appl. Environ. Microbiol. 72 , 638 644. (36) Sutka, R. L., Adams, G. C., Ostrom, N. E., and Ostrom, P. H. (2008) Isotopologue fractionation during N 2 O production by fungal denitrification. Rapid Commun. Mass Spectrom. 22 , 3989 3996. (37) Haslun, J. A., Ostrom, N. E., Hegg, E. L., and Ostrom, P. H. (2017) Estimation of isoto pologue variation of N 2 O during denitrification by Pseudomonas aureofaciens and Pseudomonas chlororaphis : Implications for N 2 O source apportionment. Biogeosciences Discuss. in review. 78 (38) E vironmental P rotection A gency . (2015) Nitrous Oxide Emissions. https://www.epa.gov/ghgemissions/overview - greenhouse - gases (39) Frey, S. D., Elliott, E. T., and Paustian, K. (1999) Bacterial and fungal abundance and biomass in conventional and no - tillage agroecosystems along two climatic gradients. Soil Biol. Biochem. 31 , 573 585. (40) Hendriks, J., Oubrie, A., Castresana, J., Urbani, A., Gemeinhardt, S., and Saraste, M. (2000) Nitric oxide reductases in bacteria. Biochim. Biophys. Acta - Bioenerg. 1459 , 266 273. (41) Gomes, C. M., Giuffrè, A., Forte, E., Vicente, J. B. , Saraiva, L. M., Brunori, M., and Teixeira, M. (2002) A novel type of nitric - oxide reductase: Escherichia coli flavorubredoxin. J. Biol. Chem. 277 , 25273 25276. (42) Wang, J., Vine, C. E., Balasiny, B. K., Rizk, J., Bradley, C. L., Tinajero - Trejo, M., Poo le, R. K., Bergaust, L. L., Bakken, L. R., and Cole, J. A. (2016) The roles of the hybrid cluster protein, Hcp and its reductase, Hcr, in high affinity nitric oxide reduction that protects anaerobic cultures of Escherichia coli against nitrosative stress. Mol. Microbiol. 100 , 877 892. (43) Romão, C. V., Vicente, J. B., Borges, P. T., Victor, B. L., Lamosa, P., Silva, E., Pereira, L., Bandeiras, T. M., Soares, C. M., Carrondo, M. A., Turner, D., Teixeira, M., and Frazão, C. (2016) Structure of Escherichia coli Flavodiiron Nitric Oxide Reductase. J. Mol. Biol. 428 , 4686 4707. (44) Khatua, S., and Majumdar, A. (2015) Flavodiiron nitric oxide reductases: Recent developments in the mechanistic study and model chemistry for the catalytic reduction of NO. J. Inor g. Biochem. 142 , 145 153. (45) Heiss, B., and Frunzke Zumft, K. W. G. (1989) Formation of the N - N bond from nitric oxide by a membrane - bound cytochrome bc complex of nitrate - respiring (denitrifying) Pseudomonas stutzeri . J. Bacteriol. 171 , 3288 3297. (46) Field, S. J., Thorndycroft, F. H., Matorin, A. D., Richardson, D. J., and Watmough, N. J. (2008) The Respiratory Nitric Oxide Reductase (NorBC) from Paracoccus denitrificans . Methods Enzymol. Elsevier Masson SAS. (47) van der Oost, J., de Boer, a P., de G ier, J. W., Zumft, W. G., Stouthamer, a H., and van Spanning, R. J. (1994) The heme - copper oxidase family consists of three distinct types of terminal oxidases and is related to nitric oxide reductase. FEMS Microbiol. Lett. 121 , 1 9. (48) Shiro, Y., Sugim oto, H., Tosha, T., Nagano, S., and Hino, T. (2012) Structural basis for nitrous oxide generation by bacterial nitric oxide reductases. Philos. Trans. R. Soc. B - Biological Sci. 367 , 1195 1203. (49) Hino, T., Matsumoto, Y., Nagano, S., Sugimoto, H., Fukumor i, Y., Murata, T., Iwata, S., and Shiro, Y. (2010) Structural Basis of Biological N 2 O Generation by Bacterial Nitric Oxide Reductase. Science (80 - . ). 330 , 1666 1670. 