Flavins are arguably one of the most versatile cofactors in biology, resulting from their ability to facilitate both 1e- and 2e- transfer reactions, as well as their widely-varying reduction potentials that can be tuned by the local protein environment. Flavins have three redox states: oxidized (OX), 1e--reduced semiquinone (SQ), and 2e--reduced hydroquinone (HQ). Their reactivity in redox processes is determined by E’OX/SQ and E’SQ/HQ, which is affected by electrostatic and H-bonding interactions in the flavin binding site. Free in solution, flavins exhibit “inverted potentials”, where the 1e– reduced state is more unstable than the fully-reduced state. The flavin binding site of flavodoxins, a class of electron transfer flavoproteins, alters the 1e- reduction potentials such that they become “normally” ordered, stabilizing E’OX/SQ relative to E’SQ/HQ. In other cases, the protein environment can increase potential inversion. A dramatic example can be found in flavin-based electron bifurcation (FBEB), which utilizes significant potential separation (ΔE) to conserve energy from exergonic redox reactions by concomitantly driving endergonic ET. The properties of the flavin binding site that induce this inversion are unknown, largely due to the limited mutagenesis space in authentic EB-ases and lack of physical methods for measuring highly-inverted 1e- reduction potentials. Since potential separation is fundamental to the mechanism of energy conservation in FBEB, understanding its cause is both interesting from a basic science perspective, and valuable to the field of electrocatalysis. To move towards this goal, it is essential to develop minimalist models for potential inversion, since the study of this phenomenon in native EB-ases is nontrivial. We have demonstrated that iLOV can be used as a model flavoprotein to evidence the effects of near-flavin mutations on redox behavior and potential inversion, substituting Lys to illustrate the influence of an ionizable and H-bonding functional group on semiquinone stability. We have designed expression and reconstitution methods to recombinantly produce iLOV WT, Q104K, and Q104A in high holoprotein yield. The 2e- reduction potential of iLOV WT and Q104K was measured by protein film voltammetry, and equilibration studies with a redox dye were conducted to confirm that protein-film electrochemistry corresponded to bulk properties. The semiquinone protonation state was characterized by EPR and UV-vis. Using spectral data to infer concentrations of the three flavin redox states at electrochemical equilibrium, ΔE was calculated for iLOV WT, iLOV Q104K and iLOV Q104A. The results suggest that iLOV WT has the most inverted 1e- potentials, followed by Q104A, with Q104K having the least potential separation. The substitution of Ala at the N5 position appeared to stabilize the SQ, although this is likely due to exposure to ambient light. The addition of Lys at N5 appears to influence the thermodynamic properties of the flavin such that E’OX/SQ, and seemingly E’SQ/HQ, is more positive. This can be rationalized by its relative acidity and closer pKa matching between the flavin and Lys, as compared to Gln in iLOV WT. Our discoveries on the crucial elements of potential inversion in flavin redox chemistry may shed light on the mechanisms of potential tuning in natural systems, particularly extreme potential inversion of EB-ases.

