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

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. Respiratory complex I (R-CI) is a major contributor to reactive oxygen species (ROS) in mitochondria. By reconstituting R-CI into artificial membrane vesicles and in conjunction with EPR spectroscopy, the effects of ROS are investigated. The activity of R-CI is reduced because of ROS, not only by damaging the enzyme but also by making the membrane more polar and protic. image