79 (50) Moënne - Loccoz, P. (2007) Spectroscopic characterization of heme iron nitrosyl specie s and their role in NO reductase mechanisms in diiron proteins. Nat. Prod. Rep. 24 , 610 620. (51) Hendriks, J. H. M., Jasaitis, A., Saraste, M., and Verkhovsky, M. I. (2002) Proton and electron pathways in the bacterial nitric oxide reductase. Biochemistry 41 , 2331 2340. (52) Blomberg, M. R. A. (2017) Can Reduction of NO to N 2 O in Cytochrome c Dependent Nitric Oxide Reductase Proceed through a Trans - Mechanism? Biochemistry 56 , 120 131. (53) Duarte, A. G., Cordas, C. M., Moura, J. J. G., and Moura, I. (2014) Steady - state kinetics with nitric oxide reductase (NOR): new considerations on substrate inhibition profile and catalytic mechanism. Biochim. Biophys. acta - Bioenergetics 1837 , 375 84. (54) Cordas, C. M., Duarte, A. G., Moura, J. J. G., and Moura, I. (2013) Electrochemical behaviour of bacterial nitric oxide reductase - Evidence of low redox potential non - heme FeB gives new perspectives on the catalytic mechanism. Biochim. Biophys. Acta - Bioenerg. 1827 , 233 238. (55) Speelman, A. L., Zhang, B., Silakov, A., Skodje, K. M., Alp, E. E., Zhao, J., Hu, M. Y., Kim, E., Krebs, C., and Lehnert, N. (2016) Unusual Synthetic Pathway for an {Fe(NO)2}9Dinitrosyl Iron Complex (DNIC) and Insight into DNIC Electronic Structure via Nuclear Resonance Vibrational Spectroscopy. Inorg. Chem. 55 , 5485 5501. (56) Girsch, P., and De Vries, S. (1997) Purification and initial kinetic and spectroscopic characterization of NO reductase from Paracoccus denitrificans . Biochim. Biophys. Acta - Bioenerg. 1318 , 202 216. (57) Moënne - Loccoz, P., and De Vries, S. (1998) Structural characterization of the catalytic high - spin heme b of nitric oxide reductase: A resonance Raman study. J. Am. Chem. Soc. 120 , 5147 5152. (58) Kum ita, H., Matsuura, K., Hino, T., Takahashi, S., Hori, H., Fukumori, Y., Morishima, I., and Shiro, Y. (2004) NO reduction by nitric - oxide reductase from denitrifying bacterium Pseudomonas aeruginosa : Characterization of reaction intermediates that appear in the single turnover cycle. J. Biol. Chem. 279 , 55247 55254. (59) Lin, Y., Yeung, N., Gao, Y., Miner, K. D., Tian, S., Robinson, H., and Lu, Y. (2010) Roles of glutamates and metal ions in a rationally designed nitric oxide reductase based on myoglobin. Pr oc. Natl. Acad. Sci. U. S. A. 107 , 8581 6. (60) Chakraborty, S., Reed, J., Sage, J. T., Branagan, N. C., Petrik, I. D., Miner, K. D., Hu, M. Y., Zhao, J., Alp, E. E., and Lu, Y. (2015) Recent Advances in Biosynthetic Modeling of Nitric Oxide Reductases and Insights Gained from Nuclear Resonance Vib rational and Other Spectroscopic Studies. Inorg. Chem. 54 , 9317 9329. (61) Matsumura, H., Hayashi, T., Chakraborty, S., Lu, Y., and Moënne - Loccoz, P. (2014) The production of nitrous oxide by the heme/nonheme diiron center of engineered myoglobins (Fe B Mbs) proceeds through a trans - iron - nitrosyl dimer. J. Am. Chem. Soc. 136 , 2420 2431. 80 (62) Matsumoto, Y., Tosha, T., Pisliakov, A. V, Hino, T., Sugimoto, H., Nagano, S., Sugita, Y., and Shiro, Y. (2012) Crystal structure of quinol - dependent nitric oxide reduct ase from Geobacillus stearothermophilus . Nat. Struct. Mol. Biol. 19 , 238 245. (63) Suharti, Heering, H. A., and de Vries, S. (2004) NO Reductase from Bacillus azotoformans Is a Bifunctional Enzyme Accepting Electrons from Menaquinol and a Specific Endogenous Membrane - Bound Cytochrome c 551 . Biochemistry 43 , 13487 13495. (64) Al - Attar, S., and De Vries, S. (2015) An electrogenic nitric oxide reductase. FEBS Lett. 589 , 2050 2057. (65) Park, S., Shimizu, K., Addachi, S., Nakagawa, A., Tanaka, I., Nakahara, K., Shoun, H., Obayashi, E., Nakamura, H., Lizauka, T., and