Significance The plant cysteine oxidases (PCOs) have been identified as oxygen-sensing enzymes in plants, controlling hypoxia-dependent processes, including adaptive responses to flooding. As such, they are potential targets for engineering plants with enhanced flood tolerance. To approach this in a rational manner requires an understanding of how PCO structure relates to their ability to trigger hypoxic adaptation. We report the structures of two PCOs from Arabidopsis and show that the effects of mutagenesis of key amino acids at their active site are seen both in vitro and, importantly, in planta. This work provides a platform for further efforts to manipulate PCO structure and function to improve the ability of crops to withstand future climate extremes.