<p> In this work the redox properties of cytochrome c nitrite reductase (CcNiR), a decaheme homodimer that was isolated from <i>S. oneidensis,</i> were determined in the presence and absence of the strong-field ligands cyanide and nitrite. Four hemes per CcNiR protomer are hexa-coordinate with tightly bound axial histidines, while the fifth (active site) has one tightly bound lysine and a distal site that can be open, or contain exogenous ligands such as the substrate nitrite. Controlled potential electrolysis in combination with UV/visible absorption (UV-vis) and electron paramagnetic resonance (EPR) spectroscopies allowed for assignment of all heme midpoint potentials under each set of conditions. The studies show that the active-site heme is the first to be reduced under all conditions. The midpoint redox potential of that heme shifts approximately 70mV to the positive upon binding a strong field ligand such as nitrite or cyanide. When controlled potential electrolysis was carried out in the presence of nitrite, a concerted two electron reduction was observed by UV-vis, and a {Fe(NO)}<sup>7</sup> reduced product was revealed in EPR. In addition, an asymmetry in ligand binding between active sites was revealed. This information is relevant for the interpretation of planned and ongoing mechanistic studies of CcNiR. </p><p> Over-expression, partial purification and characterization of another <i> S. oneidensis</i> multiheme enzyme, known as octaheme tetrathionate reductase (OTR), is also described herein. Though of unknown cellular function, OTR was previously reported to have tetrathionate reductase activity, in addition to nitrite and hydroxylamine reductase activities. The new results indicate that the expression of OTR has no effect on tetrathionate or nitrite reductase activities in the whole cell lysate, and only hydroxylamine reductase activity was substantially elevated in the overexpressing bacteria. OTR was stable in buffered solutions, but substantial activity loss during all attempts at column chromatography was a major obstacle to the complete purification. OTR also proved quite hydrophobic, so possible membrane association should be considered in future attempts to purify this protein. </p><p> Finally, this dissertation also reports attempts to improve <i> S. oneidensis'</i> ability to express foreign proteins. Though ideally suited to expressing c-hemes, it proved difficult to express carboxy his-tagged proteins in <i>S. oneidensis</i> because of persistent tag degradation. Attempts to knock out lon protease, a cytoplasmic carboxypeptidase, as well as the result of redirecting ccNiR from the SecA to the possibly more protected signal particle recognition (SRP) secretion pathway, are described. </p><p> Iron heme cofactors are single-electron transport moieties that play a crucial role in respiration. While oxygen is the electron acceptor of choice in aerobic atmospheres, microorganisms that live in anaerobic environments utilize other molecules with similarly high reduction potentials. <i> S. oneidensis</i> can utilize numerous terminal electron acceptors, including nitrite, dimethylsulfoxide and even uranium, thanks to a particularly rich array of multi c-heme respiratory proteins. Understanding of how the midpoint potentials and heme arrangements within the proteins influence these exotic respiratory processes is of interest in the fields of bioremediation and fuel development.</p>
| Identifer | oai:union.ndltd.org:PROQUEST/oai:pqdtoai.proquest.com:3685085 |
| Date | 10 March 2015 |
| Creators | Stein, Natalia |
| Publisher | The University of Wisconsin - Milwaukee |
| Source Sets | ProQuest.com |
| Language | English |
| Detected Language | English |
| Type | thesis |
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