Kinetic characterization of respirasomes and free complex I from Yarrowia lipolytica

The mitochondrion is a highly dynamic organelle capable of adapting to external stimuli and the energetic demands of the cell. As the primary source of cellular ATP, generating approximately 90 % of the total, mitochondrion facilitates the association of respiratory complexes I, III₂, and IV into supramolecular structures called respirasomes. This supramolecular organization enhances protein density within the mitochondrial inner membrane, enabling homogenous energy production. In this study, we investigate the subunits composition and the kinetic characterization of digitonin-solubilized respirasomes and the free complex I from Yarrowia lipolytica as well as their role in reactive oxygen species (ROS) production. The NADH:DBQ oxido reductase activity of respirasome and free complex I was similar. Respiration by respirasome was inhibited with rotenone, antimycin A, or cyanide, simultaneously to an increase in the ROS production. A value of 1.6 ± 0.2 for the NADH oxidized/oxygen reduced ratio was determined for the respirasome activity. The role of interaction between complexes in the function of the respirasome is discussed.