Effect of Artificial and Natural Phospholipid Membranes on Rate of Sperm Whale Oxymyoglobin Autooxidation

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G. B. Postnikova^* and E. A. Shekhovtsova

Institute of Cell Biophysics, Russian Academy of Sciences, ul. Institutskaya 3, 142290 Pushchino, Moscow Region, Russia; fax: (496) 733-0509; E-mail: gb­­_post@icb.psn.ru

^* To whom correspondence should be addressed.

Received June 15, 2012; Revision received October 1, 2012

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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 (K_dis) can be detected by changes of the rate of MbO₂ autooxidation, i.e. spontaneous turning it into metMb (k_ox), as far as a direct correlation between K_dis and k_ox 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, k_ox was found to increase due to interaction of the protein with phospholipid membranes. The effect of negatively charged liposomes from POPG on k_ox 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, k_ox is only ~10 times higher (10 mM buffer, pH 7.2, 22°C). With the same phospholipid/MbO₂ ratio of 100 : 1, k_ox is 7 times higher for the POPG than for lecithin liposomes. In the presence of mitochondria inhibited by antimycin A, k_ox 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 k_ox 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 K_dis) 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.

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KEY WORDS: myoglobin, autooxidation, phospholipid membranes, mitochondria

DOI: 10.1134/S0006297913030085

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