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. 2020 May 5:8:387.
doi: 10.3389/fchem.2020.00387. eCollection 2020.

Comparison Between O and OH Intermediates of Cytochrome c Oxidase Studied by FTIR Spectroscopy

Elena Gorbikova  1 , Ruslan Kalendar  2   3
Affiliations

Comparison Between O and OH Intermediates of Cytochrome c Oxidase Studied by FTIR Spectroscopy

Elena Gorbikova et al. Front Chem. .

Abstract

Cytochrome c oxidase is terminal enzyme in the respiratory chain of mitochondria and many aerobic bacteria. It catalyzes reduction of oxygen to water. During its catalysis, CcO proceeds through several quite stable intermediates (R, A, PR/M, O/OH, E/EH). This work is concentrated on the elucidation of the differences between structures of oxidized intermediates O and O H in different CcO variants and at different pH values. Oxidized intermediates of wild type and mutated CcO from Paracoccus denitrificans were studied by means of static and time-resolved Fourier-transform infrared spectroscopy in acidic and alkaline conditions in the infrared region 1800-1000 cm-1. No reasonable differences were found between all variants in these conditions, and in this spectral region. This finding means that the binuclear center of oxygen reduction keeps a very similar structure and holds the same ligands in the studied conditions. The further investigation in search of differences should be performed in the 4000-2000 cm-1 IR region where water ligands absorb.

Keywords: FTIR spectroscopy; cell respiration; cytochrome c oxidase; oxidized state; oxidoreduction.

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Figures

Figure 1
Figure 1
The catalytic cycle of CcO with structures of resolved intermediates. Protons taken by the BNC for the reaction chemistry are shown in magenta. Electrons taken by the BNC are marked in light green. Time constants of the appearance of reaction intermediates are also shown in green. The proposed structures of the catalytic site represent only the BNC structure and provide no information about the other two redox centers (CuA and heme a).
Figure 2
Figure 2
Comparison of the R → O transitions in three different conditions. Time-resolved mode for N131V at pH 6.5 in dark blue and for WT at pH 6.0 in dark green; equilibrium mode for WT at pH 6.0 in red. All spectra normalized to 1 mM (kinetic—by amplitude of 1,965 cm-1 band, equilibrium—by 1661/1641 peaks difference).
Figure 3
Figure 3
Comparison of R → O transitions in four different conditions at pH 9.0. Time- resolved spectra for D124N (in dark blue), N131V (in dark green), and WT (in red); equilibrium spectrum for WT (light green). All spectra were measured at pH 9.0 and normalized to 1 mM (kinetic—by amplitude of 1,965 cm-1 band, equilibrium—by 1661/1641 peaks difference).
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