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In situ Raman study of redox state changes of mitochondrial cytochromes in a perfused rat heart.

Brazhe NA, Treiman M, Faricelli B, Vestergaard JH, Sosnovtseva O - PLoS ONE (2013)

Bottom Line: Excitation at 532 nm was used to obtain Raman scattering of the myocardial surface of the isolated heart at normal and hypoxic conditions.Raman spectra of the heart under normal pO2 demonstrate unique peaks attributable to reduced c-and b-type cytochromes and oxymyoglobin (oMb).The cytochrome peaks decreased in intensity upon FCCP treatment, as predicted from uncoupling mitochondrial respiration.

View Article: PubMed Central - PubMed

Affiliation: Biophysics Department, Biological faculty, Moscow State University, Moscow, Russia.

ABSTRACT
We developed a Raman spectroscopy-based approach for simultaneous study of redox changes in c-and b-type cytochromes and for a semiquantitative estimation of the amount of oxygenated myoglobin in a perfused rat heart. Excitation at 532 nm was used to obtain Raman scattering of the myocardial surface of the isolated heart at normal and hypoxic conditions. Raman spectra of the heart under normal pO2 demonstrate unique peaks attributable to reduced c-and b-type cytochromes and oxymyoglobin (oMb). The cytochrome peaks decreased in intensity upon FCCP treatment, as predicted from uncoupling mitochondrial respiration. Conversely, transient hypoxia causes the reversible increase in the intensity of peaks assigned to cytochromes c and c1, reflecting electron stacking proximal to cytochrome oxidase due to the lack of terminal electron acceptor O2. Intensities of peaks assigned to oxy- and deoxyhemoglobin were used for the semiquantitative estimation of oMb deoxygenation that was found to be of approximately 50[Formula: see text] under hypoxia conditions.

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Peak assignment.(A): Assignment of peaks in Raman spectra of perfused heart, cardiomyocytes (CMs) and isolated CM mitochondria in partially oxidized state and after reduction with sodium dithionite (SDT). (B): Assignment of peaks in Raman spectra of reduced (cyt.c(Fe2+)) and oxidized (cyt.c(Fe3+)) cytochrome c, oxymyoglobin (oMb), deoxymyoglobin (dMb) and metmyoglobin (metMb). Vertical scale bars show Raman intensity, a.u. In Fig. 2.A vertical scale bars are the same (2000 a.u.) for heart and mitochondria with and without SDT and the scale bar is equal to 500 a.u. for cardiomyocytes with and without SDT. In Fig. 2.B scale bar (2000 a.u.) is the same for all spectra. Numbers above red dashed lines indicate peak positions, cm−1. Arrows with numbers show position of peaks that are shifted relatively to the dashed line. Numbers indicating positions of peaks corresponding to cytochromes c, c1 and b are shown in bold font, to myoglobin (oMb and dMb) are shown in regular font. Peak with position at 1658 cm−1 (shown in red-colored font) corresponds to  bond vibration in peptide backbones of protein -helixes.
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pone-0070488-g002: Peak assignment.(A): Assignment of peaks in Raman spectra of perfused heart, cardiomyocytes (CMs) and isolated CM mitochondria in partially oxidized state and after reduction with sodium dithionite (SDT). (B): Assignment of peaks in Raman spectra of reduced (cyt.c(Fe2+)) and oxidized (cyt.c(Fe3+)) cytochrome c, oxymyoglobin (oMb), deoxymyoglobin (dMb) and metmyoglobin (metMb). Vertical scale bars show Raman intensity, a.u. In Fig. 2.A vertical scale bars are the same (2000 a.u.) for heart and mitochondria with and without SDT and the scale bar is equal to 500 a.u. for cardiomyocytes with and without SDT. In Fig. 2.B scale bar (2000 a.u.) is the same for all spectra. Numbers above red dashed lines indicate peak positions, cm−1. Arrows with numbers show position of peaks that are shifted relatively to the dashed line. Numbers indicating positions of peaks corresponding to cytochromes c, c1 and b are shown in bold font, to myoglobin (oMb and dMb) are shown in regular font. Peak with position at 1658 cm−1 (shown in red-colored font) corresponds to bond vibration in peptide backbones of protein -helixes.

Mentions: Laser excitation at 532 nm evokes resonance Raman scattering of heme-containing molecules such as cytochromes and myoglobin (Mb). Since the heme structure in cytochromes and Mb is almost the same their Raman spectra possess sets of peaks with similar positions of frequency shifts. However, since the surrounding protein in these molecules differs and since myoglobin is cytosolic molecule and cytochromes are transmembrane (cytochromes b and c1) or membrane-bound proteins (cytochrome c), the relative input of peak intensities into overall Raman spectrum is also different [12], [15]–[17]. To identify the main peaks in spectra from the heart, we also recorded Raman spectra of isolated CMs and isolated heart mitochondria (Fig. 2A), as well as of purified oxidized and reduced cytochrome c, oxy-, deoxy-, and methemoglobin (oMb, dMb and metMb, respectively) (Fig. 2B). Under 532 nm excitation conditions, the contribution of the oxidized cytochromes b and c to Raman scatter is negligible [17]. Therefore, to aid cytochromal peak identification we used sodium dithionite in some experiments to achieve maximal cytochrome reduction. Likewise, SDT aids in an assignment of Mb peaks, due to a frequency shift upon transition from oMb to dMb.


