Chen, W.; Nelson, O. D.; Li, X.; Zhang, M.; Lu, X.; Yu, T.; Yao, C.-H.; Zhang, S.; Zhang, Y.; Francisco, A.; Ahn, B.; Lundberg, E.; Song, M.; Rahbani, J. F.; Chen, Z.; Lin, H.

Coenzyme A (CoA) is an essential cofactor required for numerous metabolic reactions, yet its ability to bind and regulate proteins remains poorly defined. Using a proteomic approach, we identified malic enzyme 2 (ME2) as a CoA-binding protein. ME2 uses NAD(P)+ to convert malate to pyruvate, generating NAD(P)H to support energy production and redox homeostasis. ME2 binds CoA at an allosteric site previously thought to bind NAD(P)+. Reduced CoA has minimal effect on ME2 activity, but the oxidized form, CoA disulfide, strongly activates ME2 by promoting ME2 tetramerization and a catalytically efficient closed conformation. Under oxidative stress, ME2 facilitates CoA disulfide formation, enhancing NADPH production and cellular defense against reactive oxygen species (ROS). Mice with ME2 mutation that cannot bind CoA show impaired muscle performance, elevated ROS, and mitochondrial dysfunction. These findings establish CoA as a redox-sensing cofactor, allowing cells to respond to ROS and promote mitochondrial metabolism, and expanding the function of this essential cofactor.