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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- 作者：Yaowu Xiao ; M. Shane Hutson ; Marina Belenky ; Judith Herzfeld ; and Mark S. Braiman
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：October 12, 2004
- 年：2004
- 卷：43
- 期：40
- 页码：12809 - 12818
- 全文大小：304K
- 年卷期：v.43,no.40(October 12, 2004)
- ISSN：1520-4995
文摘
Arginine-82 has long been recognized as an important residue in bacteriorhodopsin (bR), becauseits 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+-releasestep, 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 with 15N at thetwo 
-nitrogens (15N-Arg-bR). Comparison of the spectra from the two isotopic variants shows that a1556 cm-1 vibrational difference band due to the M photocycle intermediate of 14N-Arg-bR loses substantialintensity in 15N-Arg-bR. However, this isotope-sensitive arginine vibrational difference band is onlyobserved at pH 7 and not at pH 4 where fast H+ release is blocked. These observations support the earlierconclusion, based on site-directed mutagenesis and chemical labeling, that a strong C-N stretch vibrationof Arg-82 can be assigned to a highly perturbed frequency near 1555 cm-1 in the M state of wild-type bR[Hutson et al. (2000) Biochemistry 39, 13189-13200]. Furthermore, alkylguanidine model compoundspectra indicate that the unusually low arginine C-N stretch frequency in the M state is consistent witha nearly stoichiometric light-induced deprotonation of an arginine side chain within bR, presumably arginine-82.
-nitrogens (15N-Arg-bR). Comparison of the spectra from the two isotopic variants shows that a1556 cm-1 vibrational difference band due to the M photocycle intermediate of 14N-Arg-bR loses substantialintensity in 15N-Arg-bR. However, this isotope-sensitive arginine vibrational difference band is onlyobserved at pH 7 and not at pH 4 where fast H+ release is blocked. These observations support the earlierconclusion, based on site-directed mutagenesis and chemical labeling, that a strong C-N stretch vibrationof Arg-82 can be assigned to a highly perturbed frequency near 1555 cm-1 in the M state of wild-type bR[Hutson et al. (2000) Biochemistry 39, 13189-13200]. Furthermore, alkylguanidine model compoundspectra indicate that the unusually low arginine C-N stretch frequency in the M state is consistent witha nearly stoichiometric light-induced deprotonation of an arginine side chain within bR, presumably arginine-82.