Coleman M, Nilsson A, Russell T S, Rath P, Pandey R, Rothschild K J
Physics Department, Boston University, Massachusetts 02215, USA.
Biochemistry. 1995 Nov 28;34(47):15599-606. doi: 10.1021/bi00047a027.
Bacteriorhodopsin functions as a light-driven proton pump in the purple membrane of Halobacterium salinarium. A variety of studies have established that a proton is transferred over an approximately 10 A distance from Asp 96 to the retinylidene Schiff base during the M --> N transition of the bR photocycle. In order to further explore the mechanism of this Schiff base reprotonation, we compared the properties of the double mutant Thr 46 --> Asp/Asp 96 --> Asn (T46D/D96N), the single mutants Asp 96 --> Asn (D96N) and Thr 46 --> Asp (T46D), and wild-type bR. In contrast to D96N, which exhibits a very slow M decay, T46D/D96N has an M decay close to that of wild-type bR. FTIR difference spectroscopy detects bands in the carboxyl and carboxylate stretch region of T46D/D96N consistent with the deprotonation of Asp 46 during the M --> N transition. In addition, bands associated with structural changes of Asn 96 in the mutant D96N are absent in T46D/D96N. Resonance Raman spectroscopy provides evidence that both T46D/D96N and T46D have a long-lived N-like species in their photocycles. These data demonstrate that Asp 46 can substitute for Asp 96 as the proton donor group in the reprotonation pathway of the Schiff base during the M --> N transition. However, N decay is delayed in comparison to wild-type bR. This may be due to a partial block in the proton pathway leading from the cytoplasmic medium to Asp 46.
细菌视紫红质在盐生盐杆菌的紫膜中作为光驱动质子泵发挥作用。各种研究已经证实，在细菌视紫红质光循环的M→N转变过程中，质子从天冬氨酸96转移到视黄叉席夫碱的距离约为10埃。为了进一步探索这种席夫碱再质子化的机制，我们比较了双突变体苏氨酸46→天冬氨酸/天冬氨酸96→天冬酰胺（T46D/D96N）、单突变体天冬氨酸96→天冬酰胺（D96N）和苏氨酸46→天冬氨酸（T46D）以及野生型细菌视紫红质的特性。与表现出非常缓慢的M衰减的D96N不同，T46D/D96N的M衰减与野生型细菌视紫红质相近。傅里叶变换红外差光谱法在T46D/D96N的羧基和羧酸盐伸缩区域检测到与M→N转变过程中天冬氨酸46去质子化一致的谱带。此外，在T46D/D96N中不存在与突变体D96N中天冬酰胺96结构变化相关的谱带。共振拉曼光谱提供的证据表明，T46D/D96N和T46D在其光循环中都有一个长寿命的类似N的物种。这些数据表明，在M→N转变过程中，天冬氨酸46可以替代天冬氨酸96作为席夫碱再质子化途径中的质子供体基团。然而，与野生型细菌视紫红质相比，N衰减有所延迟。这可能是由于从细胞质介质到天冬氨酸46的质子途径存在部分阻断。
