Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2008. / Vita. / Includes bibliographical references. / Here, we describe our attempts to harness the exquisite specificity of natural protein and RNA enzymes to develop improved methods to study protein localization and protein-protein interactions in live cells. We first attempted to detect endogenous protein-protein interactions (PPIs) in live cells by means of a ribozyme complementation assay, but we found that the strategy was limited by the interaction affinity constraints and by low ribozyme activity in cells. We then sought to still detect interactions among endogenous proteins but in fixed cells. We devised an improved immunofluorescence (IF) technique, in which the antibodies are conjugated to an enzyme-substrate pair. We chose E. coli biotin ligase (BirA), which catalyzes the covalent ligation of biotin to a 15amino acid recognition sequence (AP). Only upon PPI would BirA be in close enough proximity to biotinylate the AP. Although the use of proximity biotinylation within the IF scheme proved challenging because of the geometric rigidity of the antibody conjugates, we later successfully applied the concept to the study of recombinant proteins in live cells, where BirA and AP were each genetically fused to the proteins of interest. We demonstrated that this method offers a combination of high spatial and temporal resolution with a low rate of false positives. We engineered the BirA/AP affinity to reduce background and eliminate false positives, while still allowing robust detection of relatively transient PPIs (half-life > 1 minute). We demonstrated that the methodology exhibits high specificity for the detection of PPIs in living mammalian cells, with a fold induction in the detected signal upon PPI of - 5-25. Using FRB-FKBP12 system as a model, the BirA/AP(-3) pair was also able to quantitatively predict interaction KIds. / (cont.) Importantly, we showed that proximity biotinylation can detect the subcellular localization of the PPI under study. We also developed a new method for site-specific labeling of proteins in live cells. Through rational design, we re-directed E. coli lipoic acid ligase (LplA) to specifically ligate an unnatural alkyl azide substrate to an engineered 22-amino acid LplA acceptor peptide (LAP) tag. The alkyl azide can then be selectively derivatized with a cyclooctyne conjugated to any probe of interest. We first demonstrated that LplA can be used to label LAP-tagged proteins with Cy3, AlexaFluor568, and biotin at the surface of living mammalian cells, and we then applied the methodology to one- and two-color cellsurface receptor labeling. Finally, we also showed that LplA can site-specifically label intracellular proteins, although the signal/background ratio still needs to be improved. / by Marta Fernández Suárez. / Ph.D.

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2013. / Cataloged from PDF version of thesis. / Includes bibliographical references. / The NV center is becoming a very hot topic in many areas of science, including, Physics, Chemistry, Biology, and Quantum Information. The Degen group has focused on a specific application of the NV center, namely scanning magnetometry. In my time in the group, I focused on building a microscope and studying NV centers in bulk and nanocrystalline diamond. I began by building a confocal microscope which was capable of observing and determine single NV centers. I made measurements on the photon statistics of different defects to determine if they were single emitters or multiple emitters. I also made microwave frequency magnetic measurements to determine the spin properties of single NV centers by measuring their couplings to neighboring paramagnetic nuclei as well as to a spin bath. Through these efforts, I was able to successfully confirm that the microscope was capable of identifying and measuring single NV centers and their properties. Lastly, I worked on the first steps of improving our understanding of NV centers in bulk diamond crystals. The goal of magnetometry involves putting the NV center as close to the diamond surface as possible. I made measurements that were aimed at studying the spin and coherence properties of the NV when it was within 10 nm of the diamond surface. These studies provided insight into the interactions of the NV center with the diamond surface. / by Benjamin Kwasi Ofori-Okai. / S.M.

