Kataoka M, Kamikubo H, Tokunaga F, Brown L S, Yamazaki Y, Maeda A, Sheves M, Needleman R, Lanyi J K
Department of Earth and Space Science, Faculty of Science, Osaka University, Japan.
J Mol Biol. 1994 Nov 4;243(4):621-38. doi: 10.1016/0022-2836(94)90037-x.
The active site of an ion pump must communicate alternately with the two opposite membrane surfaces. In the light-driven proton pump, bacteriorhodopsin, the retinal Schiff base is first the proton donor to D85 (with access to the extracellular side), and then it becomes the acceptor of the proton of D96 (with access to the cytoplasmic side). This "reprotonation switch" has been associated with a protein conformation change observed during the photocycle. When D85 is replaced with asparagine, the pKa value of the Schiff base is lowered from above 13 to about 9. We determined the direction of the loss or gain of the Schiff base proton in unphotolyzed and in photoexcited D85N, and the D85N/D96N and D85N/D96A double mutants, in order to understand the intrinsic and the induced connectivities of the Schiff base to the two membrane surfaces. The influence of D96 mutations on proton exchange and on acceleration of proton shuttling to the surface by azide indicated that in either case the access of the Schiff base on D85N mutants is to the cytoplasmic side. In the wild-type protein (but with the pKa of the Schiff base lowered by 13-trifluoromethyl retinal substitution) the results suggested that the Schiff base can communicate also with the extracellular side. Raising the pH without illumination of D85N so as to deprotonate the Schiff base caused the same, or nearly the same, change of X-ray scattering as observed when the Schiff base deprotonates during the wild-type photocycle. The results link the charge state of the active site to the global protein conformation and to the connectivity of the Schiff base proton to the membrane surfaces. Their relationship suggests that the conformation of the unphotolyzed wild-type protein is stabilized by coulombic interaction of the Schiff base with its counter-ion. A proton is translocated across the membrane after light-induced transfer of the Schiff base proton to D85, because the protein assumes an alternative conformation that separates the donor from the acceptor and opens new conduction pathways between the active site and the two membrane surfaces.
离子泵的活性位点必须交替地与两个相对的膜表面进行沟通。在光驱动质子泵细菌视紫红质中，视黄醛席夫碱首先是向D85供质子（可接触细胞外侧），然后它成为D96质子的受体（可接触细胞质侧）。这种“再质子化开关”与光循环过程中观察到的蛋白质构象变化有关。当用天冬酰胺取代D85时，席夫碱的pKa值从13以上降至约9。我们确定了未光解和光激发的D85N以及D85N/D96N和D85N/D96A双突变体中席夫碱质子的得失方向，以便了解席夫碱与两个膜表面的内在和诱导连接性。D96突变对质子交换以及叠氮化物将质子加速转运到表面的影响表明，在任何一种情况下，D85N突变体上席夫碱的可接触侧都是细胞质侧。在野生型蛋白质中（但席夫碱的pKa因13 - 三氟甲基视黄醛取代而降低），结果表明席夫碱也可以与细胞外侧进行沟通。在不光照D85N的情况下提高pH以使席夫碱去质子化，会导致与野生型光循环过程中席夫碱去质子化时观察到的相同或几乎相同的X射线散射变化。这些结果将活性位点的电荷状态与整体蛋白质构象以及席夫碱质子与膜表面的连接性联系起来。它们之间的关系表明，未光解的野生型蛋白质的构象通过席夫碱与其抗衡离子的库仑相互作用而得以稳定。在光诱导席夫碱质子转移到D85后，一个质子跨膜转运，因为蛋白质呈现出一种替代构象，该构象将供体与受体分开，并在活性位点和两个膜表面之间开辟了新的传导途径。
