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. 2006 Apr 18;45(15):4998-5009.
doi: 10.1021/bi0525573.

Peroxidase activity and structural transitions of cytochrome c bound to cardiolipin-containing membranes

Natalia A Belikova  1 , Yury A Vladimirov, Anatoly N Osipov, Alexandr A Kapralov, Vladimir A Tyurin, Maksim V Potapovich, Liana V Basova, Jim Peterson, Igor V Kurnikov, Valerian E Kagan
Affiliations

Peroxidase activity and structural transitions of cytochrome c bound to cardiolipin-containing membranes

Natalia A Belikova et al. Biochemistry. .

Abstract

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.

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Figures

Figure 1
Figure 1. Native agarose gels of CL/cyt c complexes at different CL/cyt c ratios
A: TOCL: B: Bovine heart CL (BHCL). TOCL or BHCL dissolved in methanol-chloroform (∼4:1 mixture, less than 2%) were added to cyt c solution in HEPES buffer (25 mM, pH=7.4 with 100 μM DTPA) and incubated for 15 min. Then, the samples were loaded onto 0.8% aragose gel (as indicated on the figure) and the electrophoresis was run for 15 min in HEPES-imidazole buffer (35 mM HEPES, 43 mM imidazole, pH = 7.4). The gels were stained with Coomassie Brilliant Blue R-250. Cyt c - 720 pmol (120 μM, 6 μl), CL concentration was changed to achieve CL/cyt c ratios from 0:1 up to 20:1 molar ratios.
Figure 2
Figure 2. Native PAGE of CL/cyt c complexes in the presence of NAO
Typical PAGE stained for protein (upper panel) and for NAO fluorescence (lower panel). Before staining of gels with Coomassie Brilliant Blue R-250 for protein, fluorescence of NAO was measured using a 520 nm band pass filter. Note that NAO and cyt c have different mobility in the gel.
Figure 3
Figure 3. Optical spectra of CL/cyt c complexes corresponding to absorbance of Fe-S(Met80) bond
Adjusted 695 nm spectra of CL/cyt c complexes in 50 mM phosphate buffer (A) and 25 mM HEPES buffer (B, C): (a) cyt c, (b) DOPC liposomes with cyt c after 15 min incubation, (c) TOCL/DOPC liposomes with cyt c after 15 min incubation, (d) TOCL/DOPC with cyt c after 15 min incubation in high-ionic buffer (25 mM HEPES plus 1 M KCl), (e) cyt c incubated with KCN for 15 min, (f) TOCL/DOPC liposomes with cyt c and KCN after 15 min incubation. Liposomes 1 mM total lipids, cyt c 50 μM, KCN 500 μM.
Figure 4
Figure 4. Peroxidase activity of lipid/cyt c complexes at different ratios as assessed by the etoposide phenoxyl radical ESR assay
A - Formation of phenoxyl radical as a result a CL/cyt c peroxidase activity measured by ESR. Cyt c 40 μM, TOCL/DOPC liposomes 1:1, etoposide 700 μM, H2O2 1 mM. Inset: typical ESR signal of etoposide phenoxyl radical. (●) - TOCL, (▲) - TMCL, (□) DOPC. B - Second order rate constant of interaction of cyt c and H2O2 and second order rate constant of recombination of etoposide radicals were used as adjustable parameters in the fit (see Methods for details). (●) - TOCL, (▲) - TMCL.
Figure 5
Figure 5. Cyt c tryptophan (Trp) fluorescence induced upon its binding with CL
A. Typical cyt c Trp59 fluorescence emission spectra obtained after excitation at 293 nm. (N, native state cyt c) cyt c, 5 μM; (1) TOCL/DOPC liposomes incubated for 15 min with cyt c (at 50:1 molar ratio) in 25 mM HEPES buffer, pH=7.4; (2) same as 1 plus 150 mM KCl after the incubation, (3) same as 1, plus 1 M KCl after the incubation; (U, unfolded state of cyt c) cyt c after addition of 6.0 M GdmCl. B. Dependence of the emission of cyt c Trp59 fluorescence (340 nm) on the lipids:cyt c ratio (25 mM HEPES, pH=7.4), (●) - TOCL, (▲) - TMCL, (□) DOPC.
Figure 6
Figure 6. Peroxidase activity of CL/cyt c complexes in buffers with different ionic strengths