Thesis (M.S.)--Massachusetts Institute of Technology, Dept. of Chemistry, 1927. / Includes bibliographical references (leaf 30). / by Charles J. Pedersen. / M.S.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2004. / Vita. / Includes bibliographical references (leaves 222-230). / Optically pure (R)-5,5',6,6',7,7',8,8'-octahydro-1,1'-bi-2-napthol was derivatized with mesityl groups in the 3 and 3' positions to give (R)-MES₂BitetH₂ 1.43. Addition of the di-potassium salt of 1.43 to (Ar[sub]prN)Mo(CHCMe₂Ph)(OTf)₂(DME) yielded [(Ar[sub]prN)Mo((R)-MES₂Bitet)(CHCMe₂Ph)(THF), (R)-1.44a. This material was studied in detail by variable temperature ¹H NMR spectroscopy with and without solvent additives (THF, DMF and MeCN). An X-ray study of (R)-1.44a showed it to crystallize as the THF adduct of an anti-alkylidene, in a distorted trigonal bipyramid. Complex (R)-1.44a was shown to be an active and selective catalyst for olefin metathesis for representative ARCM transformations to form dihydrofurans. CHAPTER 2: The first catalytic asymmetric ring-closing metathesis (ARCM) method for the synthesis of nitrogen-containing heterocycles was developed; this was accomplished via Mo-catalyzed desymmetrization of unsaturated prochiral amines to afford tetrahydropyridines (2.68a-e). The selectivity of this transformation was sensitive to the olefin substitution pattern of the prochiral amine. However, this novel method was also applicable for the formation seven-(2.84), and eight-membered (2.84) rings in high yield, with exceptional enantioselectivity. Importantly, this method remained highly effective when performed without solvent. Several chiral benzoazepines (2.95a-c) were also prepared via Mo- catalyzed ARCM. In these latter cases, the steric size of groups attached to the prochiral carbon was observed to affect both the rate and selectivity of ARCM. Related benzoazocine precursors (2.97 and 2.100) could not be successfully desymmetrized to cyclic amines. / (cont.) A prochiral tetraene (2.101) was rapidly transformed into a spirocyclic-benzazepine (2.103) by tandem ARCM/RCM with good enantioselectivity. Carbocyclic amines (2.105a-f) were also efficiently synthesized via ARCM with good enantioselectivity. Several of these enantioselective transformations were equally effective when catalyzed by catalysts prepared in situ. Three new poly(styrene) supported chiral Mo-based catalysts were prepared. Two of the supported complexes were biphenolate-based ((S)-3.75 and (S)-3.76) while the third was binapnatholate-based ((R)-3.77). Additionally, a new support was developed, wherein the Mo-alkylidene could be supported in tandem with polymer generation. Several poly(norbornene) supported catalysts were also prepared, with various cross- linking levels. It was found that the activity and selectivity of these poly(norbornene) supported systems was greatest with low cross-linking levels (8 %), such as for (S)-3.88d. Two more lightly cross-linked poly(norbornene) complexes were prepared with distinct imido groups, (S)-3.89 and (S)-3.90. The ability of these polymer-bound chiral complexes to promote an assortment of asymmetric ring-closing (ARCM) and ring-opening (AROM) metathesis reactions was studied. In many instances, the levels of reactivity and enantioselectivity observed were competitive with the analogous homogeneous catalysts. In all cases, the optically enriched products obtained through the use of the above supported complexes, after simple filtration and removal of the polymeric chiral Mo complexes, were found to contain significantly lower levels of metal impurities ... / by Sarah Jennifer Dolman. / Ph.D.

