Copper is a trace element utilized by organisms as a cofactor involved in redox chemistry, electron transport, photosynthesis, and oxidation reactions. In excess, copper is toxic; it can generate reactive oxygen species causing cellular damage, or poison other metalloproteins by replacing native metal cofactors. Gram-negative bacteria have developed homeostatic mechanisms to maintain the intracellular copper concentration in the face of changing environmental conditions. For Gram-negative enteric bacteria, like Esherichiacoli and Salmonella enterica serovar typhimurium, copper is encountered in industrial and institutional settings, where the metal is used as a broad-spectrum biocide. For environmental bacteria, such as the marine cyanobacterium Synechococcus sp. WH8102, copper stress occurs because human activity changes the concentration of copper in the ocean. This dissertation contains six chapters, relating four stories of our investigations into the molecular mechanisms of copper homeostasis in Gram-negative bacteria. Chapter I contains literature review and background on the implications of bacterial copper homeostasis. Chapter II reports our work investigating the expression of two E. coli proteins, CusF and CusB, upon copper stress; we show that CusF expresses at a ~10-fold molar excess over CusB. Chapter III describes a collaboration between our lab and Jose Argüello's lab at Worcester Polytechnic Institute, and we show that CusF can acquire Cu(I) from CopA. Our results from Chapters II and III show that CusF functions as a major copper chaperone in the periplasm of E. coli. Chapter IV details our work characterizing a novel protein from marine cyanobacteria, Synw_0921. Although Synw_0921 is believed to be involved in copper homeostasis, we show that it is an iron-sulfur cluster protein. Bioinformatic analysis suggests that Synw_0921 represents a new family of proteins that help marine cyanobacteria adapt to copper changes in their unique environment. Chapter V relates our work on CueR and GolS, two homologous sensor proteins with distinct metal-dependent transcriptional activation; we find that the activity cannot be explained by binding affinity differences. Chapter VI concludes with final thoughts on the intersection of biochemistry and molecular biology in the important process of understanding copper homeostasis.

A series of conformationally constrained 2,6-bisferrocenylphenyl thioethers were synthesized via Suzuki-Miyaura cross coupling reactions. Structural information was obtained using X-ray crystallography and dynamic ¹H NMR spectroscopic studies, showing highly constrained m-terphenyl systems. Interaction of the ferrocene moieties through space mediated by the sulfur were studied by ultra-violet photoelectron spectroscopy (UPS), cyclic voltammetry, differential pulse voltammetry, UV-Vis-NIR spectroscopy and DFT computations. Electrochemical results show two, fully reversible 1e⁻ redox processes for the ferrocenes where the separation of peaks is affected by both solvent and supporting electrolyte, suggesting significant electrostatic interaction which is further confirmed in the gas phase by UPS studies. To determine if these interactions could be observed at greater distances, extended m-terphenyl complexes were shown in which 2-sulfur and 3-aromatic moieties were synthesized using a developed selective Suzuki-Miyaura monocoupling procedure in good yields. In these systems, interaction was not observed by electrochemistry or UPS. This suggests the distance between redox centers (~16 Å) is too great for electrostatic interaction, even though there is enhanced interactions observed in the truncated systems. Two new bisferrocenylphenylsulfoxides were also synthesized and studied to determine the effect of the polar sulfoxide bond on through space interaction between the ferrocene moieties. The electronic and redox properties of these compounds were studied by ultra-violet photoelectron spectroscopy, cyclic voltammetry, differential pulse voltammetry, and DFT computations. Electrochemical results for 2,6-bis(ferrocenyl) thioanisole S-oxide show two, fully reversible one electron redox processes. The initial oxidation shows a 62 mV negative shift compared to the sulfide analog 2,6-bis(ferrocenyl)thioanisole, and an increased peak separation for the oxidations of 160 mV. No peak separation is observed in the extended sulfoxide system. No intervalence charge transfer band was observed in the truncated sulfoxide complex by monitoring the UV-Vis/NIR spectroscopy of the mixed valence complex, ruling out electronic communication. Thus, the through space electrostatic interactions of the sulfoxide causes the non-equivalent ferrocenes in the truncated system to have different oxidation potentials. Synthesis was developed towards the synthesis of 1,8-bisferrocenyl-9-(alkylthio) anthracene complexes. It was observed that due to steric congestion at the C9 position of the anthracene scaffold, standard thionation reactions did not proceed as expected. Instead, the reaction of 1,8-dibromo-9-anthrone with Lawesson reagent afforded the intramolecular nucleophilic aromatic substitution cyclization product in quantitative yields. The reaction of the same anthrone under studied dithioketal formation conditions led to sulfur-rearrangement, giving the undesired 1,8-bisferrocenyl-10-(ethylthio)anthracene derivative, as confirmed by X-ray crystallography. Attempted Newman-Kwart rearrangement of 1, 8-dibromoanthracen-9-yl) dimethylcarbamothioate afforded no significant observed product formation, and decomposition of starting materials when heated for extended times. 1,8-bisferrocenyl-9-(methoxy)anthracene was synthesized and structurally characterized by dynamic X-ray crystallography to confirm connectivity. Electrochemical experiments show 2 reversible redox processes separated by 115 mV. Chemical oxidation experiments show unexpected, strong electronic coupling in the mixed valence complex. This coupling was characterized by near-IR absorption at 941 nm, indicating intervalence charge transfer (IVCT). Single electron reduction of 1,8-bisferrocenyl-9-(methoxy)anthracene, followed by quenching with various electrophiles afforded an inseparable mixture of products, one of which was identified by mass spectrometry as the desired 1,8-bisferrocenyl-9-(methylthio)anthracene product. However, this complex was not separable from the mixture and further characterization was not possible. All other routes attempted to incorporate sulfur into the system afforded no conversion of starting materials or decomposition of the reaction mixture.

