The axial ligands of the iron porphyrin in Cytochrome c, an electron transfer protein, are an imidazole group of a histidine residue and a methionine thioether. This ligand coordination sphere has been difficult to model and consequently the influence of these ligands on the properties of cytochrome c has been problematic. The electrochemical and spectroscopic study of a novel strapped porphyrin has been addressed toward this problem. Spectroscopic studies have demonstrated the ability of this porphyrin to hold a thioether ligand near the central metal atom. The influence of the thioether is not seen in the UV/visible spectrum of the iron complex of this porphyrin. The coordination of N-methyl imidazole to the iron complexes of several porphyrins has been studied by UV/visible spectroscopy. These studies indicate a reduced affinity of the strapped porphyrin for this ligand. Also, the oxidation products of several porphyrins were monitored by thin-layer spectroelectrochemistry. Cyclic voltammetry has been used to demonstrate the influence of the thioether on the Fe('+3)/Fe('+2) electron transfer reaction. It was found that the thioether stabilizes the lower oxidation state causing an anodic shift in the half-wave potential for the reaction. However, the stabilization seen with this model system is not sufficient to account for the large positive redox potential of Cytochrome c. The oxidations of a selected group of free base and metallo- porphyrins were also studied. It was found that the oxidation of strapped porphyrins was similar in many respects to those of non-strapped porphyrins. The notable acception to this generalization was the instability of the cation radical of the strapped porphyrins used in this work.

by Lee Fung Yee Janet. / Thesis (M.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. 257-316). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

Redox chemistry is the basis for biological energy generation and anabolism. Redox conditions also serve as critical cues that modulate the development of many organisms. Roles for redox chemistry in the control of gene expression have been well characterized in multicellular eukaryotes, where oxygen availability in particular is a major developmental cue. As a gaseous metabolic substrate, oxygen becomes limiting as cellular communities grow, and can act as an indicator of aggregate size or developmental stage. In many of these cases, there are dedicated sensory and signal transduction networks that link oxygen and other redox signals to changes in gene expression and morphogenesis. The opportunistic pathogen Pseudomonas aeruginosa, like many species of microbes, forms multicellular structures called biofilms. Cells in biofilms can assume physiological states that differ from cells grown in well-mixed, homogeneous liquid cultures. They often exhibit increased resistance to environmental stresses and antibiotics, rendering biofilm physiology an important focus in the study of microbial pathogens. Biofilm development and architecture are tuned by environmental conditions. In turn, growth and survival in the community, and the specific structure of that community, give rise to internal microenvironments that are experienced by cells within a biofilm. Mechanisms that tune biofilm developmental programs in response to redox conditions are not well understood. This is due to challenges presented by most popular laboratory models of biofilm formation, which are not amenable to perturbation, characterization at the microscale, or high-throughput screening or analysis. In this thesis, I describe a standardized colony morphology assay for the study of P. aeruginosa PA14 biofilm development and use this model to address fundamental questions about the relationships between electron acceptor availability, biofilm cell physiology, and the regulation of biofilm morphogenesis. In the colony morphology assay, PA14 grows as ~1cm-diameter biofilms on agar-solidified media under controlled conditions, and displays a developmental pattern that is predictably influenced by changes in redox conditions. Microscale heterogeneity in chemical ecology can be profiled using microelectrodes, and the effects of specific mutations on development can be rigorously tested through high-throughput screening and the application of metabolic assays directly to biofilm samples. Prior to the work described here, application of the colony morphology assay had revealed that endogenous redox-active antibiotics called phenazines influence PA14 biofilm development such that defects in phenazine production promote colony wrinkling and the formation of a distinct wrinkle pattern. As phenazines can act as alternate electron acceptors for cellular metabolism, this provided an early clue to the role of redox conditions in determining biofilm architecture. The introduction to this thesis (Chapter 1) provides an overview of observations in P. aeruginosa and other microbes, drawing parallels between the physiology of colony biofilm development across phylogeny and highlighting specific preliminary studies that hint at redox-sensing mechanisms and signaling pathways that drive community morphogenesis. The associated Appendix A examines the effects of CORM-2, a synthetic compound that releases the respiratory poison carbon monoxide, on P. aeruginosa biofilm development. The inhibitory effects of CORM-2 are ameliorated by reducing agents and increased availability of electron donors for P. aeruginosa metabolism. Chapter 2 describes the foundational characterization of the P. aeruginosa PA14 colony morphology assay model, which showed that colony wrinkling is invoked under high intracellular NADH levels and electron acceptor-limiting conditions, suggesting that it is an adaptive strategy to increase access to electron acceptor. The associated Appendices B and C describe (i) a mathematical modeling approach demonstrating that wrinkle geometry is indeed optimized for efficient access to electron acceptors, and (ii) a study investigating the effects of phenazine antibiotics on the multicellular development of a eukaryotic microbe. Chapter 3 details the identification and characterization of a candidate mediator of the multicellular response to electron acceptor availability in PA14 called RmcA. RmcA contains domains that have been implicated in redox-sensitive developmental control in eukaryotic systems and domains that modulate intracellular levels of cyclic di-GMP (c-di-GMP). C-di-GMP is an important secondary messenger that controls social behaviors, including the secretion of factors required for colony biofilm structure formation, in diverse bacteria. RmcA thus bridges the gap between sensing of redox signals and colony morphogenesis. Appendix D outlines my approaches to purification and attempts to crystallize this and one other protein contributing to PA14 redox-driven colony morphogenesis. Finally, Appendix E describes the role of another protein that modulates c-di-GMP in response to metabolite-dependent signaling and physiological effects during interactions between P. aeruginosa and the fungus C. albicans. Together, the findings presented in this thesis have expanded our knowledge about the role that redox chemistry plays in biofilm development.

