School of Chemistry - Theses
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ItemNo Preview AvailableBiochemical Characterisation of C. elegans Ferritins( 2022)Ferritin plays an important role in maintaining optimum cellular iron concentrations. Consisting of 24 protein subunits in a spherical architecture, it creates an interior cavity that stores iron atoms in the form of a ferric oxy-hydroxide. Extensive in-vitro enzymological studies have greatly assisted our understanding but it is not yet fully understood how iron is released from ferritin in-vivo. A clearer understanding could aid iron related disorders, including iron overload or deficiency. The use of the model organism Caenorhabditis elegans will allow genetic manipulation to further understand the mechanisms involved in iron metabolism within a whole organism. C. elegans expresses two types of ferritin, FTN-1 and FTN-2 which have not been biochemically characterised. The aim of this thesis is to establish the mechanism of iron storage for these proteins to develop strategies to control and manipulate the protein for in vivo and ex vivo studies. C. elegans ferritins have been expressed, purified, and characterised through a range of techniques. Both ferritins show ferroxidase activity but with L-type ferritin nucleation properties. Even with identical ferroxidase active sites, FTN-2 is shown to react ~10 times faster than FTN-1. Combined structural and stopped-flow spectroscopy studies revealed the source of the difference in rates to be iron transport to the ferroxidase site. Both FTN-1 and FTN-2 react initially to form a mu-1,2-peroxo diferric intermediate that absorbs at 595 nm. Structural comparison showed an asparagine residue (Asn106) near the ferroxidase active site, which is a valine in most other ferritins including human H ferritin. The mu-1,2-peroxo diferric intermediate of human H ferritin absorbs at 665 nm and replacement of Asn106 to valine in FTN-2 shows a similar absorption. This may explain the variation in wavelength of the peroxide-to-iron(III) charge transfer band observed for ferritin across taxa.
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ItemStructural and functional characterisation of bacterial proteins that act in the utilisation of arsenic oxyanions for respiration( 2022)Arsenic is a toxic metalloid present as a contaminant in drinking water, affecting more than 140 million people worldwide. Ingestion of arsenic as the soluble oxyanions arsenite and arsenate, is linked to neurological, reproductive and respiratory disorders, cancer and diabetes mellitus. Although toxic to human health, arsenite and arsenate can be utilised by some prokaryotes as sources of energy from the environment. Such prokaryotes are Pseudorhizobium banfieldiae sp. str. NT-26 (NT-26) and Chrysiogenes arsenatis (C.arenatis) respire using these oxyanions by the actions of arsenite oxidase (AioAB) and arsenate reductase enzymes (ArrAB), respectively. The focus of the present work details structural and functional characterisation of proteins that allow these bacteria to use arsenic oxyanions for respiration. This includes characterisation of periplasmic binding proteins, ArrX and AioX that bind to arsenic oxyanions, the interaction of these proteins with the sensor histidine kinase, AioS and the respiratory arsenite oxidase enzyme, AioAB, and its interaction with its electron acceptor, cytochrome c552. The substrate specificity of periplasmic binding proteins, AioX and ArrX to arsenite and arsenate, from NT-26 and C.arsenatis, respectively were investigated. The aims of this study were to establish how both these proteins distinguish between arsenite and arsenate, and to determine the structure of ArrX protein in an apo- and arsenate-bound state. The X-ray crystal structure of ArrX with arsenate was determined to 1.74 Angstrom resolution. Structural comparison of the AioX and the ArrX proteins and isothermal titration calorimetry (ITC) analyses of mutant proteins identified a conserved Cys residue in their substrate binding sites that play a key role in the discrimination between arsenite and arsenate for both proteins. Structural analyses also revealed that the nature of neighbouring residues (Gly in AioX and Thr in ArrX) may provide varied structural flexibilities that contribute to the differential interaction of the conserved Cys residue to arsenic oxyanions. The biophysical characterisation of the interaction between the AioX protein and its sensor histidine kinase AioS was performed to investigate the processes that control the expression of the AioAB enzyme in NT-26. Size exclusion chromatography (SEC) and analytical ultracentrifugation (AUC) experiments revealed that AioX and AioS proteins can form a stable heterodimer complex in the absence of arsenite. These findings also revealed that the oligomeric state of the complex does not change in the presence of arsenite. A loop in the AioX protein, which was proposed to be involved in the interaction with AioS was shown experimentally not to directly participate in the interface between the two proteins. Structural and functional characterisation of the interaction between the AioAB enzyme with its electron acceptor cytochrome c552 was carried out to investigate the structural basis of the electron transfer process that underpins the respiration of Pseudorhizobium banfieldiae sp. str. NT-26. The crystal structure of the AioAB/cytc552 was determined to 2.25 Angstrom resolution. The structure showed two AioA2B2/(cytc552)2 complexes per asymmetric unit. Three of the four AioAB/cytc552 complexes revealed that the cytc552 molecule docks in a cleft at the interface of between the AioA and AioB subunits. The positioning of the cytc552 proteins in these complexes revealed an edge-edge distance of 7.5 Angstrom between the heme of cytc552 and the Rieske 2Fe-2S cluster in the AioB subunit, which is considered an ideal distance for fast electron transfer between proteins. The interface of the AioAB and the cytc552 protein also shows electrostatic interactions and is stabilised by two salt bridges, a classic example of transient complex formation for fast electron transfer. Additionally, the structure also highlights the unique positioning of one of the four cytc552 proteins that sit at a long distance from the redox cofactors of AioAB subunits and presumably aid in crystallisation. Enzyme kinetics analysis also revealed the AioAB/cytc552 system to be catalytically very efficient.