Phosphorus is most commonly found in its fully oxidized state (P+5) of phosphate and phosphate esters, which are incorporated into biomolecules such as nucleotides, nucleic acids, and phospholipids. Phosphite, a reduced form of phosphorus (P+3), is found in rivers, lakes, sediments, and oceans, and is of suspected biological origin. However, there is no conclusive evidence of phosphite production in axenic culture, nor any clear mechanistic pathway for phosphite biosynthesis. Given that the phosphate/phosphite redox couple heavily favors oxidation (-690 mV vs. NHE), phosphite has been proposed to be a downstream product in the biosynthesis or degradation of phosphonates, molecules containing a direct phosphorus-carbon bond. This hypothesis forms the basis of my investigations, which primarily aimed to detail biogenic phosphite production in a phosphonate synthesizing organism using an array of optimized analytical techniques for phosphite detection.I developed an enzymatic, fluorometric assay for phosphite sensing. The method is reliant on the activity of a NAD+-dependent phosphite dehydrogenase, which through a series of electron transfer reactions, reduces resazurin to the highly fluorescent resorufin. Through the use of a thermostable variant of phosphite dehydrogenase, novel sample preparation methods, and phosphite standard additions, stable fluorescence readout can be read in 20 minutes with a 3 M limit of detection. The assay’s limit of detection and phosphite recovery were assessed in a wide array of biologically- and environmentally-relevant matrices to determine the sensor’s robustness. The sensor, along with 1H-coupled and 1H-decoupled 31P-NMR, were employed to observe phosphite production in Streptomyces viridochromogenes, a gram-positive soil bacterium. Through phosphonate feeding experiments in cell culture and soluble cell lysate, as well as the recombinant expression and purification of CTP-phosphonoformate nucleotidyltransferase (PhpF), phosphite was determined to be the decomposition product of activated phosphonate phosphonoformate-CMP. This work challenged the preexisting assumption that phosphite was a decomposition product of phosphonoformate at biologically-relevant temperatures and pHs. Finally, phosphite was investigated as a mobile, selective P-source for toluene-degrading inoculant Pseudomonas veronii in a synthetic soil community (SynCom) as a means of enhancing bioremediation efficiency. A phosphite-oxidizing (ptxD+) mutant of Pseudomonas veronii was constructed that can use phosphite as a sole P source. Soil microcosms with all iterations of the presence or absence of inoculant, SynCom, toluene, and phosphite were propagated, and were monitored for community composition with 16S rRNA gene amplicon sequencing and toluene degradation rates using GC-MS. Phosphite was determined to enhance inoculant abundance most prominently in toluene-free soil microcosm, likely due to the growth substrate being carbon limited. These investigations provide new insights into the role of phosphite in the biogeochemical P cycle, elucidating both a biological production mechanism of phosphite as well as its bioavailability in soil microbial communities.

Hydroxyacid oxoacid transhydrogenase (HOT) and succinate semialdehyde dehydrogenase (SSADH) are a pair of oxidoreductases that operate along pathways adjacent to the Szent–Györgyi–Krebs cycle and together catalyze the oxidation of γ hydroxybutyrate (GHB) to succinate. GHB was initially synthesized to study the neurotransmitter GABA but has become infamous as a date rape drug. Succinate is a well-known Krebs Cycle intermediate.HOT is an iron-dependent group III alcohol dehydrogenase (ADH) and the only member of this little-known, microbial-associated group found in animals. Despite its discovery in 1988, few studies on HOT have been published and the enzyme remains poorly characterized. HOT is proposed to oxidize GHB to succinate semialdehyde with concomitant reduction of a tightly bound NAD+ cofactor that does not exchange with the solvent NAD+ pool. The NAD+ cofactor is regenerated in the active site by reduction of an α ketoglutarate co-substrate to D 2 hydroxyglutarate (D2HG). Since D2HG has been identified as an oncometabolite involved in tumor progression, and changes in HOT expression levels have been detected in some cancers, HOT appears to be a potential drug target worthy of continued study. In published research on HOT, animal organs have been primary source of the enzyme, with cultured animal cells being secondary. However, structural (X ray) studies demand a relatively good amount of pure enzyme, while functional (enzyme kinetics) studies would benefit from the ability to mutate active site residues. Recombinant HOT containing an affinity purification tag, transformed into E. coli or stably transfected into a mammalian cell line, would ideally produce large amounts of pure and mutable HOT for structural and functional studies. This manuscript describes the heterologous expression of human HOT in E. coli, resulting in: 1) soluble and nearly pure recombinant HOT containing an N terminal maltose binding protein; and 2) soluble and partially purified recombinant HOT with an N terminal His-tag. Also described is the stable transfection of PC 12 cells (rat pheochromocytoma) with a vector encoding recombinant HOT with a C terminal HA tag. Integration of this sequence into the cell’s genome resulted in a line of cells that constitutively expressed this construct and, upon lysis and chromatography, yielded moderately pure recombinant HOT. Lastly, because the HOT catalyzed reaction produces no measurable change in absorbance or fluorescence under steady state conditions, the isolated reaction cannot be conveniently followed by UV-Vis or fluorescence spectroscopy. However, the HOT reaction may be coupled to the SSADH reaction since the succinate semialdehyde produced by HOT is the substrate for SSADH and the SSADH reaction produces NAD(P)H. Because NAD(P)H has a maximal absorbance at 340 nm, or fluorescence emission at 460 nm, the coupled reaction can be conveniently followed using a UV Vis spectrophotometer or a spectrofluorometer. E. coli SSADH was chosen as the auxiliary enzyme for the coupled assay, though the enzyme kinetics of this SSADH had yet to be thoroughly investigated. Thus, this dissertation also describes the expression, purification and steady-state enzyme kinetics for recombinant E. coli SSADH containing an N terminal His tag. This effort resulted in the determination of kinetic constants and pH-rate profile for the SSADH-catalyzed oxidation of succinic semialdehyde to succinate.

