* To whom correspondence should be addressed.
Received June 15, 2012; Revision received October 1, 2012
We were the first to show that MbO2 deoxygenation in the cell occurs only upon interaction of myoglobin with mitochondrial membrane, which must be accompanied by changes in the heme cavity conformation of the protein and its affinity for the ligand. Under aerobic conditions, some changes in the equilibrium O2 dissociation constant (Kdis) can be detected by changes of the rate of MbO2 autooxidation, i.e. spontaneous turning it into metMb (kox), as far as a direct correlation between Kdis and kox is experimentally shown. In this work, we studied the effect on MbO2 autooxidation rate of phospholipid liposomes from neutral soybean phosphatidylcholine (lecithin) and from negatively charged 1-palmitoyl-2-oleylphosphatidylglycerol (POPG) at various phospholipid/MbO2 ratios from 25 : 1 to 100 : 1, and also the effect of rat liver mitochondria at concentration of 1 and 2 mg/ml mitochondrial protein (at 22 and 37°C). In all cases, kox was found to increase due to interaction of the protein with phospholipid membranes. The effect of negatively charged liposomes from POPG on kox is significantly greater than that of neutral lecithin liposomes. At the POPG/MbO2 molar ratio of 25 : 1, MbO2 autooxidation rate is almost 25-fold increased compared to the control, whereas in the presence of 50-fold molar excess of lecithin, kox is only ~10 times higher (10 mM buffer, pH 7.2, 22°C). With the same phospholipid/MbO2 ratio of 100 : 1, kox is 7 times higher for the POPG than for lecithin liposomes. In the presence of mitochondria inhibited by antimycin A, kox grows proportionally to their concentration (about 10-fold per 1 mg/ml of mitochondrial protein), and practically does not change after adding superoxide dismutase in the reaction mixture. The kox value decreases markedly at high ionic strength, thus suggesting an important role of coulombic electrostatics in the myoglobin–mitochondrial interaction. The increase in the autooxidation rate of MbO2 (and hence its Kdis) due to the interaction with phospholipid membranes points to decreasing affinity of myoglobin for oxygen, which facilitates O2 detachment from MbO2 at physiological p02 values.
KEY WORDS: myoglobin, autooxidation, phospholipid membranes, mitochondria