Abstract
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 with 15N at the two η-nitrogens (15N-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 14N-Arg-bR loses substantial intensity in 15N-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) 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.