Phosphorus is a key element for life. This work reviews the role of phosphorus in life. Theories on the origin of life are confounded by a lack of reactive phosphorus, and attempts to overcome the dearth of reactive phosphorus must employ unrealistic phosphorus compounds, energetic organic compounds, or unusual physical conditions.Meteoritic schreibersite provided an abundant source of reactive phosphorus for the early Earth. Water corrodes schreibersite to form a mixed valence series of phosphorus compounds. Schreibersite corrosion was studied by a variety of techniques, including NMR, MS, XRD, and EPR. Reduced phosphorus in schreibersite corrodes through release of phosphite radicals which react with other radicals to form the phosphorus compounds observed. These radicals are also capable of phosphorylating simple organic compounds to form P-C and P-O-C linkages.The meteoritic mass flux was calculated using the mass frequency distribution of several meteorite collections. Much of the meteoritic mass that falls to the Earth is composed of metallic material which supplies abundant reactive phosphorus. Meteorites are a comparatively poorer source of carbon. Craters concentrate both reduced phosphorus and organic compounds through geomorphologic processes.Phosphorus and sulfur biochemistry are intricately linked in metabolism. The cosmochemistry of sulfur was studied in depth using changing C/O ratios, sulfide formation kinetics, and gas diffusion. The results have implications for meteorites, studies of Jupiter, and of protoplanetary disks.

Anaerobic digestion is a common process for treatment of wastewater sludge from municipal sewage systems. Volatile sulfur compounds, including volatile organic sulfur compounds (VOSCs) and hydrogen sulfide, have been reported as the most odorous compounds in digestion emissions and impurities which can damage facilities for generation, transportation, storage, and utilization of biogas. There has been no comprehensive study on biological generation and degradation kinetics of VOSC or modeling VOSC behaviors through anaerobic sludge digestion. The goal of the present study was to establish a model for VOSC conversions in anaerobic sludge digestion which could facilitate quantitative analysis of VOSC emissions in anaerobic digestion. VOSCs and methionine were employed in dosed batch tests. VOSC conversion processes in anaerobic methionine digestion have been identified. The kinetics for the identified VOSC degradation and conversion processes were determined at 35 and 55 °C respectively. Mixed-second order kinetics were found to best fit the conversion processes. A model was established based on the identified processes and estimated kinetic constants. To extend the model to VOSC release in anaerobic sludge digestion, mesophilic and thermophilic incubations were conducted with four different sludge samples. The effects of temperature and sludge source on VOSC release patterns were assessed. It was found that an unidentified DMS generation mechanism was triggered in the mesophilic incubation of activated sludge in which iron was dosed. To apply the model which was established based on methionine degradation in sludge digestion, hydrolysis of particulate materials was incorporated. The model simulations for VOSC behavior in thermophilic batch incubation were able to represent the observed VOSC releases. However, the simulations could not well fit the observed VOSC release at 35 ° because the model did not include the unidentified DMS generation mechanism. Application of the model to bench-scale digesters was lack-of-fit. It may have been due to imprecise estimation of the degradable sulfur in the feed sludge. In addition, in the batch tests and digester operation the ratios of the raw and digested sludge were different. This might have resulted in different concentrations of the microorganisms which mediated biotransformations and hence resulted in different kinetic constants.

Chronic subclinical levels of disease occur frequently in intensive swine production and compromise nutrient utilization efficiency. Sulfur amino acids (methionine plus cysteine; M+C) have been implicated in improving the animal’s response to immune system stimulation (ISS). Research objectives were to determine the effects of ISS on the optimal dietary methionine to methionine plus cysteine ratio (M:M+C) and on the fractional synthesis rate (FSR) of albumin, fibrinogen and total protein in plasma, liver, and small intestine (SI) of growing pigs. A nitrogen balance study showed that the optimal M:M+C was increased during ISS and greater than 0.62. In a flooding dose infusion study it was determined that total plasma protein FSR was increased during ISS and tended to decrease with reduced M+C intake. Plasma albumin FSR decreased with reduced M+C intake. The data implicates M+C as important nutrients involved in the immune response and careful dietary supplementation during ISS is necessary. / Funding sponsored by Evonik Degussa, Ontario Pork, the Ontario Ministry of Agriculture, Food and Rural Affairs and the Natural Sciences and Engineering Research Council of Canada.

