Abstract Body: Mitochondrial matrix Ca²⁺ concentration ([_matrixCa²⁺]) is theorized to be an essential regulator of cardiac metabolism by positively regulating key mitochondrial dehydrogenases housed within the mitochondrial matrix. However, ablation or functional inhibition of the mitochondrial calcium uniporter channel (mtCU) fails to significantly perturb basal metabolism and is largely phenotypically silent in the absence of stress. This begs the question, what are the primary molecular mechanisms regulating calcium-dependent changes in cardiac metabolism? The primary function of MICU proteins (MICU1, MICU2, and MICU3) is reported to be gatekeeping of the mtCU and regulating mitochondrial Ca²⁺ uptake. Here, we demonstrate that MICU proteins function in coordination to impart Ca²⁺-dependent regulation to FADH₂-dependent mitochondrial dehydrogenases through metabolon formation independent of the mtCU and [_matrixCa²⁺]. Our results demonstrate that MICU proteins differentially localize to mitochondrial microdomains and form heterodimers and interactomes in response to intermembrane space Ca²⁺ binding their respective EF-hand domains. Utilizing an equimolar expression platform coupled with unbiased proteomics we reveal unique interactomes for MICU1/2 versus MICU1/3 heterodimers and demonstrate that MICU proteins control coupling of Mitochondrial Glycerol-3-Phosphate Dehydrogenase with Succinate Dehydrogenase/Complex II and impart Ca^2+-dependent changes in activity. We propose that MICU-mediated mitochondrial metabolons are a fundamental system facilitating matching of mitochondrial energy production with cellular demand and is the primary physiological Ca²⁺ signaling mechanism regulating homeostatic energetics – not mtCU-dependent changes in [_matrixCa²⁺].