Structural Basis for Inhibition of ROS‐Producing Respiratory Complex I by NADH‐OH

NADH:ubiquinone oxidoreductase, respiratory complex I, plays a central role in cellular energy metabolism. As a major source of reactive oxygen species (ROS) it affects ageing and mitochondrial dysfunction. The novel inhibitor NADH‐OH specifically blocks NADH oxidation and ROS production by complex I in nanomolar concentrations. Attempts to elucidate its structure by NMR spectroscopy have failed. Here, by using X‐ray crystallographic analysis, we report the structure of NADH‐OH bound in the active site of a soluble fragment of complex I at 2.0 Å resolution. We have identified key amino acid residues that are specific and essential for binding NADH‐OH. Furthermore, the structure sheds light on the specificity of NADH‐OH towards the unique Rossmann‐fold of complex I and indicates a regulatory role in mitochondrial ROS generation. In addition, NADH‐OH acts as a lead‐structure for the synthesis of a novel class of ROS suppressors. The novel inhibitor NADH‐OH binds with high affinity to respiratory complex I, thereby blocking NADH oxidation and the production of reactive oxygen species. The tight interaction is mediated by residues conserved in complex I but lacking in other enzymes containing a Rossmann‐fold. Accordingly, it is a lead structure for the development of new drugs for fighting oxygen stress.