Role of arginine-82 in fast proton release during the bacteriorhodopsin photocycle: a time-resolved FT-IR study of purple membranes containing 15N-labeled arginine.
Role of arginine-82 in fast proton release during the bacteriorhodopsin photocycle: a time-resolved FT-IR study of purple membranes containing 15N-labeled arginine.
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精氨酸 82 在细菌视紫红质光循环过程中快速质子释放中的作用：对含有 15N 标记精氨酸的紫色膜进行时间分辨 FT-IR 研究。
DOI：
10.1021/bi049238g
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Braiman,MarkS
中科院分区：
文献类型：
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作者：
Xiao,Yaowu;Hutson,MShane;Belenky,Marina;Herzfeld,Judith;Braiman,MarkS
精氨酸-82是细菌视紫红质（bR）中的一个重要残基，它的突变通常导致H+快速释放的丧失，而H+快速释放是正常光诱导H+转运机制中的一个重要步骤。为了帮助阐明与H+释放步骤相关的Arg-82的结构变化，我们测量了含有天然丰度同位素（14 N-Arg-bR）或在两个η-氮上选择性地和均匀地用15 N标记的所有七种精氨酸（15 N-Arg-bR）的野生型bR的时间分辨FT-IR差光谱。两种同位素变体的光谱比较表明，15 N-Arg-bR的1556 cm-1振动差带由于14 N-Arg-bR的M光循环中间体而在15 N-Arg-bR中损失了大量强度。然而，这种同位素敏感的精氨酸振动差带仅在pH 7下观察到，而不是在pH 4下，其中快速H+释放被阻断。这些观察结果支持了基于定点诱变和化学标记的早期结论，即Arg-82的强C-N伸缩振动可以归因于野生型bR M状态中接近1555 cm-1的高度扰动频率[Hutson et al.（2000）Biochemistry 39，13189−13200]。此外，烷基胍模型化合物的光谱表明，在M状态下，精氨酸C−N伸缩频率异常低，这与bR内精氨酸侧链（可能是精氨酸-82）的近化学计量光诱导去质子化一致。
Arginine-82 has long been recognized as an important residue in bacteriorhodopsin (bR), because its mutation usually results in loss of fast H+release, an important step in the normal light-induced H+transport mechanism. To help to clarify the structural changes in Arg-82 associated with the H+-release step, we have measured time-resolved FT-IR difference spectra of wild-type bR containing either natural-abundance isotopes (14N-Arg-bR) or all seven arginines selectively and uniformly labeled with15N at the two η-nitrogens (15N-Arg-bR). Comparison of the spectra from the two isotopic variants shows that a 1556 cm-1vibrational difference band due to the M photocycle intermediate of14N-Arg-bR loses substantial intensity in15N-Arg-bR. However, this isotope-sensitive arginine vibrational difference band is only observed at pH 7 and not at pH 4 where fast H+release is blocked. These observations support the earlier conclusion, based on site-directed mutagenesis and chemical labeling, that a strong C−N stretch vibration of Arg-82 can be assigned to a highly perturbed frequency near 1555 cm-1in the M state of wild-type bR [Hutson et al. (2000)Biochemistry39, 13189−13200]. Furthermore, alkylguanidine model compound spectra indicate that the unusually low arginine C−N stretch frequency in the M state is consistent with a nearly stoichiometric light-induced deprotonation of an arginine side chain within bR, presumably arginine-82.