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ItemThe Synthesis, Structure and Properties of Anionic Metal–Tetraoxolene Coordination Polymers( 2021)Electrically conductive, magnetically ordered, and electrochemically active materials are important components for data processing technologies, energy conversion and storage devices, and sensors and optoelectronics. The synthesis of compounds with these properties is therefore an important scientific endeavour to meet the progressively more demanding sustainability and performance targets. Crystalline coordination polymers have recently been proposed as candidate materials to achieve these aims. Coordination polymers can exhibit synergy between electrical conductivity, magnetic ordering and electroactivity, coupled with porosity, flexibility, and tuneable architecture and functionality, not achievable using traditional inorganic materials. This thesis describes studies into anionic tetraoxolene coordination polymers constructed using ligands derived from 3,6-disubtitued-2,5-dihydroxy-1,4-benzoquinones (H2Xan, X = F, Cl, Br; anilic acids), and redox-active viologen countercations. Despite the desirable properties of tetraoxolene coordination polymers previously described in literature, the impact that viologen countercations have upon the structure and properties of tetraoxolene coordination polymers has never been explored. Chapter 2 first presents structural studies on a range of Mn(II) and Cd(II) coordination polymers synthesised using diamagnetic fluoranilate (Fan2-) and chloranilate (Clan2-) ligands and redox-active N,N'-dimethyl-4,4'-bipyridinium (MeV2+) countercations. The structural disparity observed between Fan2- and Clan2- compounds demonstrates the inherent sensitivity of reactions to supramolecular interactions between cations, metals, ligands, and solvents. It also highlights the structural unpredictability associated with the use of viologen cations, compared to redox-inactive alkylammonium countercations. Structural studies on mixed-valence Fe(III)-tetraoxolene coordination polymers containing viologen countercations are subsequently presented, showing resultant anionic tetraoxolene network topology remains difficult to predict. However localisation of the tetraoxolene radical trianion (Xan3-) state is consistently observed, caused by charge-transfer (CT) and Coulombic interactions between the pi-electron deficient viologen cations, and the pi-electron rich anionic networks. This reveals the potential for viologens to modulate the charge-state distribution in mixed-valence tetraoxolene coordination polymers. In Chapter 3, 5,6-dihydropyrazino[1,2,3,4-lmn][1,10]-phenanthrolinediium (PhenQ2+) countercations are incorporated into an isostructural family of mixed-valence 2D Fe(III)-tetraoxolene frameworks (PhenQ)[Fe2(Xan)3] (X = F, Cl, Br). As for the mixed-valence networks obtained in Chapter 2, CT interactions between PhenQ2+ cations and the anionic [Fe2(Xan)3]2- networks cause localisation of the Xan3- state and partial ligand charge-ordering. Spectroscopic studies on (PhenQ)[Fe2(Xan)3].yDMF showed the network-cation interactions impact upon the electronic structure of the material. The inclusion of PhenQ2+ gives rise to additional framework redox states and modifies the conductive and magnetic properties of the material, compared to tetraoxolene frameworks with only redox-inactive countercations. Chapter 4 investigates a family of mixed-valence Fe-fluoranilate frameworks containing MeV2+ countercations, (MeV)[Fe2(Fan)3].solvent (solvent = MeCN, DMF). X-ray diffraction studies reveal the compounds undergo a range of slight structural distortions upon changing the guest solvent. Remarkably, these guest-induced structure distortions cause partial switching of the Fe valence state, never before observed in Fe-tetraoxolene coordination polymers. The guest-induced Fe valence fluctuation in turn modifies the electronic spectra and structure of the frameworks, with resultant switching of the transport properties. In Chapter 5, the mixed-valence Fe-tetraoxolene framework (MeV)[Fe2(Clan)3] undergoes a surprising structural transformation upon lattice solvent loss, causing the isolated compound to exhibit a previously unobserved cation-network packing arrangement. This dramatic structure transformation results in changes to the electronic spectrum of the compound, as well as the electrochemical response and transport properties of the material, compared to topologically similar Fe-tetraoxolene frameworks containing redox-inactive cations. These results demonstrate the impact of structure packing and supramolecular interactions on the network electronic structure. This provides encouragement that the physicochemical properties of the compounds may be tailored by controlling these supramolecular interactions. Finally, Chapter 6 presents a summary of the results obtained in this thesis, highlights the overarching themes of this research, and puts forth possible future research directions for the field.