The Effect of Mitochondrial and Artificial Bilayer Phospholipid Membranes on Conformation of Myoglobin and Its Affinity for Oxygen
We have first shown that oxygen release from MbO2 at near-zero O2 concentrations (p02) only proceeds when interacting the protein with respiring mitochondria, but no deoxygenation of MbO2 occurs, if they are separated from MbO2 solution by a semi permeable membrane. The rates of O2 uptake by mitochondria from solution in the presence of MbO2 (V1) and MbO2 deoxygenation (V2) completely coincide for different mitochondrial preparations, the native, frozen and uncoupled by FCCP, as both V1 and V2 are determined by respiratory activity of mitochondria. However, V1 and V2 reflect different processes, because they are differently affected by the proteins like lysozyme, competing with MbO2 for binding to mitochondria. It is found that myoglobin non-specifically interacts with phospholipid sites of the outer mitochondrial membrane, while any specific proteins or protein channels for the binding to myoglobin are lacking. Physiologically active MbO2 and not active metmyoglobin (metMb) adsorb on the surface of the artificial BLMs from neutral lecithin and negatively charged 1-palmitoyl-2-oleilphosphatidylglycerol (POPG), the adsorption activity is being much higher for negatively charged BLM and two–three fold larger for metMb compared to MbO2. The pronounced ionic strength dependence of the binding implies significant contribution of coulombic electrostatics into the formation of myoglobin–mitochondrial complex, most probably due to local electrostatic interactions between oppositely charged groups of phospholipids (the heads) and polar myoglobin residues (invariant Lys and Arg) near the heme cavity. Interaction of metMb with POPG liposomes leads to conformational changes first of all in the heme cavity, while secondary and tertiary structures of the protein are preserved. Significant increasing of MbO2 autooxidation rate under aerobic conditions, much more pronounced in the presence of mitochondria and negatively charged POPG liposomes, as well as some shift of MbO2 / Mb(2) equilibrium towards ligand-free Mb(2) under anaerobic ones, both evidence in favor of decreased myoglobin affinity for O2, induced by its interaction with phospholipid membranes, which must facilitate O2 detachment from MbO2 at physiological p02 values in the cell.
Myoglobin, Mb, Hemoglobin, Hb, Mitochondria, MC, Oxymyoglobin, MbO2, Oxyhemoglobin, HbO2, Bilayer Phospholipid Membrane, BLM, Partial Pressure of Oxygen, pO2, Affinity for O2, p50
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