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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.


Related in: MedlinePlus

SERS spectra of mitochondria.(A) (1) Spectrum of mitochondrial buffer on AgNSS; (2) spectrum of mitochondria suspension used in SERS experiments placed into the Petri dish with smooth silver plate without nanostructures; (3) Raman spectrum of concentrated mitochondrial aggregate placed into ordinary Petri dish; excitation power 1.5 mW, objective x63, NA 0.9, registration time 20 s; (4) SERS spectrum of mitochondria suspension placed on AgNSS after pyruvate, succinate, ADP and MgCl2 application; (5) SERS spectrum of mitochondria suspension placed on AgNSS after application of sodium dithionite. SERS spectra were recorded from the diluted mitochondria sample with excitation power 1.5 mW; objective x5, NA 0.15, registration time 20 s. (B) SERS spectra of mitochondria recorded from different places of AgNSS shown schematically by colored spots in Fig. D. (C) SERS spectra of mitochondria recorded from the same place on AgNSS with time lapse between spectra acquisition of 30 s. Accumulation time for each spectrum is 20 s. Dotted vertical lines indicate positions of maxima of the most intensive peaks. Vertical scale bars are equal to 200 a.u in all figures. (D) Optical microphotograph of Ag nanostructured surface in the transmitted light. Horizontal length of the microphotograph is 100 μm. Detailed morphology of AgNSSs is shown in Fig. 4.
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f2: SERS spectra of mitochondria.(A) (1) Spectrum of mitochondrial buffer on AgNSS; (2) spectrum of mitochondria suspension used in SERS experiments placed into the Petri dish with smooth silver plate without nanostructures; (3) Raman spectrum of concentrated mitochondrial aggregate placed into ordinary Petri dish; excitation power 1.5 mW, objective x63, NA 0.9, registration time 20 s; (4) SERS spectrum of mitochondria suspension placed on AgNSS after pyruvate, succinate, ADP and MgCl2 application; (5) SERS spectrum of mitochondria suspension placed on AgNSS after application of sodium dithionite. SERS spectra were recorded from the diluted mitochondria sample with excitation power 1.5 mW; objective x5, NA 0.15, registration time 20 s. (B) SERS spectra of mitochondria recorded from different places of AgNSS shown schematically by colored spots in Fig. D. (C) SERS spectra of mitochondria recorded from the same place on AgNSS with time lapse between spectra acquisition of 30 s. Accumulation time for each spectrum is 20 s. Dotted vertical lines indicate positions of maxima of the most intensive peaks. Vertical scale bars are equal to 200 a.u in all figures. (D) Optical microphotograph of Ag nanostructured surface in the transmitted light. Horizontal length of the microphotograph is 100 μm. Detailed morphology of AgNSSs is shown in Fig. 4.

