Hexokinase II (HKII) plays a critical role in ATP production by catalyzing the initial step in glucose metabolism and maintaining the gradient for glucose entry into the cell. HKII also closely interacts with the mitochondrial outer membrane protein, voltage dependent anion channel (VDAC), to permit the channeling of mitochondrial generated ATP, its substrate, for use in cellular metabolism. It has been reported that adding protease to mitochondrial isolation buffer may improve mitochondrial respiration. However, proteases could potentially degrade the HKII‐VDAC protein interaction at the interphase between mitochondrial outer membrane and extra‐matrix space. We hypothesize that protease interferes with the association of HKII to VDAC and this alters mitochondrial bioenergetics. To test this, we isolated mitochondria from brain and heart of guinea pigs to determine the consequences of HKII binding and unbinding to VDAC on mitochondrial respiration and membrane potential (ΔΨm). Mitochondria were isolated by differential centrifugation using the same isolation buffers except one with and one without the presence of protease. We examined the HKII binding to mitochondria, specifically to VDAC, between the two mitochondrial groups using Western blot (WB) and immunoprecipitation (IP). We measured the rate of O2 consumption using mitochondrial substrates before and after adding ADP and evaluated changes in ΔΨm using TMRM fluorescent dye. WB showed, in both heart and brain mitochondria, binding of HKII to mitochondria in the absence of protease was evident, but this was not detected in mitochondria isolated in the presence of protease. Moreover, IP experiments with cross‐linking revealed HKII‐VDAC association in the protease‐free isolation buffer, but not in the buffer containing protease. Mitochondrial respiration as determined by states 3 (ADP respiration) and 4 (ADP depleted respiration) respiration was significantly slower when HKII was bound to VDAC compared to when it was not bound to VDAC. ΔΨm repolarization following ADP‐induced depolarization (state 3 respiration) was substantially delayed when mitochondria were isolated in the absence, in contrast to the presence, of protease. Our results indicate that HKII binding to isolated mitochondria directly modulates oxidative phosphorylation, possibly by regulating the transfer of substrates and metabolites, including ADP/ATP, via VDAC. Overexpression of HKII has been implicated in cytoprotection; however, the underlying mechanisms is not well understood. The implications of the increased association or dissociation of HKII‐VDAC in cell physiology (survival) and/or pathophysiology (cell death) will be further explored in intact cardiac and brain cells.Support or Funding InformationThis work was supported in part by the Veterans Administration (BX‐002539‐01) and the National Institutes of Health (R01 HL131673‐01A1 and P01‐GM066730).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.