The goal of this project is to characterize the mechanisms by which cardiolipin (CL) binding affects the structure and function of two mitochondrial electron transport complexes: cytochrome C oxidase (COX) and cytochrome bc1 (Cyt. bc1). The specific goals during the project period are: Functional and Structural Association of Cardiolipin with Cytochrome C Oxidase 1. Identify amino acid sequences within COX subunits VIa, VIb, VIla and VIc that bind cardiolipin. 2. Determine the effect of CL removal upon COX conformational flexibility and/or binuclear center reactivity 3. Establish whether CL is Directly Involved in Subunit VIa & VIb Binding to COX 4. Determine the mechanism by which CL removal inhibits proton translocation by COX Functional and Structural Association of Cardiolipin with Cytochrome bci 1. Identify subunit(s) and amino acid sequence(s) of cyt. bc1 that participate in CL binding 2. Measure affinity and stoichiometry of cardiolipin binding to cytochrome bc1 These specific aims are each a logical extension of our previous studies. They also take advantage of new experimental approaches developed during the past funding period, i.e. 1) synthesis of CL derivatives that contain a variety of chemically reactive functional groups; 2) new sensitive HPLC and ESI/MS methods for analyzing subunit content and molecular masses of each subunit; 3) new methods for accurately measuring the self-association of each complex. With our unique CL analogues and approaches, we can map the CL binding sites within each complex and determine mechanisms by which tightly bound CL influences enzyme function and/or structure. Results obtained with these two complexes will allow us to determine whether CL has unique or universal roles in coupled electron transport.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM024795-23
Application #
6476334
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Chin, Jean
Project Start
1978-01-01
Project End
2004-11-30
Budget Start
2001-12-01
Budget End
2002-11-30
Support Year
23
Fiscal Year
2002
Total Cost
$289,000
Indirect Cost
Name
University of Texas Health Science Center San Antonio
Department
Biochemistry
Type
Other Domestic Higher Education
DUNS #
800772162
City
San Antonio
State
TX
Country
United States
Zip Code
78229
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; Varha?, Rastislav; Hosler, Jonathan P et al. (2016) Delipidation of cytochrome c oxidase from Rhodobacter sphaeroides destabilizes its quaternary structure. Biochimie 125:23-31
Musatov, Andrej; Robinson, Neal C (2014) Bound cardiolipin is essential for cytochrome c oxidase proton translocation. Biochimie 105:159-64
Musatov, Andrej; Fabian, Marian; Varha?, 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
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
Sedlák, Erik; Robinson, Neal C (2009) Sequential dissociation of subunits from bovine heart cytochrome C oxidase by urea. Biochemistry 48:8143-50
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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