Myoglobin (Mb), besides its roles as an oxygen (O₂) carrier/storage protein and nitric oxide NO scavenger/producer, may participate in lipid trafficking and metabolite binding. Our recent findings have shown that O₂ is released from oxy-Mb upon interaction with lactate (LAC, anerobic glycolysis end-product). Since pyruvate (PYR) is structurally similar and metabolically related to LAC, we investigated the effects of PYR (aerobic glycolysis end-product) on Mb using isothermal titration calorimetry, circular dichroism, and O₂-kinetic studies to evaluate PYR affinity toward Mb and to compare the effects of PYR and LAC on O₂ release kinetics of oxy-Mb. Similar to LAC, PYR interacts with both oxy- and deoxy-Mb with a 1:1 stoichiometry. Time-resolved circular dichroism spectra revealed that there are no major conformational changes in the secondary structures of oxy- or deoxy-Mb during interactions with PYR or LAC. However, we found contrasting results with respect to binding affinities and substrate preference, where PYR has higher affinity toward deoxy-Mb when compared with LAC (which prefers oxy-Mb). Furthermore, PYR interaction with oxy-Mb releases a significantly lower amount of O₂ than LAC. Taken together, our findings support the hypothesis that glycolytic end-products play a distinctive role in the Mb-rich tissues by serving as novel regulators of O₂ availability, and/or by impacting other activities related to oxy-/deoxy-Mb toggling in resting vs. exercised or metabolically activated conditions.

Myoglobin (Mb)-mediated oxygen (O₂) delivery and dissolved O₂ in the cytosol are two major sources that support oxidative phosphorylation. During intense exercise, lactate (LAC) production is elevated in skeletal muscles as a consequence of insufficient intracellular O₂ supply. The latter results in diminished mitochondrial oxidative metabolism and an increased reliance on nonoxidative pathways to generate ATP. Whether or not metabolites from these pathways impact Mb-O₂ associations remains to be established. In the present study, we employed isothermal titration calorimetry, O₂ kinetic studies, and UV-Vis spectroscopy to evaluate the LAC affinity toward Mb (oxy- and deoxy-Mb) and the effect of LAC on O₂ release from oxy-Mb in varying pH conditions (pH 6.0-7.0). Our results show that LAC avidly binds to both oxy- and deoxy-Mb (only at acidic pH for the latter). Similarly, in the presence of LAC, increased release of O₂ from oxy-Mb was detected. This suggests that with LAC binding to Mb, the structural conformation of the protein (near the heme center) might be altered, which concomitantly triggers the release of O₂. Taken together, these novel findings support a mechanism where LAC acts as a regulator of O₂ management in Mb-rich tissues and/or influences the putative signaling roles for oxy- and deoxy-Mb, especially under conditions of LAC accumulation and lactic acidosis.

Myoglobin (Mb) interaction with the outer mitochondrial membrane (OMM) promotes oxygen (O₂) release. However, comprehensive molecular details on specific contact regions of the OMM with oxygenated (oxy-) and deoxygenated (deoxy-)Mb are missing. We used molecular dynamics (MD) simulations to explore the interaction of oxy- and deoxy-Mb with the membrane lipids of the OMM in two lipid compositions: (a) a typical whole membrane on average, and (b) specifically the cardiolipin-enriched cristae region (contact site). Unrestrained relaxations showed that on average, both the oxy- and deoxy-Mb established more stable contacts with the lipids typical of the cristae contact site, then with those of the average OMM. However, in steered detachment simulations, deoxy-Mb clung more tightly to the average OMM, and oxy-Mb strongly preferred the contact sites of the OMM. The MD simulation analysis further indicated that a non-specific binding, mediated by local electrostatic interactions, existed between charged or polar groups of Mb and the membrane, for stable interaction. To the best of our knowledge, this is the first computational study providing the molecular details of the direct Mb-mitochondria interaction that assisted in distinguishing the preferred localization of oxy- and deoxy-Mb on the OMM. Our findings support the existing experimental evidence on Mb-mitochondrial association and shed more insights on Mb-mediated O₂ transport for cellular bioenergetics.