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Probing cytochrome c in living mitochondria with surface-enhanced Raman spectroscopy.

Brazhe NA, Evlyukhin AB, Goodilin EA, Semenova AA, Novikov SM, Bozhevolnyi SI, Chichkov BN, Sarycheva AS, Baizhumanov AA, Nikelshparg EI, Deev LI, Maksimov EG, Maksimov GV, Sosnovtseva O - Sci Rep (2015)

Bottom Line: Here we suggest a novel label-free approach based on the surface-enhanced Raman spectroscopy (SERS) to monitor the redox state and conformation of cytochrome c in the electron transport chain in living mitochondria.We demonstrate that SERS spectra of living mitochondria placed on hierarchically structured silver-ring substrates provide exclusive information about cytochrome c behavior under modulation of inner mitochondrial membrane potential, proton gradient and the activity of ATP-synthetase.Mathematical simulation explains the observed enhancement of Raman scattering due to high concentration of electric near-field and large contact area between mitochondria and nanostructured surfaces.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics, Biological Faculty, Moscow State University, Leninskie gory 1/12, Moscow, 119234, Russia.

ABSTRACT
Selective study of the electron transport chain components in living mitochondria is essential for fundamental biophysical research and for the development of new medical diagnostic methods. However, many important details of inter- and intramembrane mitochondrial processes have remained in shadow due to the lack of non-invasive techniques. Here we suggest a novel label-free approach based on the surface-enhanced Raman spectroscopy (SERS) to monitor the redox state and conformation of cytochrome c in the electron transport chain in living mitochondria. We demonstrate that SERS spectra of living mitochondria placed on hierarchically structured silver-ring substrates provide exclusive information about cytochrome c behavior under modulation of inner mitochondrial membrane potential, proton gradient and the activity of ATP-synthetase. Mathematical simulation explains the observed enhancement of Raman scattering due to high concentration of electric near-field and large contact area between mitochondria and nanostructured surfaces.

No MeSH data available.


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Dependence of SERS spectra of mitochondria on proton-motive force and activity of ATP synthesis.(A) SERS spectra of mitochondria before and after application of FCCP (0,5 μM) (lower and upper spectra, respectively). (B) SERS spectra of mitochondria before and after application of oligomycin (10 μM) (lower and upper spectra, respectively). (C) Ratios of peak intensities calculated for SERS spectra of mitochondria in control (after application of pyruvate, succinate, ADP and MgCl2, black bars) and in 2 min after application of protonofor FCCP (0,5 μM) (red bars, number of experiments n = 3) or oligomycin (10 μM) (blue bars, number of experiments n = 3). Ratios were calculated by dividing intensities of peak maxima at 750 by 1638 cm−1 (I750/I1638), 1170 by 1638 cm−1 (I1170/I1638) and 1371 by 1638 cm−1 (I1371/I1638). Ratios in control measurements were taken as 100%. Vertical bars show SE value, %. SE value of control ratios were calculated from 10 independent measurements of control SERS spectra from the same spot.
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f3: Dependence of SERS spectra of mitochondria on proton-motive force and activity of ATP synthesis.(A) SERS spectra of mitochondria before and after application of FCCP (0,5 μM) (lower and upper spectra, respectively). (B) SERS spectra of mitochondria before and after application of oligomycin (10 μM) (lower and upper spectra, respectively). (C) Ratios of peak intensities calculated for SERS spectra of mitochondria in control (after application of pyruvate, succinate, ADP and MgCl2, black bars) and in 2 min after application of protonofor FCCP (0,5 μM) (red bars, number of experiments n = 3) or oligomycin (10 μM) (blue bars, number of experiments n = 3). Ratios were calculated by dividing intensities of peak maxima at 750 by 1638 cm−1 (I750/I1638), 1170 by 1638 cm−1 (I1170/I1638) and 1371 by 1638 cm−1 (I1371/I1638). Ratios in control measurements were taken as 100%. Vertical bars show SE value, %. SE value of control ratios were calculated from 10 independent measurements of control SERS spectra from the same spot.