Thesis: Ph. D. in Inorganic Chemistry, Massachusetts Institute of Technology, Department of Chemistry, 2015. / Vita. Cataloged from PDF version of thesis. / Includes bibliographical references. / Chapter 1. Component Interactions in Three- and Four-Component Bacterial Multicomponent Monooxygenases. Bacterial multicomponent monooxygenases (BMMs) catalyze oxidation of hydrocarbon substrates through activation of dioxygen. Each BMM utilizes a diiron active site housed within a catalytic hydroxylase protein. This diiron active site is responsible for activation of dioxygen and oxidation of hydrocarbons. Additional component proteins modify the structure of the hydroxylase regulating substrate access and pre-organizing the diiron site for reactivity. The relationships between structure and reactivity that provide this control are reviewed for both three- and four-component BMMs, including soluble methane monooxygenase, phenol hydroxylase, toluene 4-monooxygenase, and toluene/oxylene monooxygenase. Comparisons between three- and four-component BMMs are highlighted to demonstrate how nature preserves the control over reactivity within the BMM superfamily. Chapter 2. A Flexible Glutamine Regulates the Catalytic Activity of Toluene/o- Xylene Monooxygenase. Toluene/o-xylene monooxygenase (ToMO) belongs to the enzyme superfamily of bacterial multicomponent monooxygenases (BMMs) and is capable of oxidizing aromatic substrates. The carboxylate-rich diiron active site is located 12 A below the surface of the catalytic hydroxylase component (ToMOH). The shortest opening between the surface of the protein and the diiron active site is a small hydrophilic pore. Here we examine the function of residues lining this pore, N202 and Q228, within ToMOH from Pseudomonas sp. OX1. Through characterization of the steady-state turnover of WT ToMOH and three mutant enzymes, N202A, Q228A, and Q228E, we demonstrate that these residues are critical for turnover. Mechanistic analysis reveals that these residues are critical for water egress and efficiently consuming NADH to hydroxylate product. We propose that this activity results from movement of these residues, opening and closing the pore during catalysis. In addition, N202 and Q228 are important for interaction of two component proteins, the diiron-reducing protein and the regulatory protein, suggesting that these two proteins bind competitively to the hydroxylase. The function of the pore region in other BMMs is discussed in light of these results. Chapter 3. Component Interactions and Electron Transfer in Toluene/o-Xylene Monooxygenase. Toluene/o-xylene monooxygenase (ToMO) activates dioxygen to oxidize aromatic hydrocarbons. Prior to dioxygen activation, the diiron active site must acquire two electrons. This process requires three redox active proteins, a hydroxylase (ToMOH), a Rieske protein (ToMOC), and an NADH oxidoreductase (ToMOF). A fourth, regulatory component with no redox active cofactors is also required to achieve catalysis (ToMOD). Through pre-steady-state kinetics, we demonstrate that ToMOD alters electron transfer from ToMOC to ToMOH. Under steady-state conditions, ToMOD increases the rate of turnover up to one equivalent of ToMOD to ToMOH. At excess ToMOD concentrations, the regulatory protein inhibits steady-state catalysis in a manner that depends upon the concentration of ToMOC. Protein-binding studies, computational docking, and rapid electron transfer kinetics indicate that this inhibitory function results from competition between ToMOD and ToMOC for binding to ToMOH. These results are discussed in the context of additional proteins in the bacterial multicomponent monooxygenase superfamily. Chapter 4. Oxygen Activation by the Hydroxylase of Toluene/o-Xylene Monooxygenase in the Presence of its Redox Partners. To hydroxylate arene substrates, toluene/o-xylene