title: Structural Changes and Proapoptotic Peroxidase Activity of Cardiolipin-Bound Mitochondrial Cytochrome c.
abstract: The cellular process of intrinsic apoptosis relies on the peroxidation of mitochondrial lipids as a critical molecular signal. Lipid peroxidation is connected to increases in mitochondrial reactive oxygen species, but there is also a required role for mitochondrial cytochrome c (cyt-c). In apoptotic mitochondria, cyt-c gains a new function as a lipid peroxidase that catalyzes the reactive oxygen species-mediated chemical modification of the mitochondrial lipid cardiolipin (CL). This peroxidase activity is caused by a conformational change in the protein, resulting from interactions between cyt-c and CL. The nature of the conformational change and how it causes this gain-of-function remain uncertain. Via a combination of functional, structural, and biophysical experiments we investigate the structure and peroxidase activity of cyt-c in its membrane-bound state. We reconstituted cyt-c with CL-containing lipid vesicles, and determined the increase in peroxidase activity resulting from membrane binding. We combined these assays of CL-induced proapoptotic activity with structural and dynamic studies of the membrane-bound protein via solid-state NMR and optical spectroscopy. Multidimensional magic angle spinning (MAS) solid-state NMR of uniformly (13)C,(15)N-labeled protein was used to detect site-specific conformational changes in oxidized and reduced horse heart cyt-c bound to CL-containing lipid bilayers. MAS NMR and Fourier transform infrared measurements show that the peripherally membrane-bound cyt-c experiences significant dynamics, but also retains most or all of its secondary structure. Moreover, in two-dimensional and three-dimensional MAS NMR spectra the CL-bound cyt-c displays a spectral resolution, and thus structural homogeneity, that is inconsistent with extensive membrane-induced unfolding. Cyt-c is found to interact primarily with the membrane interface, without significantly disrupting the lipid bilayer. Thus, membrane binding results in cyt-c gaining the increased peroxidase activity that represents its pivotal proapoptotic function, but we do not observe evidence for large-scale unfolding or penetration into the membrane core.
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
authors.forename: Abhishek
authors.initials: A
authors.lastname: Mandal
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
authors.forename: Cody L
authors.initials: CL
authors.lastname: Hoop
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
authors.forename: Maria
authors.initials: M
authors.lastname: DeLucia
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
authors.forename: Ravindra
authors.initials: R
authors.lastname: Kodali
authors.affiliation: Departments of Environmental and Occupational Health, Chemistry, Pharmacology, and Chemical Biology and Center for Free Radical and Antioxidant Health, University of Pittsburgh, Pittsburgh, Pennsylvania.
authors.forename: Valerian E
authors.initials: VE
authors.lastname: Kagan
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
authors.forename: Jinwoo
authors.initials: J
authors.lastname: Ahn
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania. Electronic address: pvdwel@pitt.edu.
authors.forename: Patrick C A
authors.initials: PC
authors.lastname: van der Wel
mesh_terms: D017209:Apoptosis / Q000502:physiology*; D066241:Carbon-13 Magnetic Resonance Spectroscopy; D002308:Cardiolipins / Q000378:metabolism*; D045304:Cytochromes c / Q000378:metabolism*; D004926:Escherichia coli; D008051:Lipid Bilayers / Q000737:chemistry; D008928:Mitochondria / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D009195:Peroxidase / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000737:chemistry; D011487:Protein Conformation; D017550:Spectroscopy, Fourier Transform Infrared; D013057:Spectrum Analysis
chemical_list: D002308:Cardiolipins; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D045304:Cytochromes c; D009195:Peroxidase; C017251:1,2-oleoylphosphatidylcholine