Shiro, Y. (1997) Crystal structure of nitric oxide reductase from denitrifying fungus Fusarium oxysporum . Nature 4 , 827 832. (66) Lehnert, N., Praneeth, V. K. K., and Paulat, F. (2006) Electronic Structure of Iron(II) - Porphyrin Nitroxyl Complexes: Molecular Mechanism of Fungal Nitric Oxide Reductase (P450nor). J. Comput. Chem. 27 , 1338 1351. (67) McQuarters, A. B., Wirgau, N. E., and Lehnert, N. (2014) Model complexes of key intermediates in fungal cytochrome P450 nitric oxide reductase (P450nor). Curr. Opin. Chem. Biol. 19 , 82 89. (68) Baillie, T. A. (1981) The Use of Stable Isotopes in Pharm acological Research. Pharmacol. Rev. 33 , 81 32. - Catalyzed Carboxylation of Ribulose Bisphosphate. Biochemistry 23 , 6275 6284. (70) Huskey, P. W. (1991) Origins and Interpret ations of Heavy - Atom Isotope Effects, in Enzyme Mechanism from Isotope Effects (Cook, P. F., Ed.), pp 38 70. CRC Press, Inc., Boca Raton. (71) Lewis, B. E., and Schramm, V. L. (2006) Enzymatic Binding Isotope Effects and the Interaction of Glucose with Hex okinase, in Isotope Effects in Chemistry and Biology (Kohen, A., and Limbach, H. - H., Eds.), pp 1020 1050. CRC Press, Inc., New York. (72) Westheimer, F. H. (1961) The magnitude of the primary kinetic isotope effect for compounds of hydrogen and deuterium. Chem. Rev. 61 , 265 273. (73) Forster, P., V. Ramaswamy, P. Artaxo, T. Berntsen, R. Betts, D.W. Fahey, J. Haywood, J. Lean, D.C. Lowe, G. Myhre, J. Nganga, R. P., and G. Raga, M. S. and R. V. D. (2007) Changes in Atmospheric Constituents and in Radiative Fo rcing, in Climate Change 2007: The Physical Science Basis. Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change (Solomon, S., D. Qin, M. Manning, Z. Chen, M. Marquis, K.B. Averyt, M. T. and H. L. M., Ed.), pp 131 217. Cambridge University Press, New York. 81 (74) Crow, A., Matsuda, Y., Arata, H., and Oubrie, A. (2016) Structure of the Membrane - intrinsic Nitric Oxide Reductase from Roseobacter denitrificans . Biochemistry 55 , 3198 3203. (75) Shiro, Y., Sugimoto, H., Tosha, T., Nagano, S., and Hino, T. (2012) Structural basis for nitrous oxide generation by bacterial nitric oxide reductases. Source Philos. Trans. Biol. Sci. 367 , 1195 1203. (76) Schneider, J. L. (2011) How Kinetic Isotope Effects Can Be Us ed to Understand Enzymatic Transition States. Einstein J. Biol. Med. 27 , 18 19. (77) Flock, U., Watmough, N. J., and Ädelroth, P. (2005) Electron/proton coupling in bacterial nitric oxide reductase during reduction of oxygen. Biochemistry 44 , 10711 10719. (78) Toyoda, S., and Yoshida, N. (1999) Determination of nitrogen isotopomers of nitrous oxide on a modified isotope ratio mass spectrometer. Anal. Chem. 71 , 4711 4718. (79) Yu, C., and Yao, W. (2017) Robust linear regression: A review and comparison. Comm un. Stat. - Simul. Comput. 46 , 6261 6282. - catalyzed reactions. Annu. Rev. Biochem. 58 , 377 401. (81) Singh, P., Islam, Z., and Kohen, A. (2016) Examinations of the Chemical Step in Enzyme Cataly sis. Methods Enzymol. 1st ed. Elsevier Inc. (82) Toyoda, S., Yoshida, N., and Koba, K. (2017) Isotopocule Analysis of Biologically Produced Nitrous Oxide in Various Environments. Mass Spectrom. Rev. 36 , 135 160. (83) Kool, D. M., Wrange, N., Oenema, O., Do lfing, J., and Van Groenigen, J. W. (2007) Oxygen exchange between (de)nitrification intermediates and H 2 O and its implications for source determination of NO 3 - and N 2 O: a reviw. Rapid Commun. Mass Spectrom. 