monooxygenase (ToMO) utilizes four protein components, a catalytic hydroxylase (ToMOH), a regulatory protein (ToMOD), a Rieske protein (ToMOC), and a reductase (ToMOF). Within ToMOH, this chemistry is achieved through the activation of dioxygen. Previous dioxygen activation studies of ToMO have utilized a simplified protein system comprising ToMOH and ToMOD, but with dithionite and methyl viologen supplying the electrons. Here, we revisit the dioxygen activation experiments but with ToMOC, ToMOF, and NADH. The use of these proteins and NADH dramatically alters dioxygen activation chemistry and subsequent arene hydroxylation in single turnover studies. Chapter 5. Oxygen Activation in the T201S Variant of Toluene/o-Xylene Monooxygenase. The secondary coordination spheres in diiron proteins influence reactivity at the active site. In the diiron protein toluene/o-xylene monooxygenase, a threonine residue (T201) near the diiron site modulates steady-state turnover and dioxygen activation chemistry in the presence and absence of substrate. Previous oxygen-activation studies, reveal that mutation of this residue to a serine (T201S) yields diiron-0 2 adducts different from those observed for of WT ToMOH. These oxygen-activation experiments were conducted using dithionite and methyl viologen as the reducing agents. As in Chapter 4, we re-examine oxygen activation by T201S in the presence of the redox proteins, ToMOC and ToMOF. Stopped-flow UVvisible spectroscopy reveals that the use of these component proteins changes the number and identity of diiron-02 adducts formed during the dioxygen activation steps in T201S. Appendix A. Heterologous Expression and Purification of Components of Toluene/o-Xylene Monooxygenase from Pseudomonas sp. OXI. Here we provide the detailed protocols for expression and purification of the component proteins of toluene/o-xylene monooxygenase. Gene sequences and miscellaneous purification and handling notes are also provided. / by Alexandria Deliz Liang. / Ph. D. in Inorganic Chemistry

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2006. / Vita. / Includes bibliographical references. / The attempted syntheses of molybdenum imido alkylidene complexes of the type Mo(NArc,)(CH-t-Bu)[Biphen] and Mo(N-2-CF3C6H4)(CHCMe2Ph)[Biphen] (Biphen2 = 3,3'-di-t-butyl-5,5',6,6'-tetramethyl- 1,1'-biphenyl-2,2'-diolate) from Mo(NArcl)(CH-t-Bu)(OTf)2(dme) and [Biphen]K2 have sporadically afforded mixtures containing the desired products along with the corresponding amido alkylidyne complexes, Mo(NHArcl)(C-t-Bu)[Biphen] and Mo(NH-2-CF3C6H4)(CCMe2Ph)[Biphen], respectively. The reaction of [Biphen]K2 with Mo(NArc,)(CH-t-Bu)(OTf)2(dme) and 10 equivalents of triethylamine reproducibly gave Mo(NHArc,)(C-t-Bu)[Biphen] in 40% yield. An X-ray crystal structure of a related complex, Mo(NHArc,)(CCMe2Ph)[S-Biphen] confirmed the proposed structure and also revealed that one ortho chloride approaches within 2.93 A of the metal approximately trans to the alkylidyne ligand. Attempts to prepare three other amido alkylidyne complexes in an analogous manner from Mo(NR")(CH-t-Bu)(OTf)2(dme) (NR" = N-2-CF3C6H4, N-2,6-i-Pr2C6H5, N-2,6-Me2C6H5) with [Biphen]K2 in the presence of 10-20 equivalents of triethylamine failed. / (cont.) Chapter 2 The reaction between Mo(NAr)(CH-t-Bu)(CH2-t-Bu)2 (Ar = 2,6-i-Pr2C6H3) and various alcohols (1-AdamantylOH, t-BuOH, ArOH, (CF3)2CHOH, (CF3)2MeCOH, (CF3)3COH, C6F5OH) in pentane or toluene yielded either complexes of the type Mo(NAr)(CH-t-Bu)(CH2-t-Bu)(OR) through direct addition of ROH across a Mo-C bond, or complexes of the type Mo(NAr)(CH2-t-Bu)3(OR) through direct addition of ROH across a Mo=C bond. The trineopentyl species appear to be formed when the alcohol has a relatively low pKa. The outcome also can depend upon whether the alcohol is employed neat, or in