氧自由基曾被認為是有氧代謝过程的無用副產物。然而，在正常條件下，氧自由基也可以修飾蛋白質的結構與功能，同時能在多種細胞代謝過程中作為重要的信號分子。半胱氨酸的巰基極易被氧自由基、氮自由基以及其它的親電子分子所氧化，且其可逆的氧化反應對於與氧化還原相關的調控與信號傳遞是極其重要的。雖然僅有特定的一小部分半胱氨酸可以被氧化修飾，我們至今對於影響半胱氨酸對氧化還原敏感性的决定因素所知甚少。對於蛋白质半胱氨酸氧化修饰的比較分析及預測，不仅可以提高我們對半胱氨酸氧化還原敏感性的决定因素的認識，同時也能對後續关于重要的氧化還原敏感蛋白的實驗驗證起指導作用。 / 本研究中，本人主要做了如下三部分的工作：第一部分， RedoxDB數據庫的構建。这也是首個針對已被实验验证的蛋白氧化還原修飾的數據庫。第二部分，氧化還原敏感的半胱氨酸位點的特征分析及預測。本人分析了基於序列的各種可能與半胱氨酸氧化還原敏感性相關的特征，並發現其中三個特征可用於預測氧化還原敏感的半胱氨酸位點。基於這三個特征，我開發了RSCysPred 一个基於支持向量機且只依赖蛋白序列的預測氧化還原敏感的半胱氨酸位點的新方法。第三部分，可逆二硫鍵的分析。可逆二硫鍵的形成是蛋白質可逆氧化還原修飾的最主要的類型之一。我从RedoxDB中获取相关数据，然后針對功能位點標簽以及靜電特征等方面的特征，對可逆二硫鍵與結構二硫鍵進行了詳盡的比較分析。結果表明相比結構二硫鍵，可逆二硫鍵具有一些显著不同的特征。进一步的分析显示這些特征可用於可逆二硫鍵的預測。 / Reactive oxygen species (ROS) has been regarded as unwanted by-product of aerobic metabolism. However, under normal conditions, ROS can modify the structure and function of proteins and may also act as important signaling molecules in various cellular processes. Cysteine thiol groups of proteins are particularly susceptible to oxidation by ROS/RNS and other electrophilic molecules, and their reversible oxidation is of critical roles for redox regulation and signaling. Despite the fact that only a small fraction of cysteine residues undergone oxidative modification, the determinants of cysteine redox-sensitivity is poorly understood to date. Comparative analyses and computational prediction of protein cysteine oxidative modification will not only enhance our understanding about the determinants of cysteine redox-sensitivity, it may also facilitate further experimental investigation of important redox proteins. / This thesis includes the following three parts of work. Part I: Construction of RedoxDB - a curated database of protein oxidative modification. It is the first database for experimentally verified protein oxidative modification events. Part II: Feature analysis and computational prediction of redox-sensitive cysteines. I analyzed various sequence-based features potentially related to cysteine redox-sensitivity, and identified three features for efficient computational prediction of redox-sensitive cysteine. Based on these features, I developed RSCysPred, a SVM-based tool for predicting redox-sensitive cysteine using primary protein sequence only. Part III: Study on reversible disulfide formation, which is the major type of protein oxidative modification. By using data retrieved from RedoxDB, I performed extensive comparison between reversible disulfide-bonded cysteines and structural disulfide-bonded cysteines with regard to the functional site signatures and electrostatic properties. The results support that reversible disulfides show several remarkable differences compared with structural disulfides. Further analyses indicated that these features could be used for efficient prediction of reversible disulfide. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Sun, Mingan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 77-89). / Abstract also in Chinese. / Thesis/Assessment Committee --- p.I / Statement: --- p.I / Abstract of thesis entitled --- p.II / 摘要 --- p.IV / Acknowledgements --- p.V / Table of contents --- p.VI / List of Tables --- p.IX / List of Figures --- p.X / List of Abbreviations --- p.XI / CHAPTER 1 --- p.1 / General Introduction --- p.1 / Chapter 1.1 --- Generation of ROS and oxidative stress --- p.2 / Chapter 1.2 --- Protein cysteine oxidative modification --- p.3 / Chapter 1.2.1 --- Protein oxidative modification --- p.3 / Chapter 1.2.2 --- Special properties of cysteine --- p.3 / Chapter 1.2.3 --- Reversible cysteine oxidation and its function --- p.4 / Chapter 1.2.4 --- Specificity of cysteine oxidative modification --- p.5 / Chapter 1.3 --- Experimental approaches for detecting protein oxidative modification --- p.6 / Chapter 1.4 --- Computational prediction of protein oxidative modification --- p.7 / Chapter 1.5 --- Project Objectives --- p.8 / CHAPTER 2 --- p.11 / RedoxDB: a Curated Database of Experimentally Verified Protein Oxidative Modification --- p.11 / Chapter 2.1 --- Introduction --- p.12 / Chapter 2.2 --- Database concept --- p.13 / Chapter 2.3 --- Database interface and tools --- p.15 / Chapter 2.4 --- Discussion --- p.16 / CHAPTER 3 --- p.23 / Computational Prediction of Redox-Sensitive Cysteines Using Sequential Distance and Other Sequence-based Features --- p.23 / Chapter 3.1 --- Introduction --- p.24 / Chapter 3.2 --- Methods and Materials --- p.26 / Chapter 3.2.1 --- Datasets --- p.26 / Chapter 3.2.2 --- Feature extraction --- p.28 / Chapter 3.2.3 --- Support vector machines (SVMs) implementation and parameter optimization --- p.29 / Chapter 3.2.4 --- Performance assessment --- p.30 / Chapter 3.2.5 --- Statistical analyses --- p.30 / Chapter 3.3.6 --- Webserver implementation --- p.30 / Chapter 3.3 --- Results --- p.31 / Chapter 3.3.1 --- Performance evaluation by 10-fold cross-validation --- p.31 / Chapter 3.3.2 --- Evaluation of the most efficient features --- p.33 / Chapter 3.3.3 --- Comparison with current structure-based method --- p.34 / Chapter 3.3.4 --- Performance evaluation using OSCTdb --- p.36 / Chapter 3.4 --- Discussion --- p.37 / CHAPTER 4 --- p.49 / Comparative Analysis of Reversible and Structural Disulfides to Reveal Their Distinct Characteristics --- p.49 / Chapter 4.1 --- Introduction --- p.50 / Chapter 4.2 --- Materials and methods --- p.52 / Chapter 4.2.1 --- Datasets --- p.52 / Chapter 4.2.2 --- Functional site signatures --- p.53 / Chapter 4.2.3 --- Electrostatic properties --- p.55 / Chapter 4.2.4 --- Support vector machines (SVMs) implementation and parameter optimization --- p.56 / Chapter 4.2.5 --- Performance assessment --- p.56 / Chapter 4.2.6 --- Statistical analyses --- p.56 / Chapter 4.3 --- Results --- p.56 / Chapter 4.3.1 --- General features of the functional site signatures for reversible-SS Cys and structural-SS Cys --- p.56 / Chapter 4.3.2 --- Differences in amino acid composition are related to several physical-chemical properties --- p.58 / Chapter 4.3.3 --- Electrostatic characteristics of disulfide-bonded Cys --- p.60 / Chapter 4.3.4 --- Comparison of S-S distance --- p.61 / Chapter 4.3.5 --- Predictive power of newly identified features --- p.62 / Chapter 4.4 --- Discussion --- p.63 / Chapter 4.4.1 --- Reversible disulfides show distinct characteristics compared with structural disulfides --- p.63 / Chapter 4.4.2 --- Chances and challenges for computational prediction of reversible disulfides --- p.64 / CHAPTER 5 --- p.79 / Conclusions and Perspectives --- p.79 / Chapter 5.1 --- Contributions and conclusions from this thesis research --- p.80 / Chapter 5.2 --- Future perspectives --- p.81 / Reference --- p.82 / List of Publications --- p.95

