Reductive nitrosylation of the cardiolipin-ferric cytochrome c complex

Area of Science:

• Biochemistry
• Chemical Kinetics
• Molecular Biology

Background:

• Native horse heart cytochrome c (cytc) exhibits limited ligand reactivity and lacks catalytic activity.
• Bovine cardiolipin (CL) binding confers myoglobin-like properties to cytc.
• Nitric oxide (NO) signaling is crucial in various cellular processes, including apoptosis.

Purpose of the Study:

• To investigate the kinetics and mechanisms of nitric oxide (NO) binding to cardiolipin-cytochrome c (CL-cytc) across a physiological pH range.
• To elucidate the pH-dependent transformations of CL-cytc upon NO interaction.
• To explore the potential role of CL-cytc nitrosylation in apoptosis regulation.

Main Methods:

• Stopped-flow spectroscopy to monitor NO binding kinetics.
• UV-Vis spectroscopy to characterize spectral changes.
• Kinetic analysis of nitrosylation and denitrosylation reactions at varying pH and NO concentrations.

Main Results:

• CL-cytc undergoes reversible nitrosylation at pH 7.2 and irreversible reductive nitrosylation between pH 7.9 and 9.5, forming CL-cytc(II)-NO.
• NO binding to CL-cytc(III) is limited by heme-Fe bond cleavage, with a first-order rate constant of 9.3 s⁻¹.
• A pH-dependent conversion of nitrosylated CL-cytc(III) to CL-cytc(II) (rate constant 3.0 × 10³ M⁻¹s⁻¹) and subsequent nitrosylation of CL-cytc(II) (rate constant 1.4 × 10⁷ M⁻¹s⁻¹) were observed.

Conclusions:

• Cardiolipin binding significantly alters cytc's reactivity towards NO, enabling distinct nitrosylation pathways.
• The observed nitrosylation and denitrosylation kinetics suggest a potential regulatory role for CL-cytc in apoptosis.
• Understanding these molecular mechanisms provides insights into NO signaling and cellular fate determination.