benzene, and mixtures are observed in some circumstances. The conversion of Mo(NAr)(CH2-t-Bu)3(OR) into Mo(NAr)(CH-t-Bu)(CH2-t-Bu)(OR) was shown to be unimolecular in several examples. Mo(NAr)(CH-t-Bu)(CH2-t-Bu)(OR) complexes have been found to be surprisingly active catalysts for various metathesis reactions. In contrast, M(NAr)(CH-t-Bu)(CH2-t-Bu)2 species are virtually inactive for metathesis. X-ray structures are reported for Mo(NAr)(CH2-t-Bu)3(OC6F5), Mo(NAr)(CH-t-Bu)(CH2-t-Bu)IOSi(O-t-Bu)3], [Mo(NAr)(CH-t-Bu)(CH2-t-Bu)(OC6F)12, and Mo(NAr)(CH-t-Bu)(CH2-t-Bu)(OC6F5)(PMe3). / (cont.) Chapter 3. Complexes of the type Mo(NR")(CHR')(N(R)3,5-C6H3Me2)2 (NR" = N-2,6-i-Pr2C6H, N-2,6-Me2C6Hs; R' = t-Bu, CMe2Ph; R' = i-Pr, t-Bu) and Mo(NR")(CHR')(NR2)2 (NR" = N-2,6-i-Pr2C6H,, N-2,6-Me2C6H5; R' = t-Bu, CMe2Ph; R = Me, Ph) can be isolated as orange-red solids in 30-35% yields or oils by reacting Mo(NR")(CHR')(OTf)2(dme) with LiN(R')(3,5-C6H3Me2)(ether) or with LiNR2. The synthesis of Mo(NR")(CHCMe2Ph)(NPh2)2 can be improved to 70-90% isolated yields when Mo(NR")(CHCMe2Ph)[OCMe(CF3)212 is used with LiNPh2(ether). Mo(NAr)(CHCMe2Ph)(NPh2)2 has been crystallographically characterized. Mo(NR")(CHR')(N(R)3,5-C6H3Me2)2 species reacted with t-BuOH and Me(CF3)2COH in benzene to give Mo(NR")(CHR')(OR)2 (OR = O-t-Bu, OCMe(CF3)2) in situ. However, no reactions of Mo(NR")(CHR')(N(R')3,5-C6H3Me2)2 were observed with enantiomerically pure diols such as [R-TRIP]H2 (3,3'-2,4,6-i-Pr3C6H2-binaphthol), [R-Ph]H2 (3,3'-C6H5-binaphthol), [rac-Mesitylbinap]H2 (3,3'-2,4,6-Me3C6H2-binaphthol) and [R-TMSbinapJH2 (3,3'-SiMe3-binaphthol). / (cont.) Bisamido complexes of the type Mo(NR")(CHR')(NPh2)2 were found to react with the aforementioned alcohols and diols to give Mo(NR")(CHR')(diolate) species in situ, which were further reacted in a catalytic fashion with two substrates to give the corresponding ring-closed products. Preliminary :results of the in situ catalysis demonstrated here compare fairly well with the analogous catalytic reactions reported with isolated catalysts. Appendix A. Mo(NAr)(CH-t-Bu)(CH2-t-Bu)(OC6F5) (Ar = 2,6-i-Pr2C6H3) can be reacted with 5-10 equivalents of trans-3-hexene to give a crystallographically characterized dimeric complex, [Mo(NAr)(CH2-t-Bu)(OC6F5)]2 that contains an unbridged Mo=Mo bond (2.410(8) A) in high yields. The above complex can also be prepared by treating Mo(NAr)(CH-t-Bu)(CH2-t-Bu)(OC6F5) with 0.5 equivalents of divinylbenzene. IMo(NAr)(CH2-t-Bu)(OC6F5)]2 will slowly catalyze the metathesis reactions of simple substrates, although less than 5% of the catalyst seems to be activated in such reactions. / (cont.) It was observed that catalytically active species for metathesis reactions can be generated by another Mo (d2) species, Mo(NArcl)(Biphen)(H2C=CH2)(ether) (NArc, = N-2,6-C12C6H3, Biphen2 = 3,3'-di-t-butyl-5,5',6,6'-tetramethyl-1,1'-biphenyl-2,2'-diolate) that could effect the ring-opening metathesis polymerization of norbornene. A mixture of Mo(NArcl)(Biphen)(H2C=CH2)(ether) and 20 equivalents of diallylether in benzene-d6 when treated with 10 equivalents of norbornene gives 54% conversion to dihydrofuran in 10 days. / by Amritanshu Sinha. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2007. / Vita. / Includes bibliographical references. / The observation that mutations in the Escherichia coli genes umuC+ and umuD+ abolish mutagenesis induced by UV-light strongly supported the counterintuitive notion that such mutagenesis is an active rather than passive process. Biochemical studies have revealed that umuC+ and its homolog dinB+ encode novel, low to moderate fidelity DNA polymerases with the ability to catalyze synthesis on imperfect DNA templates in a process termed translesion synthesis (TLS). Similar enzymes exist in nearly all organisms, constituting the Y-superfamily of DNA