Molecular basis for the coupling ion selectivity of F1F0 ATP synthases: Probing the liganding groups for Na+ and Li+ in the c subunit of the ATP synthase from Propionigenium modestum
Molecular basis for the coupling ion selectivity of F1F0 ATP synthases: Probing the liganding groups for Na+ and Li+ in the c subunit of the ATP synthase from Propionigenium modestum
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DOI：
10.1021/bi970831q
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发表时间：
1997-07-29
期刊：
影响因子：
2.9
通讯作者：
Dimroth, P
中科院分区：
文献类型：
--
作者：
Kaim, G;Wehrle, F;Dimroth, P
在Propionigenium modestum c亚基的位置65处的保守谷氨酸残基直接参与Na+跨膜的结合和易位。cQ 32 I和cS66 A取代在推定的邻近cE 65的位点特异性引入抑制生长的单位点突变体的琥珀酸最低琼脂，表明这两个氨基酸残基是重要的氧化磷酸化系统的适当功能。然而，如果在P. modestum c亚基中另外存在cF 84 L/cL 87 V双突变，则这种生长抑制被消除。新构建的大肠杆菌菌株MPC 848732 I，具有cQ 32 I/cF 84 L/cL 87 V三重突变，揭示了从Na+到H+的偶联离子特异性的变化。因此，这种酶的ATP水解不被NaCl激活，ATP驱动的H+运输不受这种碱金属盐的影响。这两种活动的影响，但是，氯化锂。这些数据证明了在该突变ATP酶内Na+结合位点的损失和Li+和H+结合位点的保留。在急诊coli菌株MPC 848766 A（cS66 A/cF 84 L/cL 87 V）的ATP酶的专一性进一步被限制为H+作为唯一的偶联离子。因此，Na+和Li+均不刺激ATP酶活性，也没有观察到ATP驱动的Li+转运。E.大肠杆菌突变株MPC 32 N（cQ 32 N）经NaCl和LiCl激活后，突变体ATP酶对NaCl的Km值比亲本菌株高5倍，但对LiCl的Km值没有变化。这些结果表明Na+与P. modestum的c亚基的结合需要由Q32、E65和S66提供的配体基团。对于Li+的配位，两个配体E65和S66是足够的，并且H+易位仅由E65介导。
The conserved glutamate residue at position 65 of the Propionigenium modestum c subunit is directly involved in binding and translocation of Na+ across the membrane. The site-specific introduction of the cQ32I and cS66A substitutions in the putative Vicinity to cE65 inhibited growth of the single-site mutants on succinate minimal agar, indicating that both amino acid residues are important for proper function of the oxidative phosphorylation system. This growth inhibition was abolished, however, if the cF84L/cL87V double mutation was additionally present in the P. modestum c subunit. The newly constructed Escherichia coli strain MPC848732I, harboring the cQ32I/cF84L/cL87V triple mutation, revealed a change in the coupling ion specificity from Na+ to H+. ATP hydrolysis by this enzyme was therefore not activated by NaCl, and ATP-driven H+ transport was not affected by this alkali salt. Both activities were influenced, however, by LiCl. These data demonstrate the loss of the Na+ binding site and retention of Li+ and H+ binding sites within this mutant ATPase. In the E. coli strain MPC848766A (cS66A/cF84L/cL87V), the specificity of the ATPase was further restricted to H+ as the exclusive coupling ion. Therefore, neither Na+ nor Li+ stimulated the ATPase activity, and no ATP-driven Li+ transport was observed. The ATPase of the E. coli mutant MPC32N (cQ32N) was activated by NaCl and LiCl. The mutant ATPase exhibited a 5-fold higher K-m for NaCl but no change in the K-m for LiCl in comparison to that of the parent strain. These results demonstrate that the binding of Na+ to the c subunit of P. modestum requires liganding groups provided by Q32, E65, and S66. For the coordination of Li+, two liganding partners, E65 and S66, are sufficient, and H+ translocation was mediated by E65 alone.