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.
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 ((14)N-Arg-bR) or all seven arginines selectively and uniformly labeled with (15)N at the two eta-nitrogens ((15)N-Arg-bR). Comparison of the spectra from the two isotopic variants shows that a 1556 cm(-1) vibrational difference band due to the M photocycle intermediate of (14)N-Arg-bR loses substantial intensity in (15)N-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(-1) in the M state of wild-type bR [Hutson et al. (2000) Biochemistry 39, 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.
Xiao
Yaowu
Y
Department of Chemistry, Syracuse University, Syracuse, New York 13244-4100, USA.
Hutson
M Shane
MS
Belenky
Marina
M
Herzfeld
Judith
J
Braiman
Mark S
MS
D001120:Arginine / Q000378:metabolism*; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009587:Nitrogen Isotopes; D010777:Photochemistry; D011522:Protons; D018717:Purple Membrane / Q000737:chemistry*; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature; D013997:Time Factors
D009587:Nitrogen Isotopes; D011522:Protons; D001436:Bacteriorhodopsins; D001120:Arginine