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Peroxynitrite and Mitochondrial Cytochromes

Lopez Manzano, Elisenda (2011) Peroxynitrite and Mitochondrial Cytochromes. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Abstract

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.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Lopez Manzano, Elisendaelisendalopez@gmail.com
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairPeterson, Jamesjpp16@pitt.eduJPP16
Committee MemberPitt, Bruce Rbrucep@pitt.eduBRUCEP
Committee MemberFabisiak, James Pfabs@pitt.eduFABS
Committee MemberPearce, Linda Llip10@pitt.eduLIP10
Committee MemberGladwin, Mark Tgladwinmt@upmc.eduMTG16
Committee MemberHendrich, Michael Phendrich@andrew.cmu.edu
Date: 29 June 2011
Date Type: Completion
Defense Date: 15 March 2011
Approval Date: 29 June 2011
Submission Date: 5 April 2011
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Institution: University of Pittsburgh
Schools and Programs: Graduate School of Public Health > Environmental and Occupational Health
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: BPAEC; carbonate radical; Complex III; complex IV; cytochrome c; cytochrome oxidase; cytochrome reductase; endothelial cells; mitochondria; oxidative stress; peroxynitrite; superoxide
Other ID: http://etd.library.pitt.edu/ETD/available/etd-04052011-110334/, etd-04052011-110334
Date Deposited: 10 Nov 2011 19:34
Last Modified: 19 Dec 2016 14:35
URI: http://d-scholarship.pitt.edu/id/eprint/6786

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