The goal of this project is to characterize mechanisms by which cardiolipin (CL) binding affects the structure and function of three mitochondrial electron transport complexes: cytochrome c oxidase, cytochrome bc1, and NADH dehydrogenase (Complex I). Our understanding of CL's role within each complex is at different stages. The purpose of our proposed experiments is to determine the function of CL within each complex and to define a possible universal binding motif. Cardiolipin binding to cytochrome c oxidase is well defined and the binding sites have been localized within the enzyme. The goal with cytochrome c oxidase is to determine whether CL facilitates proton entry into the binuclear center of cytochrome c oxidase and to identify the functional mechanism. Cardiolipin binding to cytochrome bc1 is less well defined. CL is tightly bound to cyt bc1 and is critically required for structural stability and maintenance of activity. However, the binding sites within cyt bc1 isolated from higher eukaryotes are not defined. We propose to map the 4-6 binding sites within Cyt bc1 to determine the subunits and amino acid sequences participating in CL binding. Identified binding sites will be compared with the known PL binding sites within yeast cyt bc1 and bovine heart cytochrome c oxidase. Phospholipid binding to Complex I has not been defined. We have found that 2 CL and 2 PE are tightly associated with the purified enzyme, but their structural and functional importance has not been established. We will determine if CL is an essential component of Complex I and map its location within the complex.
These specific aims are logical extensions of our previous studies. They take advantage of experimental approaches developed during past funding periods, i.e., 1) synthesis of arylazido-CL photolabels;2) analysis of subunit content using sensitive HPLC and mass spectrometry methods;and 3) selective dissociation of subunits using mild structural perturbants. With our cardiolipin analogues and experimental approaches, we are in a unique position to define the mechanisms by which tightly bound CL influences the function and/or structure of three key enzymes within the mitochondrial inner membrane.Public Health Relevance. Cardiolipin is a unique phospholipid of the mitochondrial inner membrane. Oxidative damage or age related loss of cardiolipin is associated with apoptosis and a number of diseases, e.g., ischemia/reperfusion injury and age-related degeneration. In this research project we will determine the cardiolipin requirements of three inner membrane electron transfer complexes and map the cardiolipin binding sites within each complex. Successful completion of this project will determine the absolute requirement for cardiolipin and help us understand its pathophysiological importance.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM024795-29
Application #
7993099
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Chin, Jean
Project Start
1978-01-01
Project End
2012-11-30
Budget Start
2010-12-01
Budget End
2012-11-30
Support Year
29
Fiscal Year
2011
Total Cost
$305,645
Indirect Cost
Name
University of Texas Health Science Center San Antonio
Department
Biochemistry
Type
Schools of Medicine
DUNS #
800772162
City
San Antonio
State
TX
Country
United States
Zip Code
78229
Musatov, Andrej; Varhač, Rastislav; Hosler, Jonathan P et al. (2016) Delipidation of cytochrome c oxidase from Rhodobacter sphaeroides destabilizes its quaternary structure. Biochimie 125:23-31
Musatov, Andrey; Siposova, Katarina; Kubovcikova, Martina et al. (2016) Functional and structural evaluation of bovine heart cytochrome c oxidase incorporated into bicelles. Biochimie 121:21-8
Musatov, Andrej; Robinson, Neal C (2014) Bound cardiolipin is essential for cytochrome c oxidase proton translocation. Biochimie 105:159-64
Musatov, Andrej; Fabian, Marian; Varhač, Rastislav (2013) Elucidating the mechanism of ferrocytochrome c heme disruption by peroxidized cardiolipin. J Biol Inorg Chem 18:137-44
Musatov, Andrej (2013) Dual effect of heparin on Fe²⁺-induced cardiolipin peroxidation: implications for peroxidation of cytochrome c oxidase bound cardiolipin. J Biol Inorg Chem 18:729-37
Musatov, Andrej; Robinson, Neal C (2012) Susceptibility of mitochondrial electron-transport complexes to oxidative damage. Focus on cytochrome c oxidase. Free Radic Res 46:1313-26
Sedlak, Erik; Fabian, Marian; Robinson, Neal C et al. (2010) Ferricytochrome c protects mitochondrial cytochrome c oxidase against hydrogen peroxide-induced oxidative damage. Free Radic Biol Med 49:1574-81
Sedlak, Erik; Robinson, Neal C (2009) Sequential dissociation of subunits from bovine heart cytochrome C oxidase by urea. Biochemistry 48:8143-50
Varhac, Rastislav; Robinson, Neal C; Musatov, Andrej (2009) Removal of bound Triton X-100 from purified bovine heart cytochrome bc1. Anal Biochem 395:268-70
Lemma-Gray, Patrizia; Valusova, Eva; Carroll, Christopher A et al. (2008) Subunit analysis of bovine heart complex I by reversed-phase high-performance liquid chromatography, electrospray ionization-tandem mass spectrometry, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Anal Biochem 382:116-21

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