In situ Raman study of redox state changes of mitochondrial cytochromes in a perfused rat heart.

Brazhe NA, Treiman M, Faricelli B, Vestergaard JH, Sosnovtseva O - PLoS ONE (2013)

Peak assignment.(A): Assignment of peaks in Raman spectra of perfused heart, cardiomyocytes (CMs) and isolated CM mitochondria in partially oxidized state and after reduction with sodium dithionite (SDT). (B): Assignment of peaks in Raman spectra of reduced (cyt.c(Fe2+)) and oxidized (cyt.c(Fe3+)) cytochrome c, oxymyoglobin (oMb), deoxymyoglobin (dMb) and metmyoglobin (metMb). Vertical scale bars show Raman intensity, a.u. In Fig. 2.A vertical scale bars are the same (2000 a.u.) for heart and mitochondria with and without SDT and the scale bar is equal to 500 a.u. for cardiomyocytes with and without SDT. In Fig. 2.B scale bar (2000 a.u.) is the same for all spectra. Numbers above red dashed lines indicate peak positions, cm−1. Arrows with numbers show position of peaks that are shifted relatively to the dashed line. Numbers indicating positions of peaks corresponding to cytochromes c, c1 and b are shown in bold font, to myoglobin (oMb and dMb) are shown in regular font. Peak with position at 1658 cm−1 (shown in red-colored font) corresponds to  bond vibration in peptide backbones of protein -helixes.
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Related In: Results  -  Collection

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pone-0070488-g002: Peak assignment.(A): Assignment of peaks in Raman spectra of perfused heart, cardiomyocytes (CMs) and isolated CM mitochondria in partially oxidized state and after reduction with sodium dithionite (SDT). (B): Assignment of peaks in Raman spectra of reduced (cyt.c(Fe2+)) and oxidized (cyt.c(Fe3+)) cytochrome c, oxymyoglobin (oMb), deoxymyoglobin (dMb) and metmyoglobin (metMb). Vertical scale bars show Raman intensity, a.u. In Fig. 2.A vertical scale bars are the same (2000 a.u.) for heart and mitochondria with and without SDT and the scale bar is equal to 500 a.u. for cardiomyocytes with and without SDT. In Fig. 2.B scale bar (2000 a.u.) is the same for all spectra. Numbers above red dashed lines indicate peak positions, cm−1. Arrows with numbers show position of peaks that are shifted relatively to the dashed line. Numbers indicating positions of peaks corresponding to cytochromes c, c1 and b are shown in bold font, to myoglobin (oMb and dMb) are shown in regular font. Peak with position at 1658 cm−1 (shown in red-colored font) corresponds to bond vibration in peptide backbones of protein -helixes.
Mentions: Laser excitation at 532 nm evokes resonance Raman scattering of heme-containing molecules such as cytochromes and myoglobin (Mb). Since the heme structure in cytochromes and Mb is almost the same their Raman spectra possess sets of peaks with similar positions of frequency shifts. However, since the surrounding protein in these molecules differs and since myoglobin is cytosolic molecule and cytochromes are transmembrane (cytochromes b and c1) or membrane-bound proteins (cytochrome c), the relative input of peak intensities into overall Raman spectrum is also different [12], [15]–[17]. To identify the main peaks in spectra from the heart, we also recorded Raman spectra of isolated CMs and isolated heart mitochondria (Fig. 2A), as well as of purified oxidized and reduced cytochrome c, oxy-, deoxy-, and methemoglobin (oMb, dMb and metMb, respectively) (Fig. 2B). Under 532 nm excitation conditions, the contribution of the oxidized cytochromes b and c to Raman scatter is negligible [17]. Therefore, to aid cytochromal peak identification we used sodium dithionite in some experiments to achieve maximal cytochrome reduction. Likewise, SDT aids in an assignment of Mb peaks, due to a frequency shift upon transition from oMb to dMb.

Bottom Line: Excitation at 532 nm was used to obtain Raman scattering of the myocardial surface of the isolated heart at normal and hypoxic conditions.Raman spectra of the heart under normal pO2 demonstrate unique peaks attributable to reduced c-and b-type cytochromes and oxymyoglobin (oMb).The cytochrome peaks decreased in intensity upon FCCP treatment, as predicted from uncoupling mitochondrial respiration.

View Article: PubMed Central - PubMed

Affiliation: Biophysics Department, Biological faculty, Moscow State University, Moscow, Russia.

ABSTRACT
We developed a Raman spectroscopy-based approach for simultaneous study of redox changes in c-and b-type cytochromes and for a semiquantitative estimation of the amount of oxygenated myoglobin in a perfused rat heart. Excitation at 532 nm was used to obtain Raman scattering of the myocardial surface of the isolated heart at normal and hypoxic conditions. Raman spectra of the heart under normal pO2 demonstrate unique peaks attributable to reduced c-and b-type cytochromes and oxymyoglobin (oMb). The cytochrome peaks decreased in intensity upon FCCP treatment, as predicted from uncoupling mitochondrial respiration. Conversely, transient hypoxia causes the reversible increase in the intensity of peaks assigned to cytochromes c and c1, reflecting electron stacking proximal to cytochrome oxidase due to the lack of terminal electron acceptor O2. Intensities of peaks assigned to oxy- and deoxyhemoglobin were used for the semiquantitative estimation of oMb deoxygenation that was found to be of approximately 50[Formula: see text] under hypoxia conditions.

Show MeSH
Related in: MedlinePlus