Fluorescence intensity of DCF, the product of H2DCF oxidation, with the excitation at 502 nm and emission at 522 nm is shown. Cyt c (1.25 μM), liposomes (125 μM, DOPC/CL 1:1 ratio), H2DCF 4 μM, H2O2 50 μM, spectra obtained 30 min after addition H2O2. First set of bars: cyt c was incubated for 15 min with CL/DOPC liposomes in 25 mM HEPES with 100 μM DTPA. Second set of bars: same plus 1 M KCl. Third set of bars: 1 M KCl was added after 15 min incubation of cyt c and CL/DOPC liposomes in 25 mM HEPES).
Figure 7
Figure 7. Time-course of etoposide phenoxyl radicals generated by the peroxidase activity of cyt c in the presence of DOPC (A) or TOCL (B)
Incubation conditions: cyt c - 40 μM, TOCL/DOPC liposomes 1:1, etoposide 700 μM, H2O2 1 mM. Ratios of lipids to cyt c: (◆) - 1:1, (■) - 2:1, (▲) - 5:1, (□) - 10:1, (△) - 25:1, (●) - 50:1. Concentrations of etoposide radicals were calculated based on the magnitudes of the ESR signals as detailed in Materials and Methods.
Figure 8
Figure 8. Effects of H2O2 pretreatment on the peroxidase activity of cyt c and its complexes with TOCL and DOPC
Peroxidase activity was evaluated by ESR-detectable generation of etoposide phenoxyl radicals. Conditions: cyt c (1 mM) in 10 mM PBS (pH 7.0) was incubated with 5 mM H2O2 (added three times as shown by the arrows on the inset). Aliquots of cyt c (containing 40 μM of cyt c) were taken at indicated time intervals (0.3 min, 23 min) and mixed with liposomes (10:1 lipid/cyt c ratio) to perform measurements of peroxidase activity by ESR. Inset: Changes of cyt c absorbance in the Soret band ((◇), 410 nm) and Fe-S(Met80) band ((■), 695nm) induced by H2O2. Arrows show additions of H2O2. Absorbance at 695 nm was determined under the following conditions: cyt c (1 mM), H2O2 (5 mM). Absorbance at 410 nm was determined in the diluted samples: cyt c (10 μM), H2O2 (50 μM).
Figure 9
Figure 9. Kinetic parameters of peroxidase reaction of CL/cyt c complexes
A. Time course of H2DCF oxidation. H2O2 (50 μM) was added to start peroxidase reaction. Time-courses of peroxidase activity of cyt c in the absence and presence of liposomes at different H2O2 concentrations were obtained. The slopes of each line were calculated to yield the concentration dependence shown on Figure 9C. Cyt c 1 μM, liposomes 20 μM, H2DCF 0.5 μM, H2O2 50 μM. B. Typical ESR recordings of etoposide phenoxyl radicals over 4 min period of time. The rate of growth of this signal magnitude at different H2O2 concentrations was measured and replotted as shown on Figure 9D. Cyt c 25 μM, liposomes 500 μM, etoposide 350 μM, H2O2 100 μM, record time 4 min. C. Initial reaction rates of H2DCF oxidation by cyt c (*), DOPC/cyt c (20:1) (□), TOCL/cyt c (10:10:1) (●) in 10mM PBS with 100 μM DTPA (pH=7.4). Reaction mixture contained 1 μM cyt c, 20 μM liposomes and 0.5 μM H2DCF. Extrapolation of linear dependences of the rates for cyt c, DOPC/cyt c and TOCL/cyt c interception with the X-axis provides an estimate of the initial H2O2 concentrations capable of activating the peroxidase activity: H2O2cyt c∼ 35 μM; H2O2DOPC/cyt c∼ 30 μM; H2O2TOCL/cyt c∼ 5 μM. D. Initial rate of phenoxyl radical formation, measured by ESR assay. Cyt c (*), DOPC/cyt c (20:1) (□), TOCL/cyt c (10:1) (●), TLCL/cyt c (10:1) (○) in 10mM PBS with 100 μM DTPA (pH=7.4). Reaction mixture contained 25 μM cyt c, 500 μM lipid liposomes and 350 μM etoposide. Extrapolation of linear dependences of the rates for cyt c, DOPC/cyt c and TOCL/cyt c interception with the X-axis provides an estimate of the initial H2O2 concentrations capable of activating the peroxidase activity: H2O2cyt c∼ 60 μM; H2O2DOPC/cyt c∼ 40 μM; H2O2TOCL/cyt c∼ 10 μM, H2O2TLCL/cyt c∼ 12 μM.
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