Myoglobin-Pyruvate Interactions: Binding Thermodynamics, Structure-Function Relationships, and Impact on Oxygen Release Kinetics

Abstract

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.

Keywords: myoglobin; oxygen release; pyruvate.

Figure 1

Representative ITC plots of binding of PYR with equine Mb. Top row (a–c) displays PYR interaction with oxy-Mb at (a) pH 7.0, (b) pH 6.4, and (c) pH 6.0. Bottom row (d–f) displays PYR interaction with deoxy-Mb at (d) pH 7.0 (e) pH 6.4, and (f) pH 6.0. In each sub-figure, raw data (upper panels) and integrated data (lower panels) represent titration of reactants with time (min) or molar ratios on the x-axis and the energy released or absorbed per injection on the y-axis. The solid lines in the bottom panels represent the best-fit of experimental data using ‘one-set of sites’ model provided by the manufacturer’s software (Microcal PEAQ-ITC software). The lower graphs clearly differentiate that for oxy- and deoxy-Mb pH 7.0, and deoxy-Mb at pH 6.4, Mb-PYR binding was predominantly exothermic (upward slope) driven by hydrophobic interactions (details are given in Section 2). In contrast, at acidic pH (pH 6.4 and pH 6.0), the Mb–LAC binding was endothermic (downward slopes) and mostly favored by hydrophilic interactions. All the ITC experiments were repeated 3 times (n = 3) to obtain the thermodynamic properties. Statistical analysis was performed using one-way ANOVA. Shown here is one representative dataset from a single experiment per condition. In each sub-figure, heat flow (DP) and change in enthalpy (ΔH) are shown here.

Figure 2

Representative signature plots of binding of PYR with equine Mb. Top row (a–c) displays PYR interaction with oxy-Mb at (a) pH 7.0, (b) pH 6.4, and (c) pH 6.0. Bottom row (d–f) displays PYR interaction with deoxy-Mb at (d) pH 7.0, (e) pH 6.4, and (f) pH 6.0. All the ITC experiments were repeated 3 times (n = 3) to obtain the thermodynamic properties. Statistical analysis was performed using one-way ANOVA.

Figure 3

Effect of PYR binding to oxy-Mb and O₂ release. O₂ release kinetics were studied using an Oxytherm+ respirometer. All experiments were performed in 50 mM sodium phosphate buffer (pH 7.0, pH 6.4 and pH 6.0) containing 150 μM of oxy-Mb-enriched equine Mb preparations and varying concentrations of PYR (0.5 mM to 5 mM), using oxy-Mb alone as a zero-PYR control. (a) Representative graph showing O₂ release from oxy-Mb after addition of varying concentrations of PYR at pH 7.0. No release of O₂ from oxy-Mb in acidic pH (pH 6.0–pH 6.4) was detected (Figure S1). (b) Rate of release of O₂ from Mb against PYR concentrations at pH 7.0, calculated from the linear portion of the graphs after addition of PYR to oxy-Mb. All O₂ experiments were repeated 3 times (n = 3). Statistical analysis was performed using one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

Docking structures of PYR binding with equine skeletal muscle Mb. Top row displays oxy-Mb at (a) pH 7.0, (b) pH 6.4 and (c) pH 6.0, and bottom row displays deoxy-Mb at (d) pH 7.0, (e) pH 6.4 and (f) pH 6.0, respectively. PYR (brown), heme center (pink), and the amino acid residues (cyan) interacting with PYR are displayed as sticks. Mb protein (green) is displayed as ribbon structure an oxygen (red) in spheres. Possible hydrogen bond interactions between side chains of residues and PYR are displayed as dashed yellow lines with bond length in angstroms.

Figure 5

A schematic representation displays the stoichiometric turnover events on conversion of oxy-myoglobin (oxy-Mb) to deoxy-myoglobin (deoxy-Mb) and vice versa in metabolite-bound Mb states in a normal aerobic cell (resting) at constant supply of diffused oxygen (O₂), or in working muscle characterized by reduced pO₂ and lower pH. In resting conditions (displayed in light green color background, leftward), aerobic glycolysis generates PYR that avidly binds to deoxy-Mb (shown in solids arrows). Similarly, LAC (end-product of anaerobic glycolysis), increasingly generated as workload increases and pO₂ drops (displayed in light red color background, rightward), binds avidly to oxy-Mb, but does not bind to deoxy-Mb [32]. However, in working muscle cells and at elevated lactic acid (LAC) levels (>1.0 mM), although PYR binds to both oxy-Mb and deoxy-Mb with low binding affinities, LAC strongly binds to Mb in acidic conditions and releases O₂ from oxy-Mb [32]. In all these proposed events, release of Mb-bound PYR/LAC is not clearly known. Solid arrows () display high affinity and rapid reaction rates and dashed arrows () display lower affinity and slower reaction rates. oxy-Mb: oxygenated-Mb; deoxy-Mb: deoxygenated-Mb; PYR: pyruvate; LAC: lactate; TCA cycle: tricarboxylic acid cycle; ETC: electron transport chain; MPC: mitochondrial pyruvate complex; mLOC: mitochondrial lactate oxidation complex; MCT4: monocarboxylate transporter 4.