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 fr

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• 作者：Georg Kaim ; Franziska Wehrle ; Ursula Gerike ; and Peter Dimroth
• 刊名：Biochemistry
• 出版年：1997
• 出版时间：July 29, 1997
• 年：1997
• 卷：36
• 期：30
• 页码：9185 - 9194
• 全文大小：217K
• 年卷期：v.36,no.30(July 29, 1997)
• ISSN：1520-4995

文摘

The conserved glutamate residue at position 65 of thePropionigenium modestum c subunit isdirectly involved in binding and translocation of Na⁺across the membrane. The site-specific introductionof the cQ32I and cS66A substitutions in the putative vicinity to cE65inhibited growth of the single-sitemutants on succinate minimal agar, indicating that both amino acidresidues are important for properfunction of the oxidative phosphorylation system. This growthinhibition was abolished, however, if thecF84L/cL87V double mutation was additionally present in the P.modestum c subunit. The newlyconstructed Escherichia coli strain MPC848732I, harboringthe cQ32I/cF84L/cL87V triple mutation,revealed a change in the coupling ion specificity fromNa⁺ to H⁺. ATP hydrolysis by thisenzyme wastherefore not activated by NaCl, and ATP-driven H⁺transport was not affected by this alkali salt. Bothactivities were influenced, however, by LiCl. These datademonstrate the loss of the Na⁺ binding siteand retention of Li⁺ and H⁺ binding siteswithin this mutant ATPase. In the E. coli strainMPC848766A(cS66A/cF84L/cL87V), the specificity of the ATPase was furtherrestricted to H⁺ as the exclusive couplingion. Therefore, neither Na⁺ nor Li⁺stimulated the ATPase activity, and no ATP-driven Li⁺transportwas observed. The ATPase of the E. coli mutant MPC32N(cQ32N) was activated by NaCl and LiCl.The mutant ATPase exhibited a 5-fold higherK_m for NaCl but no change in theK_m for LiCl in comparisonto that of the parent strain. These results demonstrate that thebinding of Na⁺ to the c subunit of P.modestum requires liganding groups provided by Q32, E65, and S66.For the coordination of Li⁺, twoliganding partners, E65 and S66, are sufficient, and H⁺translocation was mediated by E65 alone.