The Effect of Reactive Oxygen Species on Respiratory Complex I Activity in Liposomes

Abstract Respiratory complex I (R‐CI) is an essential enzyme in the mitochondrial electron transport chain but also a major source of reactive oxygen species (ROS), which are implicated in neurodegenerative diseases and ageing. While the mechanism of ROS production by R‐CI is well‐established, the feedback of ROS on R‐CI activity is poorly understood. Here, we perform EPR spectroscopy on R‐CI incorporated in artificial membrane vesicles to reveal that ROS (particularly hydroxyl radicals) reduce R‐CI activity by making the membrane more polar and by increasing its hydrogen bonding capability. Moreover, the mechanism that we have uncovered reveals that the feedback of ROS on R‐CI activity via the membrane is transient and not permanent; lipid peroxidation is negligible for the levels of ROS generated under these conditions. Our successful use of modular proteoliposome systems in conjunction with EPR spectroscopy and other biophysical techniques is a powerful approach for investigating ROS effects on other membrane proteins.