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Cardiolipin modulates allosterically the nitrite reductase activity of horse heart cytochrome c
Paolo Ascenzi1, Maria Marino, Fabio Polticelli
1Interdepartmental Laboratory for Electron Microscopy, University Roma Tre, Via della Vasca Navale 79, 00146, Rome, Italy, ascenzi@uniroma3.it.
Insights
Cardiolipin liposomes significantly enhance the nitrite reductase activity of ferrous cytochrome c (cytc-Fe(II)) by promoting its nitrosylation. This interaction may prevent lipid peroxidation, potentially inhibiting the cell death program.
Area of Science:
- Biochemistry
- Molecular Biology
- Cellular Biology
Background:
- Cytochrome c (cytc) undergoes structural and functional changes upon binding to cardiolipin (CL) liposomes, including altered redox potential and increased affinity for CO and NO.
- CL liposomes are known to influence cytc's peroxidase activity and facilitate peroxynitrite isomerization, suggesting a role in cellular redox signaling.
Purpose of the Study:
- To investigate the effect of cardiolipin (CL) liposomes on the nitrite reductase activity of ferrous cytochrome c (cytc-Fe(II)).
- To elucidate the mechanism by which CL liposomes modulate cytc-Fe(II) nitrosylation and its potential implications in cellular processes.
Main Methods:
- Spectrophotometric assays were used to measure the rate constants for the nitrite-mediated conversion of cytc-Fe(II) to cytc-Fe(II)-NO in the presence and absence of CL liposomes.
- The binding affinity of CL liposomes to cytc-Fe(II) was determined by measuring apparent dissociation equilibrium constants at different pH values.
- Heme coordination state changes were inferred based on functional activity modulation.
Main Results:
- In the absence of CL liposomes, ferrous cytc-Fe(II) exhibits very low nitrite reductase activity (k_on = 7.3 × 10⁻² M⁻¹s⁻¹).
- CL liposomes significantly enhance this activity in a dose-dependent manner, increasing the rate constant to 2.6 M⁻¹s⁻¹ and inducing penta-coordination of the heme-Fe(II) atom.
- Apparent dissociation equilibrium constants for CL liposomes binding to cytc-Fe(II) were in the low micromolar range (1.4–2.2 × 10⁻⁶ M) across physiological pH values.
Conclusions:
- Cardiolipin liposomes dramatically increase the nitrite reductase activity of ferrous cytochrome c by facilitating its nitrosylation.
- This enhanced activity, mediated by CL-induced structural changes in cytc-Fe(II), may play an anti-apoptotic role by inhibiting lipid peroxidation.
- The findings suggest a novel mechanism by which cytc-CL interactions can modulate cellular redox homeostasis and potentially prevent apoptosis initiation.
Abstract:
Upon cardiolipin (CL) liposomes binding, horse heart cytochrome c (cytc) changes its tertiary structure disrupting the heme-Fe-Met80 distal bond, reduces drastically the midpoint potential, binds CO and NO with high affinity, displays peroxidase activity, and facilitates peroxynitrite isomerization. Here, the effect of CL liposomes on the nitrite reductase activity of ferrous cytc (cytc-Fe(II)) is reported. In the absence of CL liposomes, hexa-coordinated cytc-Fe(II) displays a very low value of the apparent second-order rate constant for the NO2 (-)-mediated conversion of cytc-Fe(II) to cytc-Fe(II)-NO (k on = (7.3 ± 0.7) × 10(-2) M(-1) s(-1); at pH 7.4 and 20.0 °C). However, CL liposomes facilitate the NO2 (-)-mediated nitrosylation of cytc-Fe(II) in a dose-dependent manner inducing the penta-coordination of the heme-Fe(II) atom. The value of k on for the NO2 (-)-mediated conversion of CL-cytc-Fe(II) to CL-cytc-Fe(II)-NO is 2.6 ± 0.3 M(-1) s(-1) (at pH 7.4 and 20.0 °C). Values of the apparent dissociation equilibrium constant for CL liposomes binding to cytc-Fe(II) are (2.2 ± 0.2) × 10(-6) M, (1.8 ± 0.2) × 10(-6) M, and (1.4 ± 0.2) × 10(-6) M at pH 6.5, 7.4, and 8.1, respectively, and 20.0 °C. These results suggest that the NO2 (-)-mediated conversion of CL-cytc-Fe(II) to CL-cytc-Fe(II)-NO could play anti-apoptotic effects impairing lipid peroxidation and therefore the initiation of the cell death program by the release of pro-apoptotic factors (including cytc) in the cytoplasm.
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