Mentions: To demonstrate high sensitivity of SERS spectra of mitochondria to the proton gradient across IMM and mitochondrial membrane potential (ΔΦ) and to the redox state of other ETC complexes, we applied (i) protonophore FCCP and (ii) oligomycin as an inhibitor of ATP-synthetase (ETC complex V). As it was mentioned, FCCP-induced uncoupling of the electron transport and ATP synthesis leads to the decrease in the relative amount of reduced electron carriers including cytochrome c17. Oligomycin, on the contrary, due to the inhibition of ATP-synthase, causes accumulation of protons in the intermembrane space causing IMM hyperpolarization, decrease in the electron flow rate and the increase in the relative amount of the reduced electron carriers in ETC. As expected, we observed a decrease in the relative normalized intensity of cytochromal peaks at 748, 1127, 1170 and 1371 cm−1 under the FCCP (0.5 μM) treatment (Fig. 3A,C). The application of oligomycin (10 μM), oppositely, caused an increase in the relative input of peaks at 748 and 1371 cm−1 into the overall spectrum that can be seen as the increase in relative intensities of peaks at 748 and 1371 cm−1 normalized to the intensity of the peak at 1638 cm−1 (ratios I748/I1638 and I1371/I1638) (Fig. 3B,C). Importantly, that the observed effect of FCCP and oligomycin on SERS spectra was not associated with the direct interaction of FCCP or oligomycin with cytochrome c molecules in mitochondria, since both chemicals did not affect SERS spectra of purified cytochrome c (Supplementary Fig. 5). We conclude that the behavior of c-type cytochrome SERS peaks following FCCP and oligomycin treatments is in excellent agreement with the expected dependence of the reduction state of c-type cytochromes on the proton-motive force and the ETC loading with electrons.


Probing cytochrome c in living mitochondria with surface-enhanced Raman spectroscopy.

Brazhe NA, Evlyukhin AB, Goodilin EA, Semenova AA, Novikov SM, Bozhevolnyi SI, Chichkov BN, Sarycheva AS, Baizhumanov AA, Nikelshparg EI, Deev LI, Maksimov EG, Maksimov GV, Sosnovtseva O - Sci Rep (2015)

Dependence of SERS spectra of mitochondria on proton-motive force and activity of ATP synthesis.(A) SERS spectra of mitochondria before and after application of FCCP (0,5 μM) (lower and upper spectra, respectively). (B) SERS spectra of mitochondria before and after application of oligomycin (10 μM) (lower and upper spectra, respectively). (C) Ratios of peak intensities calculated for SERS spectra of mitochondria in control (after application of pyruvate, succinate, ADP and MgCl2, black bars) and in 2 min after application of protonofor FCCP (0,5 μM) (red bars, number of experiments n = 3) or oligomycin (10 μM) (blue bars, number of experiments n = 3). Ratios were calculated by dividing intensities of peak maxima at 750 by 1638 cm−1 (I750/I1638), 1170 by 1638 cm−1 (I1170/I1638) and 1371 by 1638 cm−1 (I1371/I1638). Ratios in control measurements were taken as 100%. Vertical bars show SE value, %. SE value of control ratios were calculated from 10 independent measurements of control SERS spectra from the same spot.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4561893&req=5

