Brown L S, Váró G, Hatanaka M, Sasaki J, Kandori H, Maeda A, Friedman N, Sheves M, Nedleman R, Lanyi J K
Department of Physiology and Biophysics, University of California, Irvine 92717, USA.
Biochemistry. 1995 Oct 3;34(39):12903-11. doi: 10.1021/bi00039a053.
During the L-->M reaction of the bacteriorhodopsin photocycle the proton of the retinal Schiff base is transferred to the anionic D85. This step, together with the subsequent reprotonation of the Schiff base from D96 in the M-->N reaction, results in the translocation of a proton across the membrane. The first of these critical proton transfers occurs in an extended hydrogen-bonded complex containing two negatively charged residues (D85 and D212), two positively charged groups (the Schiff base and R82), and coordinated water. We simplified this region by replacing D212 and R82 with neutral residues, leaving only the proton donor and acceptor as charged groups. The D212N/R82Q mutant shows essentially normal proton transport, but in the photocycle neither of this protein nor of the D212N/R82Q/D96N triple mutant does a deprotonated Schiff base (the M intermediate) accumulate. Instead, the photocycle contains only the K, L, and N intermediates. Infrared difference spectra of D212N/R82Q and D212N/R82Q/D96N demonstrate that although D96 becomes deprotonated in N, D85 remains unprotonated. On the other hand, M is produced at pH > 8, where according to independent evidence the L<==>M equilibrium should shift toward M. Likewise, M is restored in the photocycle when the retinal is replaced with the 14-fluoro analogue that lowers the pKa of the protonated Schiff base, and now D85 becomes protonated as in the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)
在细菌视紫红质光循环的L→M反应过程中，视黄醛席夫碱的质子转移至阴离子态的D85。这一步骤，连同随后在M→N反应中席夫碱从D96处的再质子化，导致质子跨膜转运。这些关键质子转移中的第一步发生在一个扩展的氢键复合物中，该复合物包含两个带负电荷的残基（D85和D212）、两个带正电荷的基团（席夫碱和R82）以及配位水。我们通过用中性残基取代D212和R82简化了该区域，仅留下质子供体和受体作为带电基团。D212N/R82Q突变体显示出基本正常的质子转运，但在光循环中，该蛋白以及D212N/R82Q/D96N三重突变体均未积累去质子化的席夫碱（M中间体）。相反，光循环仅包含K、L和N中间体。D212N/R82Q和D212N/R82Q/D96N的红外差光谱表明，尽管D96在N态时去质子化，但D85仍未质子化。另一方面，在pH > 8时会产生M，根据独立证据，此时L⇌M平衡应向M移动。同样，当视黄醛被14 - 氟类似物取代时，光循环中会恢复M，该类似物降低了质子化席夫碱的pKa，此时D85如野生型一样质子化。（摘要截断于250字）