polymerases. Although DinB is the only Y-family DNA polymerase conserved among all domains of life, its precise function has remained elusive. Here we show that AdinB E. coli strains are sensitive to DNA damaging agents that form lesions at the N2 position of guanine. In vitro bypass studies of an N2-guanine adduct by DinB demonstrate considerable preference for correct nucleotide insertion and an increased catalytic proficiency on the lesion-bearing template relative to undamaged DNA. Moreover, DinB and its mammalian and archaeal orthologs possess similar substrate specificities. Mutation of a single residue in the active site ofE. coli DinB suggests that its enhanced activity is coupled to lesion recognition and that its TLS function is required for resistance to DNA damaging agents in vivo. / (cont.) Regulation of the mutagenic potential of DinB is critical for maintenance of genomic integrity. We present evidence indicating that abortive TLS products generated by a DinB variant are subject to the proofreading function of DNA polymerase III. Moreover, both the TLS activity and -1 frameshift mutator potential of DinB are modulated in a highly sophisticated manner by the DNA damage-inducible proteins RecA and UmuD2. These biochemical data, coupled with genetic analyses and molecular modeling, indicate that DinB is a specialized and remarkably controlled translesion DNA polymerase. In addition, we present evidence that the umuC+participates in several novel biological functions in addition to its established role in TLS. A novel umuC gain-of-function allele confers striking resistance to hydroxyurea and umuC+ mediates the expression of genes and physiological responses under conditions of SOS induction. Taken together, these observations hint at at a largely uncharacterized function of Y-family polymerases in sculpting physiological responses, including active mechanisms of cell death, in response to environmental stress. / by Daniel F. Jarosz. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 1996. / Includes bibliographical references. / by Marc Michael Wefers. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2011. / Pages 180 and 181 blank. Cataloged from PDF version of thesis. / Includes bibliographical references. / The vanadium nitride complex [Na][NV(N['Bu]Ar) 3] reacts with CO to produce the vanadium tris-anilide complex V(N['Bu]Ar)3 and NaNCO. This is the first example of complete denitrogenation of a termainal nitride complex with generation of a lower coordinate metal complex. This reactivity contrasts sharply with the reactivity of the niobium analogue, where the nitride anion complex [Na][NNb(N['Bu]Ar) 3] is synthesized from the reductive decarbonylation of the niobium(IV) isocyanate complex (OCN)Nb(N['Bu]Ar) 3. Electrochemical studies of the niobium(IV) and vanadium(IV) isocyanate (OCN)V(N['Bu]Ar) 3 complexes are presented. The reactivity of the vanadium carbamate complex [(THF) 2Na][O2CNV(N['Bu]Ar) 3] with electrophilic reagents is presented. The carbamate complex reacts readily with silylation and alkylation reagents to form the carbamate ester complexes of the type ROC(O)NV(N['Bu]Ar) 3. The vanadium carbamate complex reacts with SO2 via a decarboxylation pathway to produce the sulforyl imido complex [Na][O 2SNV(N['Bu]Ar)3], the solid-state structure of which is presented. The reactivity of the vanadium carbamate complex with typical dehydrating reagents, e.g organic acid anhydrides, is shown to proceed cleanly when cobaltocene, acting as an in situ reductant, is present to form the vanadium(IV) isocyanate complex (OCN)V(N['Bu]Ar) 3. The synthesis and structure of the bimetallic complex (TPP)MnOC(O)NV(N['Bu]Ar) 3 (TPP = tetraphenylporphyrin) is presented. Although thermally stable, the complex undergoes a photochemical transformation that forms the vanadium isocyanate complex