f3: Dependence of SERS spectra of mitochondria on proton-motive force and activity of ATP synthesis.(A) SERS spectra of mitochondria before and after application of FCCP (0,5 μM) (lower and upper spectra, respectively). (B) SERS spectra of mitochondria before and after application of oligomycin (10 μM) (lower and upper spectra, respectively). (C) Ratios of peak intensities calculated for SERS spectra of mitochondria in control (after application of pyruvate, succinate, ADP and MgCl2, black bars) and in 2 min after application of protonofor FCCP (0,5 μM) (red bars, number of experiments n = 3) or oligomycin (10 μM) (blue bars, number of experiments n = 3). Ratios were calculated by dividing intensities of peak maxima at 750 by 1638 cm−1 (I750/I1638), 1170 by 1638 cm−1 (I1170/I1638) and 1371 by 1638 cm−1 (I1371/I1638). Ratios in control measurements were taken as 100%. Vertical bars show SE value, %. SE value of control ratios were calculated from 10 independent measurements of control SERS spectra from the same spot.
Mentions: To demonstrate high sensitivity of SERS spectra of mitochondria to the proton gradient across IMM and mitochondrial membrane potential (ΔΦ) and to the redox state of other ETC complexes, we applied (i) protonophore FCCP and (ii) oligomycin as an inhibitor of ATP-synthetase (ETC complex V). As it was mentioned, FCCP-induced uncoupling of the electron transport and ATP synthesis leads to the decrease in the relative amount of reduced electron carriers including cytochrome c17. Oligomycin, on the contrary, due to the inhibition of ATP-synthase, causes accumulation of protons in the intermembrane space causing IMM hyperpolarization, decrease in the electron flow rate and the increase in the relative amount of the reduced electron carriers in ETC. As expected, we observed a decrease in the relative normalized intensity of cytochromal peaks at 748, 1127, 1170 and 1371 cm−1 under the FCCP (0.5 μM) treatment (Fig. 3A,C). The application of oligomycin (10 μM), oppositely, caused an increase in the relative input of peaks at 748 and 1371 cm−1 into the overall spectrum that can be seen as the increase in relative intensities of peaks at 748 and 1371 cm−1 normalized to the intensity of the peak at 1638 cm−1 (ratios I748/I1638 and I1371/I1638) (Fig. 3B,C). Importantly, that the observed effect of FCCP and oligomycin on SERS spectra was not associated with the direct interaction of FCCP or oligomycin with cytochrome c molecules in mitochondria, since both chemicals did not affect SERS spectra of purified cytochrome c (Supplementary Fig. 5). We conclude that the behavior of c-type cytochrome SERS peaks following FCCP and oligomycin treatments is in excellent agreement with the expected dependence of the reduction state of c-type cytochromes on the proton-motive force and the ETC loading with electrons.

Bottom Line: Here we suggest a novel label-free approach based on the surface-enhanced Raman spectroscopy (SERS) to monitor the redox state and conformation of cytochrome c in the electron transport chain in living mitochondria.We demonstrate that SERS spectra of living mitochondria placed on hierarchically structured silver-ring substrates provide exclusive information about cytochrome c behavior under modulation of inner mitochondrial membrane potential, proton gradient and the activity of ATP-synthetase.Mathematical simulation explains the observed enhancement of Raman scattering due to high concentration of electric near-field and large contact area between mitochondria and nanostructured surfaces.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics, Biological Faculty, Moscow State University, Leninskie gory 1/12, Moscow, 119234, Russia.

ABSTRACT
Selective study of the electron transport chain components in living mitochondria is essential for fundamental biophysical research and for the development of new medical diagnostic methods. However, many important details of inter- and intramembrane mitochondrial processes have remained in shadow due to the lack of non-invasive techniques. Here we suggest a novel label-free approach based on the surface-enhanced Raman spectroscopy (SERS) to monitor the redox state and conformation of cytochrome c in the electron transport chain in living mitochondria. We demonstrate that SERS spectra of living mitochondria placed on hierarchically structured silver-ring substrates provide exclusive information about cytochrome c behavior under modulation of inner mitochondrial membrane potential, proton gradient and the activity of ATP-synthetase. Mathematical simulation explains the observed enhancement of Raman scattering due to high concentration of electric near-field and large contact area between mitochondria and nanostructured surfaces.

No MeSH data available.


Related in: MedlinePlus