Hatanaka M, Sasaki J, Kandori H, Ebrey T G, Needleman R, Lanyi J K, Maeda A
Department of Biophysics, Graduate School of Science, Kyoto University, Japan.
Biochemistry. 1996 May 21;35(20):6308-12. doi: 10.1021/bi952973v.
Arg82, one of the residues near the protonated Schiff base of bacteriorhodopsin, facilitates proton release to the medium during the L-to-M reaction of the photocycle, but retards the rate of proton transfer from the Schiff base to Asp85. In order to understand the role of Arg82 in these processes, the structural changes upon formation of the M intermediate were studied by Fourier transform infrared spectroscopy of the hydrated films of Arg82 mutants at pH 9.5. The negative band at 1700 cm-1 in the BR --> M spectrum due to the deprotonation of Glu204 was absent when Arg82 was replaced with alanine (R82A), but present with small amplitude when residue 82 was a glutamine (R82Q), or a lysine (R82K), with a shift to 1696 cm-1. The O-H stretch of water at 3643 cm-1 is shifted toward a lower frequency in R82Q, R82K, and R82A in the unphotolyzed state. However, R82Q retains a fraction of the unshifted band. Another O-H stretch is prominent in R82Q around 3625 cm-1 but absent in R82A and probably in R82K. In parallel, R82Q retains a fraction of the slow component of the formation of the M intermediate, which is almost completely absent in R82K and R82A. These results, along with previous data for the mutants of Glu204, suggest that the guanidium group of Arg82 influences the H-bonding of water molecules located close to Asp85 and Arg82-Glu204 regions, and the rate of proton transfer from the Schiff base to Asp85. The amide group of Gln82 can substitute for it but weakly.
精氨酸82是细菌视紫红质质子化席夫碱附近的残基之一，在光循环的L态到M态反应过程中促进质子释放到介质中，但会延缓质子从席夫碱转移到天冬氨酸85的速率。为了理解精氨酸82在这些过程中的作用，通过对pH 9.5下精氨酸82突变体水合膜进行傅里叶变换红外光谱研究了M中间体形成时的结构变化。当精氨酸82被丙氨酸取代（R82A）时，由于谷氨酸204去质子化，在细菌视紫红质（BR）到M态光谱中1700 cm-1处的负带消失，但当残基82为谷氨酰胺（R82Q）或赖氨酸（R82K）时该负带存在且幅度较小，且向1696 cm-1处移动。在未光照状态下，3643 cm-1处水的O-H伸缩振动在R82Q、R82K和R82A中向较低频率移动。然而，R82Q保留了一部分未移动的谱带。另一个O-H伸缩振动在R82Q中3625 cm-1附近很突出，但在R82A中不存在，可能在R82K中也不存在。同时，R82Q保留了一部分M中间体形成的慢成分，而在R82K和R82A中几乎完全没有。这些结果，连同之前谷氨酸204突变体的数据，表明精氨酸82的胍基影响靠近天冬氨酸85和精氨酸82 - 谷氨酸204区域的水分子的氢键作用，以及质子从席夫碱转移到天冬氨酸85的速率。谷氨酰胺82的酰胺基团可以部分替代它，但作用较弱。
