Zhang Y, Fillingame R H
Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison 53706, USA.
J Biol Chem. 1995 Oct 13;270(41):24609-14.
Second site suppressor mutations at position 31 of F1 subunit epsilon recouple ATP-driven H+ translocation in the uncoupled Q42E mutant of subunit c of the Escherichia coli F1F0 ATP synthase (Zhang, Y., Oldenburg, M., and Fillingame, R. H. (1994) J. Biol. Chem. 269, 10221-10224). This finding suggests a functional interaction between subunit c and subunit epsilon during the coupling of H+ transport through F0 to ATP synthesis of F1. However, the physical proximity of the two subunits remained to be defined. In this study, Cys residues were introduced into residues in the polar loop region of subunit c surrounding Gln42 and at position 31 of subunit epsilon to see whether the subunits could be cross-linked. Disulfide bridge formation between subunit c and subunit epsilon was observed in membranes of three double mutants, i.e. cA40C/epsilon E31C, cQ42C/epsilon E31C, and cP43C/epsilon E31C, but not in wild type membranes or in membranes of the cA39C/epsilon E31C double mutant. These results indicate that the polar loop of subunit c and the region around residue 31 of subunit epsilon are physically close to each other in the F1F0 complex and support the hypothesis that these two subunits interact directly in the coupling of H+ transport to ATP synthesis. Disulfide cross-linking of the Q42C subunit c and E31C subunit epsilon leads to inhibition of ATPase coupled H+ transport, as might be expected in a model where the catalytic sites of the F1 ATPase alternate during H+ transport-coupled ATP hydrolysis/synthesis. However, a quantitative relationship between the extent of inhibition of transport and the extent of cross-linking could not be established by the methods used here, and the possibility remains that the epsilon-c cross-linked F1F0 complex retains residual H+ transporting activity.
在大肠杆菌F1F0 ATP合酶c亚基的解偶联Q42E突变体中，F1亚基ε第31位的第二位点抑制突变恢复了ATP驱动的H⁺转运偶联（张，Y.，奥尔登堡，M.，和菲林盖姆，R. H.（1994年）《生物化学杂志》269，10221 - 10224）。这一发现表明，在H⁺通过F0转运与F1的ATP合成偶联过程中，c亚基和ε亚基之间存在功能相互作用。然而，这两个亚基的物理接近性仍有待确定。在本研究中，将半胱氨酸残基引入c亚基围绕Gln42的极性环区域的残基以及ε亚基的第31位，以观察这两个亚基是否可以交联。在三个双突变体的膜中观察到了c亚基和ε亚基之间二硫键的形成，即cA40C/εE31C、cQ42C/εE31C和cP43C/εE31C，但在野生型膜或cA39C/εE31C双突变体的膜中未观察到。这些结果表明，在F1F0复合物中，c亚基的极性环和ε亚基第31位残基周围的区域彼此物理上接近，并支持这两个亚基在H⁺转运与ATP合成偶联过程中直接相互作用的假说。Q42C c亚基和E31C ε亚基的二硫键交联导致ATP酶偶联H⁺转运受到抑制，正如在F1 ATP酶催化位点在H⁺转运偶联的ATP水解/合成过程中交替的模型中所预期的那样。然而，这里使用的方法无法建立转运抑制程度与交联程度之间的定量关系，并且ε - c交联的F1F0复合物保留残余H⁺转运活性的可能性仍然存在。