Digitized by Kansas Correctional Industries

Tetraphenylporphyrins can be used to absorb visible light and pass on their excitation energy to electron transfer agents. The purpose of this research has been to investigate our ability to understand and control the energetics of porphyrin derivatives in order to use their electron transfer ability to harness the energy of sunlight. Shifts in the redox (reduction and oxidation) potentials of tetraphenylporphyrins result from variations in the substituents attached at the para- position of the phenyl rings of tetraphenylporphyrins, as well as variations in the state of ionization of those substituents, and the solvent in which the reactions are carried out. To measure the effect these variations, results from cyclic voltammetric experiments were plotted versus literature values of Hammett substituent constants to confirm the validity of linear free energy relationships as a model of substituent effects. Solvent effects on reduction potentials were correlated using experimentally determined values of the empirical solvent parameter ET. Some specific conclusions are summarized. 1. The usefulness of linear free energy relationships in correlating variations in redox potentials with changes in substituent was confirmed with two exceptions. Two of the porphyrins were shown to undergo a different electrochemical oxidation mechanism than the remaining porphyrins, and another porphyrin was shown to be more difficult to reduce than predicted on the basis of its substituent constant. 2. Solvent effects, here investigated as the effect of added water on the reduction potential of tetraaminophenylporphyrin in DMSO, were demonstrated to correlate with the Dimroth-Reichardt solvent parameter, ET , determined experimentally for each water-DMSO mix. 3. Variations in the state of ionization of ionizable substituents such as carboxylic acid, amine, and hydroxyl substituents were shown to affect porphyrin electrochemistry mostly through the protonation of bulk, solution-phase porphyrin by added proton donor. An additional effect of added proton donor was noted in an alteration in the mechanism of reduction to include some of a different mechanism wherein reduced porphyrin is protonated in a chemical equilibrium and then further reduced electrochemically.

