Mitochondrial dysfunction, particularly in relation to electron transport chain (ETC) derived oxidative stress, is widely held to be important in numerous pathologies. However, mitochondrial levels of the bioenergetically critical small inorganic molecules are still debatable or unknown. Nevertheless, investigation of the behavior of the ETC components, individually and collectively, in response to varying the levels of these species is still of considerable importance. This dissertation investigated the reaction of the reduced forms of isolated bovine complex III, cytochrome c and complex IV with peroxynitrite in the presence and absence of CO2. The presence of CO2 significantly modulates the mechanisms and extent of the cofactor oxidations. The characteristics of peroxynitrite-modified ferricytochrome c, prepared in the presence and absence of CO2, was examined by a variety of spectroscopic methods. In the absence of CO2, oxidation of the methionine 80 axial heme ligand to methionine sulfoxide results. During complex IV turnover by native ferrocytochrome c at low ionic strength increased rates were observed when the peroxynitrite modified cytochrome c is added - indicating preferential binding of the modified cytochrome to a high affinity/low activity electron-entry site on the enzyme, directing native ferrocytochrome c to bind to a lower affinity/higher activity site. It is unclear that formation of small quantities of either peroxynitrite-modified cytochrome c is proapoptotic. Since the limiting reagent in the reaction of superoxide with nitric oxide is peroxynitrite, the amount of superoxide formed biologically is critical information. The commonly employed molecular probes for superoxide, hydroethidine, and its mitochondrially-targeted derivative, MitoSoxTM, were shown to undergo reactions with components of the mitochondrial ETC including reduction of complex IV and partial reduction of complex III. The reaction with complex IV accounts for an oxygen (and hence superoxide), independent fluorescent response of MitoSox™ in cultured endothelial cells. However, the cationic ethidium species formed during oxidation of the probes by the ETC enzymes inhibit the normal turnover of complex IV by blocking transfer of electrons from ferrocytochrome c to the oxidase. In the case of oxidized MitoSox™, the observed inhibition under typical assay conditions was substantial at inhibitor levels comparable to the concentration of substrate cytochrome c. Therefore, this work has special public health relevance since it not only reviews the possible mechanisms for oxidative stress in mitochondria but also reassesses the use of MitoSox™ as it is a net generator of superoxide.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-04052011-110334 |
| Date | 29 June 2011 |
| Creators | Lopez Manzano, Elisenda |
| Contributors | James Peterson, James P. Fabisiak, Michael P. Hendrich, Linda L. Pearce, Mark T. Gladwin, Bruce R. Pitt |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-04052011-110334/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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