skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Raman Spectroscopy Reveals Selective Interactions of Cytochrome c with Cardiolipin That Correlate with Membrane Permeability

Authors:
 [1];  [1]; ORCiD logo [1]; ORCiD logo [1]
  1. Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, Utah 84112, United States
Publication Date:
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1413574
Grant/Contract Number:
FG03-93ER14333
Resource Type:
Journal Article: Published Article
Journal Name:
Journal of the American Chemical Society
Additional Journal Information:
Journal Volume: 139; Journal Issue: 10; Related Information: CHORUS Timestamp: 2017-12-15 18:55:07; Journal ID: ISSN 0002-7863
Publisher:
American Chemical Society
Country of Publication:
United States
Language:
English

Citation Formats

Kitt, Jay P., Bryce, David A., Minteer, Shelley D., and Harris, Joel M. Raman Spectroscopy Reveals Selective Interactions of Cytochrome c with Cardiolipin That Correlate with Membrane Permeability. United States: N. p., 2017. Web. doi:10.1021/jacs.7b00238.
Kitt, Jay P., Bryce, David A., Minteer, Shelley D., & Harris, Joel M. Raman Spectroscopy Reveals Selective Interactions of Cytochrome c with Cardiolipin That Correlate with Membrane Permeability. United States. doi:10.1021/jacs.7b00238.
Kitt, Jay P., Bryce, David A., Minteer, Shelley D., and Harris, Joel M. Tue . "Raman Spectroscopy Reveals Selective Interactions of Cytochrome c with Cardiolipin That Correlate with Membrane Permeability". United States. doi:10.1021/jacs.7b00238.
@article{osti_1413574,
title = {Raman Spectroscopy Reveals Selective Interactions of Cytochrome c with Cardiolipin That Correlate with Membrane Permeability},
author = {Kitt, Jay P. and Bryce, David A. and Minteer, Shelley D. and Harris, Joel M.},
abstractNote = {},
doi = {10.1021/jacs.7b00238},
journal = {Journal of the American Chemical Society},
number = 10,
volume = 139,
place = {United States},
year = {Tue Mar 07 00:00:00 EST 2017},
month = {Tue Mar 07 00:00:00 EST 2017}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record at 10.1021/jacs.7b00238

Citation Metrics:
Cited by: 6works
Citation information provided by
Web of Science