In situ reduction of hydrido-tris-(3,5-dimethylpyrazolyl)borato(trioxo) rhenium(V) with triphenylphosphine or triethylphosphite leads to a reactive rhenium(V) species that catalytically deoxygenates epoxides at 75-105��C. The reaction is stereospecific, except for trans- and cis-butene oxide which formed minor amounts of the opposite isomer. A variety of different functional groups were tolerated and even epoxides that reacted slowly could be pushed to greater than 95% conversion given extended time and/or higher temperature. The absence of clustering processes shows how the choice of ligand can have a major influence on the design of the catalytic cycle. The rhenium(V) species formed from reduction of Tp'ReO��� was identified as Tp'Re(O)(OH)���. Tp'Re(O)(OH)��� reacted with ethanol and HCl to form ethoxide and hydroxo chloride complexes, respectively. In addition, Tp'Re(O)(OH)��� was an excellent catalytic and stoichiometric reagent for the deoxygenation of epoxides and sulfoxides. Loss of water from Tp'Re(O)(OH)��� to form the catalytically active species Tp'Re02 was shown to be a necessary preequilibrium process. The kinetic behavior of the catalytic system is complex. First-order behavior in [Re][subscript T], zero-order dependence in [PPh���] and saturation behavior for epoxide were observed. The reversible formation of a coordinated epoxide complex was proposed to explain the saturation behavior. The epoxide complex was shown experimentally and computationally to engage in two separate reactions: ring expansion to form a syn-diolate complex, and direct fragmentation to alkene and trioxide. A steady-state concentration of diolate is eventually reached explaining a "burst" of alkene production prior to generation of a pseudo-zero-order catalytic system. The diolate formed is the syn-isomer, which is the kinetically formed product. Direct epoxide fragmentation is the primary source of alkene. This process was determined to be four times faster than ring expansion for cis-stilbene oxide. The synthesis and characterization of a tethered-epoxide Cp* rhenium trioxide complex has been achieved. Reduction of this complex leads to an unsaturated rhenium(V) species that is immediately complexed by the tethered epoxide. Experimental data and molecular mechanics modeling support intramolecular coordination of the epoxide to the rhenium center. These results confirm that the coordinate epoxide is a viable intermediate in rhenium-catalyzed epoxide deoxygenations. / Graduation date: 2003

Graduation date: 1995

An investigation has been made of the role of the four disulfide bonds of bovine pancreatic ribonuclease in maintaining the protein in a biologically active form. Studies were carried out to determine the effect of reductive cleavage of these bonds on the ability of the enzyme to catalyze the hydrolysis of ribonucleic acid. The appearance of sulfhydryl groups was taken as evidence that reduction of the protein had occurred. <P> No significant reduction or loss of enzymic activity of ribonuclease could be demonstrated when the protein was treated with the reducing agents sodium or potassium borohydride in aqueous solution at room temperature,pH 8. <P> Thioglycolic acid was found to be an effective agent for reducing ribonuclease disulfide bonds. At room temperature, in an aqueous solution containing a Large excess of thioglycolate over protein, reduction proceeded slowly at pH 8. At the end of five hours, approximately one disulfide bond was broken, with the loss of 20 per cent of the original enzyme activity. The addition of urea greatly facilitated reduction. The rate of reduction was especially rapid in solutions of urea concentration greater than 4 molar. In 8 M urea at pH 8, treatment of ribonuclease with thioglycolate resulted in the complete loss of enzymic activity in one half hour, with the simultaneous rupture of two or three disulfide bonds. Under these conditions, maximum reduction was achieved in approximately two hours, with cleavage of between three and four disulfide bonds per molecule. In the pH range from 3 to 10, rate of activity loss was most rapid at pH 10, slightly less rapid at pH 3, and reached a minimum near pH 5. The effects of pH and urea were additive in that the maximum rate of inactivation occurred at pH 10 in 8 M urea (97 per cent activity loss in 10 minutes), and the minimum rate st pH 5 in the absence of urea (20 per cent loss in 28 hours). <P> Inactivation was markedly inhibited by phosphate ions. A solution of protein which was O.36 M in phosphate at pH 8 lost activity very slowly when treated with thioglycolic acid, even in the presence of 4 M urea. These findings, together with the observation of other workers that polyvalent ions such as phosphate reverse the denaturetion of ribonuclease in urea, suggest that phosphate inhibits reduction by stabilizing the protein in its native configuration, whereas urea facilitates reduction by denaturing the protein. <P> Air oxidation of fully or partially inactivated protein resulted, in some cases, in the recovery of up to 4O per cent of the enzyme activity which had been lost as a result of reduction. <P> The relation between loss of activity and reduction was analyzed by correlating the data obtained in those experiments in which both the sulfhydryl concentration and the enzymic activity of samples of modified protein were determined. The experiments were carried out under a variety of conditions of pH and urea concentration. On the basis of these data, it is concluded that the inactivation of thioglycolate-treated ribonuclease is probably not a unique function of extent of reduction, but depends in part on the method by which the reduction is achieved.

Thesis (Ph. D.)--University of Texas at Austin, 2003. / Vita. Includes bibliographical references. Available also from UMI Company.