Peroxidase activity and structural transitions of cytochrome c bound to cardiolipin-containing membranes.
During apoptosis, cytochrome c (cyt c) is released from intermembrane space of mitochondria into the cytosol where it triggers the caspase-dependent machinery. We discovered that cyt c plays another critical role in early apoptosis as a cardiolipin (CL)-specific oxygenase to produce CL hydroperoxides required for release of pro-apoptotic factors [Kagan, V. E., et al. (2005) Nat. Chem. Biol. 1, 223-232]. We quantitatively characterized the activation of peroxidase activity of cyt c by CL and hydrogen peroxide. At low ionic strength and high CL/cyt c ratios, peroxidase activity of the CL/cyt c complex was increased >50 times. This catalytic activity correlated with partial unfolding of cyt c monitored by Trp(59) fluorescence and absorbance at 695 nm (Fe-S(Met(80)) band). The peroxidase activity increase preceded the loss of protein tertiary structure. Monounsaturated tetraoleoyl-CL (TOCL) induced peroxidase activity and unfolding of cyt c more effectively than saturated tetramyristoyl-CL (TMCL). TOCL/cyt c complex was found more resistant to dissociation by high salt concentration. These findings suggest that electrostatic CL/cyt c interactions are central to the initiation of the peroxidase activity, while hydrophobic interactions are involved when cyt c's tertiary structure is lost. In the presence of CL, cyt c peroxidase activity is activated at lower H(2)O(2) concentrations than for isolated cyt c molecules. This suggests that redistribution of CL in the mitochondrial membranes combined with increased production of H(2)O(2) can switch on the peroxidase activity of cyt c and CL oxidation in mitochondria-a required step in execution of apoptosis.
Belikova
Natalia A
NA
Center for Free Radical and Antioxidant Health and Department of Environmental and Occupational Health, University of Pittsburgh, 100 Technology Drive, Suite 350, Pittsburgh, Pennsylvania 15219-3130, USA.
Vladimirov
Yury A
YA
Osipov
Anatoly N
AN
Kapralov
Alexandr A
AA
Tyurin
Vladimir A
VA
Potapovich
Maksim V
MV
Basova
Liana V
LV
Peterson
Jim
J
Kurnikov
Igor V
IV
Kagan
Valerian E
VE
D000165:Acridine Orange / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D002308:Cardiolipins / Q000378:metabolism* / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D045304:Cytochromes c / Q000737:chemistry* / Q000378:metabolism; D004586:Electrophoresis; D004789:Enzyme Activation; D005047:Etoposide / Q000378:metabolism / Q000494:pharmacology; D005452:Fluoresceins / Q000378:metabolism; D006736:Horses; D057927:Hydrophobic and Hydrophilic Interactions; D008055:Lipids / Q000737:chemistry / Q000494:pharmacology; D008081:Liposomes / Q000378:metabolism; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism
D002308:Cardiolipins; D005452:Fluoresceins; D008055:Lipids; D008081:Liposomes; D010713:Phosphatidylcholines; C065013:2',7'-dichlorodihydrofluorescein; D005047:Etoposide; D014364:Tryptophan; D045304:Cytochromes c; D009195:Peroxidase; C017251:1,2-oleoylphosphatidylcholine; D000165:Acridine Orange