and putative OMn(TPP), which reacts with triphenylphosphine in the reaction mixture to produce triphenylphosphine oxide. The synthesis the niobium carbamate complex [Na][O 2CNNb(N['Bu]Ar) 3] from the reaction of [Na][NNb(N[Bu]Ar) 3] with CO2 is presented. Its solid-state structure in the form of the ionpair [(12-crown-4) 2Na][O2CNNb(N['Bu]Ar) 3] has been determined. Reaction of the niobium carbamate complex with organic acid anhydrides results in the production of five-coordinate carboxylate, acetate complexes (RC(O)O)(OCN)Nb(N['Bu]Ar) 3. The reduction of these complexes by two electrons results in the regeneration of the niobium nitride complex (60-80% yield) with concomitant release of CO (30-60% yield). This three-step process represents a highly controlled conversion of CO2 to CO via a ligand-based strategy. The reactivity of CO2 with anionic complexes featuring terminal multiply bonded ligands is extended to the oxo anion complex [(Et 2O)2Li][OTi(N['Bu]Ar) 3] resulting in the formation of the carbonate complex ([Li][O 2COTi(N['Bu]Ar) 3]) 6. The binding of CO2 to the oxo complex is reversible when 12-crown-4 is bound to the lithium countercation or if the complex is dissolved in THF. The thermodynamic parameters for the CO2 binding equilibrium have been measured. Exchanging the lithium countercation for sodium or potassium results in a significant weakening of the CO2 binding ability of the oxo complex. / by Jared S. Silvia. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2013. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Cofactors are highly prevalent in biological systems and have evolved to take on many functions in enzyme catalysis. Two cofactors, flavin adenine dinucleotide (FAD) and [4Fe-4S] clusters, were originally determined to aid in electron transfer and redox chemistry. However, additional activities for these cofactors continue to be discovered. The study of FAD in the context of rebeccamycin and staurosporine biosynthesis has yielded another role for this cofactor in the enzyme StaC. A homolog of this enzyme, RebC, uses its FAD cofactor in the oxidation of 7-carboxy-K252c. StaC also uses 7-carboxy-K252 as a substrate, but its reaction does not result in a redox transformation. Biochemical and X-ray crystallographic methods were employed to determine that, indeed, the role of FAD in the StaC system is not to catalyze redox chemistry. Instead, FAD sterically drives an initial decarboxylation event. Subtle differences in the active sites of RebC and StaC promote this redox neutral decarboxylation, by activating water for a final protonation step. In another system, the characterization of the S-adenosyl-L-methionine (AdoMet) radical superfamily showed the versatility of these cofactors. In this superfamily, which includes over 40,000 unique sequences, [4Fe-4S] clusters are responsible for the initiation of radical chemistry. A recently described subclass of this superfamily, the dehydrogenases, require additional [4Fe-4S] cluster for activity. This requirement led to the hypothesis that these enzymes are catalyzing redox chemistry by directly ligating substrates to auxiliary (Aux) clusters. X-ray structures of 2-deoxy-scyllo-inosamine dehydrogenase (BtrN), required for the biosynthesis of 2-deoxystreptamine, and an anaerobic sulfatase maturating enzyme, anSMEcpe, which installs a required formylglycine posttranslational modification, refute this hypothesis. In these structures, substrate binding is distal from each enzymes' Aux clusters. However, the Aux cluster binding architecture shared between BtrN, anSMEcpe, and another AdoMet radical enzyme, MoaA, involved in molybdenum cofactor biosynthesis, suggests that the structural features will be a staple in the AdoMet radical superfamily, common to - 30% of the AdoMet radical reactions. / by Peter John Goldman. / Ph.D.