Mentions: In our experiments SERS spectra of mitochondria placed on AgNSS demonstrate a set of intensive peaks corresponding to heme molecules of cytochromes of mitochondrial ETC with the position of their main maxima at 748, 1127, 1170, 1313, 1371, 1565, 1585 and 1638 cm−1 (Fig. 2A (spectrum 4), Table 1). These peaks originate from the normal group vibrations of pyrrol rings, methine bridges and side radicals in the heme molecule (Fig. 2, Table 1, Supplementary Fig. 1). Similar spectra of the same shape and with the similar set of peaks are observed from purified oxidized cytochrome c mixed with Ag colloid (Supplementary Fig. 2A). Note, that SERS spectra of mitochondria show a peak at 1313 cm−1 as a signature of the с-type cytochrome and do not have peaks at 1300 and 1337 cm−1 which are specific of b-type cytochromes, myoglobin and hemoglobin (Fig. 2A, Supplementary Fig. 2)181924. This is a clear evidence of the origin of SERS spectra of mitochondria from the c-type heme in cytochrome, namely, the cytochrome c. It is likely that the heme of cytochrome c1 (which is located further from outer mitochondrial membrane than cytochrome c and from AgNSS surface) does not contribute much to the SERS spectra of mitochondria. Cytochromes b of the complexes II and III are localized in the IMM part, resulting in no enhancement of their Raman scattering due to a too large distance between b-hemes and the AgNSS surface. To record SERS spectra of mitochondria with fully reduced electron carriers we added sodium dithionite (SDT) to the mitochondria sample. SERS spectra of SDT-treated mitochondria correspond to that of reduced purified cytochrome c and demonstrate a signature shift of positions of peaks sensitive to the redox state of Fe atom (Fig. 2A (spectrum 5), Table 1, Supplementary Fig. 2A). Additionally, we observed increased relative intensity of the peak at 748 cm−1 in the SERS spectra of SDT-treated mitochondria and reduced cytochrome c (Fig. 2A (spectrum 5), Supplementary Fig. 2A). Such an increase is already known for Raman spectra of reduced cytochromes, SDT-treated mitochondria, cardiomyocytes and heart161718 and for SERS spectra of reduced cytochrome c19. The same mitochondrial sample placed into the Petri dish with a smooth silver plate on the bottom did not give any spectrum (Fig. 2A, spectrum 2). We could obtain ordinary Raman spectra of intact mitochondria only by focusing laser light inside dense mitochondria aggregates using much higher laser power per area (objective x63, NA 0.9 and laser power 1.5 mW per registration spot with a diameter of 400 nm) (Fig. 2A, spectrum 3). Notably, even under such high excitation power ordinary Raman spectrum of mitochondria is noisy and does not contain Raman peaks of cytochromes. Only two weak peaks of protein bond vibrations can be observed around 1450 and 1660 cm−1 (corresponding to C-H deformations and amide bond vibration, respectively). Interestingly, we never observed SERS spectra of purified cytochrome c (in the concentration range of 10−5–10−7 M) placed on AgNSS, while mitochondria gave intensive SERS spectrum almost immediately after placement of the sample into the dish with AgNSS. SERS spectra of mitochondria recorded from different AgNSS spots and from the same AgNSS spot over time are highly reproducible (Fig. 2B,C). Stability of mitochondria SERS spectra recorded from the same spot at different time points indicates absence of mitochondria photodamage under laser illumination. To check excitation condition that can cause sample damage we intentionally increased laser power. We observed photodamage-induced broadening of mitochondria SERS peaks only under 10-fold higher excitation power, than that used in all described SERS experiments (Supplementary Fig. 3).


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)