The assembly of λ tail requires the action of 11 gene products which must interact in an organized fashion to assemble infectious tail particles. GpL is an essential protein for the formation of the tail tip complex and necessary for the assembly of λ tail. The work described here has shown that gpL and its homologues contain two domains where the C-terminal domain coordinates an oxygen-sensitive [4Fe-4S] 2+ cluster using 4 highly conserved cysteines. This is the first report of a bacteriophage morphogenetic protein to coordinate a [4Fe-4S]2+ cluster. Through two individual cysteine mutants, C184A and C228A, it was determined that these mutant proteins coordinate a [2Fe-2S]2+ cluster also using 4 cysteines; the fourth cysteine being non-conserved. λ tails assembled with cysteine mutant gpL resulted in a 1000-fold decrease in the titer of active tails and tail particles could not be detected by TEM indicating that λ tails cannot be assembled with cysteine mutant gpL. I propose that the coordination of a [4Fe-4S] cluster with the four conserved cysteines maintains a conformation in gpL that can optimally interact with other tail proteins for efficient tail assembly.

The need for selective and inexpensive detectors in liquid chromatography is of considerable interest in the determination of sulfur compounds. Of the available-selective sulfur methodologies, flame photometric detector coupled to gas chromatography is the most widely used. It has proven to be a sensitive and selective method for detection of heat stable and volatile sulfur compounds. Fundamentally, this technique is not applicable to high boiling and/or thermolabile sulfur compounds. More recently, hyphenated flame photometric detector has been utilized, with limited success, to monitor sulfur species in liquid chromatography. However, existing HPLC-FPD methodologies have never been applied to real samples, due to the low population of S 2, the emitting species, and the quenching effects of the other species present in the flame. / In this work, two total consumption high-performance liquid chromatography flame photometric (HPLC-FPD) interfaces compatible with either methanolic or aqueous mobile phases are described and optimized for monitoring low volatile and thermally fragile sulfur compounds in biological samples. Each interface was fuelled either by methanol or by hydrogen. The all quartz interfaces enclosed four consecutive thermal processes: (a) thermovaporization of the HPLC effluent; (b) pyrolysis of the organic matrix (including sulfur species) in a kinetic H2/O2 flame; (c) conversion of the oxidized sulfur compounds to H2S in a reducing post-combustion stage fuelled by hydrogen; and (d) transport of the generated hydrides towards a hydrogen radical rich surrounding of an inverted hydrogen-oxygen diffusion flame. Chemiluminescence induced in the last step was integrated as a narrow beam in a light-guide positioned remotely from the analytical cool flame and oriented towards a photomultiplier unit. Radioisotopic assays demonstrated that sulfur (as H235SO4) was transferred quantitatively to the analytical flame. Indirect evidence suggested that sulfur was hydrogenated in the post-combustion step via a thermochemical hydride generation process to mediate the formation of S2. The linearity of calibration graphs (0.9950 < r < 0.9986), where r is the correlation coefficient) and unprecedented HPLC-FPD limits of detection for sulfur compounds (1.5 etag/s for 2-methylthiophene, 2.25 etag/s for carbon disulfide, and 4.5 etag/s for ethanesulfonic acid) allowed for the speciation of sulfur species in garlic extracts. Alternatively, modification of the methanol fuelled interface to a hydrogen fuelled reactor allowed detection of thiosulfinates and high molecular weight sulfur compounds in horse kidney and garlic extracts, respectively.

No description available.

The assembly of λ tail requires the action of 11 gene products which must interact in an organized fashion to assemble infectious tail particles. GpL is an essential protein for the formation of the tail tip complex and necessary for the assembly of λ tail. The work described here has shown that gpL and its homologues contain two domains where the C-terminal domain coordinates an oxygen-sensitive [4Fe-4S] 2+ cluster using 4 highly conserved cysteines. This is the first report of a bacteriophage morphogenetic protein to coordinate a [4Fe-4S]2+ cluster. Through two individual cysteine mutants, C184A and C228A, it was determined that these mutant proteins coordinate a [2Fe-2S]2+ cluster also using 4 cysteines; the fourth cysteine being non-conserved. λ tails assembled with cysteine mutant gpL resulted in a 1000-fold decrease in the titer of active tails and tail particles could not be detected by TEM indicating that λ tails cannot be assembled with cysteine mutant gpL. I propose that the coordination of a [4Fe-4S] cluster with the four conserved cysteines maintains a conformation in gpL that can optimally interact with other tail proteins for efficient tail assembly.

Phosphate and sulfate groups are important parts of biological molecules and as such take part in defining bio-interfacial interactions. However, their roles and contributions are insufficiently understood. In this thesis, plasma based techniques were used to produce polymeric coatings with such groups for surface characterization and protein adsorption studies.