21 , 3569 3578. (84) Averill, B. A. (1996) Dissim ilatory nitrite and nitric oxide reductases. Chem. Rev. 96 , 2951 2964. (85) Rex, D., Clough, T. J., Richards, K. G., de Klein, C., Morales, S. E., Samad, M. S., Grant, J., and Lanigan, G. J. (2018) Fungal and bacterial contributions to codenitrification em issions of N 2 O and N 2 following urea deposition to soil. Nutr. Cycl. Agroecosystems 110 , 135 149. (86) Yamazaki, T., Hozuki, T., Arai, K., Toyoda, S., Koba, K., Fujiwara, T., and Yoshida, N. (2014) Isotopomeric characterization of nitrous oxide produced by reaction of enzymes extracted from nitrifying and denitrifying bacteria. Biogeosciences 11 , 2679 2689. (87) Blomberg, L. M., Blomberg, M. R. A., and Siegbahn, P. E. M. (2006) Reduction of nitric oxide in bacterial nitric oxide reductase - a theoretical model study. Biochim. Biophys. Acta - Bioenerg. 1757 , 240 252. 82 (88) Hendriks, J. H. M., Jasaitis, A., Saraste, M., and Verkhovsky, M. I. (2002) Proton and electron pathways in the bacterial nitric oxide reductase. Biochemistry 41 , 2331 2340. (89) Yeung, N., Lin, Y. W., Gao, Y., Zhao, X., Russell, B., Lei, L., Miner, K. D., Robinson, H., and Lu, Y. (2009) R ational design of a structural and functional nitric oxide reductase. Nature 462 , 1079 1081. (90) Cleland, W. W. (2005) The use of isotope effects to determine enzyme mechanisms. Arch. Biochem. Biophys. 433 , 2 12. (91) Berto, T. C., Hoffman, M. B., Murata, Y., Landenberger, K. B., Alp, E. E., Zhao, J., and Lehnert, N. (2011) Structural and electronic characterization of non - heme Fe(II) - nitrosyls as biomimetic models of the Fe B center of bacterial nitric oxide reductase. J. Am. Chem. Soc. 133 , 16714 16717. ( 92) Tsubaki, M., and Yu, N. T. (1982) Resonance Raman Investigation of Nitric Oxide Bonding in Nitrosylhemoglobin A and - myoglobin: Detection of Bound N - O Stretching and Fe - NO Stretching Vibrations from the Hexacoordinated NO - Heme Complex. Biochemistry 21 , 1140 1144. (93) Burton, G. W., Sims, L. B., Wilson, J. C., and Fry, A. (1977) Calculation of Carbon - 14, Chlorine - 37, and Deuterium Kinetic Isotope Effects in the Solvolysis of tert - Butyl Chloride. J. Am. Chem. Soc. 99 , 3371 3379. (94) Williams, R. C., and Taylor, J. W. (1974) Chlorine kinetic isotope effect models. II. Vibrational analysis and KIE [kinetic isotope effects] calculations of tert - butyl chloride transition state models. J. Am. Chem. Soc. 96 , 3721 3727. (95) Davidson, E. A., and Kanter, D. (2014) Inventories and scenarios of nitrous oxide emissions. Environ. Res. Lett. 9 , 1 12. (96) Matsumura, H., Chakraborty, S., Reed, J., Lu, Y., and Moënne - Loccoz, P. (2016) Effect of Outer - Sphere Side Chain Substitutio ns on the Fate of the trans Iron - Nitrosyl Dimer in Heme/Nonheme Engineered Myoglobins (Fe B Mbs): Insights into the Mechanism of Denitrifying NO Reductases. Biochemistry 55 , 2091 2099. (97) Chakraborty, S., and Reed, J. (2014) Spectroscopic and Computational Study of a Non - heme Iron - Nitrosyl Center in a Biosynthetic Model of Nitric Oxide Reductases in Myoglobin Containing Zn - protoporphyrin IX: Support for the Trans Mechanism of NO Reduction Saumen. Angew Chem Int Ed Engl 53 , 2417 2421. (98) Hogg, N., and Kal yanaraman, B. (1998) The Use of NO Gas in Biological Systems, in Nitric Oxide Protocols (Walker, J. M., and Titheradge, M. A., Eds.), pp 231 236. Humana Press Inc., Totowa, New Jersey. (99) Fox, J. B. J., and Ackerman, S. A. (1968) Formation of Nitric Oxid J. Food Sci. 33 , 364 370.