Pyruvate formate lyase (PFL) is the paradigm of glycyl radical enzymes (GREs) and is widely distributed in anaerobic and facultative anaerobic organisms. PFL catalyzes the transformation of pyruvate into acetyl CoA and formate, connecting the glycolytic pathway with energy conservation pathways and is directly related to bacterial virulence. PFL is characterized by a post-translationally installed glycyl radical (G●), and two cysteines necessary for catalysis. G● initiate activity by activating cysteine residues by H-atom transfer and forming reactive thiyl radicals, which react with pyruvate producing a non-oxidative C-C bond cleavage. The two active site cysteines in PFL are thought to have distinct roles as thiyl radicals, but direct structural and electronic characterization of this radical chemistry has been challenging due to the instability of the radical intermediates. We employed mechanism-based inhibitors and selenocysteine substitution to gain insight into cysteinyl radical generation, kinetics and energetics. Additionally, we characterized the immune response relevant inhibition of PFL by nitric oxide.PFL reacts with the mechanism-based inhibitor methacrylate inhibiting the enzyme activity, we observed the concomitant transformation G● into a new radical specie by electron paramagnetic resonance (EPR). We assigned the new radical to a C2 tertiary methacryl radical based on spectral simulations and reactions with methacrylate isotopologues. The reaction is specific for C418 and the methacryl radical decays reforming G●. C2● is quenched, presumably by C419, exhibiting an H/D solvent isotope effect of 3.4. Acrylate also inhibits PFL irreversibly, and alkylates C418, but the acryl secondary radical is unstable and elusive to direct observation, consistent with the expected reactivity of a secondary versus tertiary carbon-centered radical. These results support the unique roles of the two active site cysteines of PFL and a C419 S−H bond dissociation energy between that of a secondary and tertiary C−H bond. We substituted the two active site cysteines of PFL for selenocysteine. Selenocysteinyl radicals are proposed to stabilize the radical chemistry long enough for direct observation. Selenocysteine incorporation reduces PFL turnover number by over 5000-fold. Although we were unable to detect selenyl radicals directly using X-band EPR, the selenocysteine mutants displayed increased solvent H/D kinetic isotope effects and changes in KM values for pyruvate and CoA, evidencing cysteinyl radical formation and the distinct roles of the two cysteines. Finally, we demonstrated that PFL is irreversible inhibited by the bacteriostatic Nitric oxide (NO) by reacting directly with G●, as demonstrated using EPR and site-directed mutagenesis. The activation of PFL by its cognate activating enzyme (PFL-AE) is also inhibited by NO by forming dinitrosyl iron complexes with the essential [Fe4S4] cluster. Whole-cell EPR and metabolic flux analyses demonstrated that PFL and PFL-AE are inhibited by NO in bacterial cell cultures, inhibiting growth and causing a metabolic shift from formate to lactate fermentation. The findings extend to class III ribonucleotide reductase (RNR), suggesting that NO targets GREs and radical SAM enzymes generally. The results implicate an immunological role of NO in inhibiting glycyl radical enzyme chemistry in the gut.