SERS spectra of mitochondria.(A) (1) Spectrum of mitochondrial buffer on AgNSS; (2) spectrum of mitochondria suspension used in SERS experiments placed into the Petri dish with smooth silver plate without nanostructures; (3) Raman spectrum of concentrated mitochondrial aggregate placed into ordinary Petri dish; excitation power 1.5 mW, objective x63, NA 0.9, registration time 20 s; (4) SERS spectrum of mitochondria suspension placed on AgNSS after pyruvate, succinate, ADP and MgCl2 application; (5) SERS spectrum of mitochondria suspension placed on AgNSS after application of sodium dithionite. SERS spectra were recorded from the diluted mitochondria sample with excitation power 1.5 mW; objective x5, NA 0.15, registration time 20 s. (B) SERS spectra of mitochondria recorded from different places of AgNSS shown schematically by colored spots in Fig. D. (C) SERS spectra of mitochondria recorded from the same place on AgNSS with time lapse between spectra acquisition of 30 s. Accumulation time for each spectrum is 20 s. Dotted vertical lines indicate positions of maxima of the most intensive peaks. Vertical scale bars are equal to 200 a.u in all figures. (D) Optical microphotograph of Ag nanostructured surface in the transmitted light. Horizontal length of the microphotograph is 100 μm. Detailed morphology of AgNSSs is shown in Fig. 4.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f2: SERS spectra of mitochondria.(A) (1) Spectrum of mitochondrial buffer on AgNSS; (2) spectrum of mitochondria suspension used in SERS experiments placed into the Petri dish with smooth silver plate without nanostructures; (3) Raman spectrum of concentrated mitochondrial aggregate placed into ordinary Petri dish; excitation power 1.5 mW, objective x63, NA 0.9, registration time 20 s; (4) SERS spectrum of mitochondria suspension placed on AgNSS after pyruvate, succinate, ADP and MgCl2 application; (5) SERS spectrum of mitochondria suspension placed on AgNSS after application of sodium dithionite. SERS spectra were recorded from the diluted mitochondria sample with excitation power 1.5 mW; objective x5, NA 0.15, registration time 20 s. (B) SERS spectra of mitochondria recorded from different places of AgNSS shown schematically by colored spots in Fig. D. (C) SERS spectra of mitochondria recorded from the same place on AgNSS with time lapse between spectra acquisition of 30 s. Accumulation time for each spectrum is 20 s. Dotted vertical lines indicate positions of maxima of the most intensive peaks. Vertical scale bars are equal to 200 a.u in all figures. (D) Optical microphotograph of Ag nanostructured surface in the transmitted light. Horizontal length of the microphotograph is 100 μm. Detailed morphology of AgNSSs is shown in Fig. 4.
Mentions: In our experiments SERS spectra of mitochondria placed on AgNSS demonstrate a set of intensive peaks corresponding to heme molecules of cytochromes of mitochondrial ETC with the position of their main maxima at 748, 1127, 1170, 1313, 1371, 1565, 1585 and 1638 cm−1 (Fig. 2A (spectrum 4), Table 1). These peaks originate from the normal group vibrations of pyrrol rings, methine bridges and side radicals in the heme molecule (Fig. 2, Table 1, Supplementary Fig. 1). Similar spectra of the same shape and with the similar set of peaks are observed from purified oxidized cytochrome c mixed with Ag colloid (Supplementary Fig. 2A). Note, that SERS spectra of mitochondria show a peak at 1313 cm−1 as a signature of the с-type cytochrome and do not have peaks at 1300 and 1337 cm−1 which are specific of b-type cytochromes, myoglobin and hemoglobin (Fig. 2A, Supplementary Fig. 2)181924. This is a clear evidence of the origin of SERS spectra of mitochondria from the c-type heme in cytochrome, namely, the cytochrome c. It is likely that the heme of cytochrome c1 (which is located further from outer mitochondrial membrane than cytochrome c and from AgNSS surface) does not contribute much to the SERS spectra of mitochondria. Cytochromes b of the complexes II and III are localized in the IMM part, resulting in no enhancement of their Raman scattering due to a too large distance between b-hemes and the AgNSS surface. To record SERS spectra of mitochondria with fully reduced electron carriers we added sodium dithionite (SDT) to the mitochondria sample. SERS spectra of SDT-treated mitochondria correspond to that of reduced purified cytochrome c and demonstrate a signature shift of positions of peaks sensitive to the redox state of Fe atom (Fig. 2A (spectrum 5), Table 1, Supplementary Fig. 2A). Additionally, we observed increased relative intensity of the peak at 748 cm−1 in the SERS spectra of SDT-treated mitochondria and reduced cytochrome c (Fig. 2A (spectrum 5), Supplementary Fig. 2A). Such an increase is already known for Raman spectra of reduced cytochromes, SDT-treated mitochondria, cardiomyocytes and heart161718 and for SERS spectra of reduced cytochrome c19. The same mitochondrial sample placed into the Petri dish with a smooth silver plate on the bottom did not give any spectrum (Fig. 2A, spectrum 2). We could obtain ordinary Raman spectra of intact mitochondria only by focusing laser light inside dense mitochondria aggregates using much higher laser power per area (objective x63, NA 0.9 and laser power 1.5 mW per registration spot with a diameter of 400 nm) (Fig. 2A, spectrum 3). Notably, even under such high excitation power ordinary Raman spectrum of mitochondria is noisy and does not contain Raman peaks of cytochromes. Only two weak peaks of protein bond vibrations can be observed around 1450 and 1660 cm−1 (corresponding to C-H deformations and amide bond vibration, respectively). Interestingly, we never observed SERS spectra of purified cytochrome c (in the concentration range of 10−5–10−7 M) placed on AgNSS, while mitochondria gave intensive SERS spectrum almost immediately after placement of the sample into the dish with AgNSS. SERS spectra of mitochondria recorded from different AgNSS spots and from the same AgNSS spot over time are highly reproducible (Fig. 2B,C). Stability of mitochondria SERS spectra recorded from the same spot at different time points indicates absence of mitochondria photodamage under laser illumination. To check excitation condition that can cause sample damage we intentionally increased laser power. We observed photodamage-induced broadening of mitochondria SERS peaks only under 10-fold higher excitation power, than that used in all described SERS experiments (Supplementary Fig. 3).

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