Ischaemia-reperfusion (IR) injury is caused paradoxically when the flow of oxygenated blood to an organ is restored after a period of restricted blood supply (ischaemia) in the course of a heart attack, stroke or organ transplantation. Mitochondria play a central role in the pathology of IR injury and are also at the heart of metabolism, which becomes dysregulated during ischaemia and causes formation of reactive oxygen species (ROS) when oxygen is resupplied. Complex I is a major source of ROS in IR injury, but also in physiological ROS signalling. The enzyme can produce ROS via reverse electron transfer (RET), which is driven by a large protonmotive force (∆p) in conjunction with a reduced coenzyme Q (CoQ) pool, with the required electrons deriving from succinate oxidation. ROS causes severe damage to cellular components, leading to cell death and ultimately to chronic inflammation. In order to expand our knowledge on the role and function of complex I in IR injury, I have developed a new mass spectrometry approach to characterise the reversible deactivation (active/deactive transition) of the complex based on the exposure of the critical Cys39 residue of its ND3 subunit. I confirmed that complex I gradually deactivates in the absence of turnover, in simplified in vitro model systems as well as during ischaemia in vivo. Conversely, reperfusion resulted in rapid reactivation of complex I. Further, declining complex I activity was found to inversely correlate with an increase in Cys39 exposure, which suggests a causal relationship. Beyond this, I developed a robust, sensitive and broadly applicable mass spectrometry-based method to determine the CoQ pool size and redox state in vivo. This method enabled me to explore the CoQ redox state during IR in heart. Remarkably, the CoQ pool appeared to be highly reduced in vivo, independent of the oxygen availability and metabolic changes throughout ischaemia. Finally, I explored RET derived ROS formation and its thermodynamic drivers in mitochondria and tissue from mice, harbouring a P25L point mutation within their ND6 subunit of complex I, which prevents the enzyme from performing RET. In a collaboration with the Hirst laboratory we combined cryoEM derived structural information from their lab with in vivo data from a myocardial infarction model that enabled us to identify the RET-blocking mechanism caused by this mutation and to confirm RET derived ROS as major cause for IR injury. In summary, my work provides new experimental tools and insights into the role and dynamics of complex I, RET-derived ROS production and its drivers during IR in vivo.