Save / Share:
  • Detergent-solubilized bovine heart cytochrome c oxidase requires 2 mol of tightly bound cardiolipin (CL) per mole of monomeric complex for functional activity. Four lines of evidence support this conclusion: (1) Phospholipid depletion shows that two tightly bound CL's must remain associated with cytochrome c oxidase in order to maintain full electron transport activity. (2) Removal of the two tightly bound CL's correlates with decreased activity that is restored by reassociation of 2 mol of exogenous CL. (3) CL-depleted cytochrome c oxidase has two high-affinity binding sites for 2-({sup 14}C)acetylcardiolipin (AcCL), K{sub d,app} <0.1 {mu}M, that are not present in enzymemore » containing endogenous CL. (4) CL, monolysocardiolipin (MLCL), and dilysocardiolipin (DLCL) complete for AcCL binding with approximately the same relative affinities as those measured by the restoration of electron transport activity (MLCL competes much better than DLCL). However, MLCL and DLCL are only 60% and 15% as effective as CL in restoring maximum activity when they are bound to high-affinity sites. The binding specificity of CL, MLCL, DLCL, and some of their acylated derivatives indicates that the apolar tails are most important for binding, not the polar head group. The authors therefore, conclude that restoration of activity to CL-depleted cytochrome c oxidase is highly specific and requires the reassociation of CL, or structurally similar compounds, with two high-affinity binding sites.« less
  • {sup 31}P NMR measurements were conducted to determine the structural and chemical environment of beef heart cardiolipin when bound to cytochrome c. {sup 31}P NMR line shapes infer that the majority of lipid remains in the bilayer state and that the average conformation of the lipid phosphate is not greatly affected by binding to the protein. An analysis of the spin-lattice (T{sub 1}) relaxation times of hydrated cardiolipin as a function of temperature describes a T{sub 1} minimum at around 25{degree}C which leads to a correlation time for the phosphates in the lipid headgroup of 0.71 ns. The relaxation behaviormore » of the protein-lipid complex was markedly different, showing a pronounced enhancement in the phosphorus spin-lattice relaxation rate. This effect of the protein increased progressively with increasing temperature, giving no indication of a minimum in T{sub 1} up to 75{degree}C. The enhancement in lipid phosphorus T{sub 1} relaxation was observed with protein in both oxidation states, being somewhat less marked for the reduced form. The characteristics of the T{sub 1} effects and the influence of the protein on other relaxation processes determined for the lipid phosphorus (spin-spin relaxation and longitudinal relaxation in the rotating frame) point to a strong paramagnetic interaction from the protein. A comparison with the relaxation behavior of samples spinning at the magic angle was also consistent with this mechanism. The results suggest that cytochrome c reversibly denatures on complexation with cardiolipin bilayers, such that the electronic ground state prevailing in the native structure of both oxidized and reduced protein can convert to high-spin states with greater magnetic susceptibility.« less
  • Deuterium NMR has been used to investigate the structure and dynamic state of cytochrome c complexed with bilayers of cardiolipin. Reductive methylation was employed to prepare (N{sup {epsilon}},N{sup {epsilon}}-C{sup 2}H{sub 3})lysyl cytochrome c, and deuterium exchange provided labeling of backbone sites to give (amide-{sup 2}H)cytochrome c or more selective labeling of just histidine residues in ({epsilon}-{sup 2}H)histidine cytochrome c. Deuterium NMR measurements on (N{sup {epsilon}},N{sup {epsilon}}-C{sup 2}H{sub 3})lysyl cytochrome c in the solid state showed restricted motions, fairly typical of the behavior of aliphatic side-chain sites in proteins. The (amide-{sup 2}H)cytochrome c provided immobile amide spectra showing that only themore » most stable backbone sites remained labeled in this derivative. Relaxation measurements on the aqueous solution of (amide-{sup 2}H)cytochrome c yielded a rotational correlation time of 7.9 ns for the protein, equivalent to a hydrodynamic diameter of 4.0 nm, just 0.6 nm greater than its largest crystallographic dimension. Similar measurements on ({epsilon}-{sup 2}H)histidine cytochrome c in solution showed that all labeled histidine residues were also immobile compared with the overall reorientational motion of the protein. A strong binding of protein with liquid-crystalline bilayers of cardiolipin disorders the lysine sites of interaction on the surface of the protein and appears to cause an extensive derangement of secondary structure, such that no stable {alpha}-helices can exist in the protein backbone with a lifetime longer than around 10{sup {minus}6}s.« less
  • Purpose: To determine whether cytochrome c (cyt c) content and associated cardiolipin oxidation can be determinants of cell sensitivity to irradiation-induced apoptosis. Methods and Materials: The small interfering RNA (siRNA) approach was used to engineer HeLa cells with lowered contents of cyt c (14%, HeLa 1.2 cells). Cells were treated by {gamma}-irradiation (in doses of 5-40 Gy). Lipid oxidation was characterized by electrospray ionization mass spectrometry analysis and fluorescence high-performance liquid chromatography-based Amplex Red assay. Release of a proapoptotic factor (cyt c, Smac/DIABLO) was detected by Western blotting. Apoptosis was revealed by caspase-3/7 activation and phosphatidylserine externalization. Results: Irradiation causedmore » selective accumulation of hydroperoxides in cardiolipin (CL) but not in other phospholipids. HeLa 1.2 cells responded by a lower irradiation-induced accumulation of CL oxidation products than parental HeLa cells. Proportionally decreased release of a proapoptotic factor, Smac/DIABLO, was detected in cyt c-deficient cells after irradiation. Caspase-3/7 activation and phosphatidylserine externalization were proportional to the cyt c content in cells. Conclusions: Cytochrome c is an important catalyst of CL peroxidation, critical to the execution of the apoptotic program. This new role of cyt c in irradiation-induced apoptosis is essential for the development of new radioprotectors and radiosensitizers.« less
  • Research highlights: {yields} Cardiolipin binding to cytochrome c. {yields} Cardiolipin-dependent peroxynitrite isomerization by cytochrome c. {yields} Cardiolipin-cytochrome c complex plays pro-apoptotic effects. {yields} Cardiolipin-cytochrome c complex plays anti-apoptotic effects. -- Abstract: Upon interaction with bovine heart cardiolipin (CL), horse heart cytochrome c (cytc) changes its tertiary structure disrupting the heme-Fe-Met80 distal bond, reduces drastically the midpoint potential out of the range required for its physiological role, binds CO and NO with high affinity, and displays peroxidase activity. Here, the effect of CL on peroxynitrite isomerization by ferric cytc (cytc-Fe(III)) is reported. In the absence of CL, hexa-coordinated cytc does notmore » catalyze peroxynitrite isomerization. In contrast, CL facilitates cytc-Fe(III)-mediated isomerization of peroxynitrite in a dose-dependent fashion inducing the penta-coordination of the heme-Fe(III)-atom. The value of the second order rate constant for CL-cytc-Fe(III)-mediated isomerization of peroxynitrite (k{sub on}) is (3.2 {+-} 0.4) x 10{sup 5} M{sup -1} s{sup -1}. The apparent dissociation equilibrium constant for CL binding to cytc-Fe(III) is (5.1 {+-} 0.8) x 10{sup -5} M. These results suggest that CL-cytc could play either pro-apoptotic or anti-apoptotic effects facilitating lipid peroxidation and scavenging of reactive nitrogen species, such as peroxynitrite, respectively.« less