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

Abstract 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.