Differences between the photocycles of halorhodopsin and the acid purple form of bacteriorhodopsin analyzed with millisecond time-resolved FTIR spectroscopy.
At pH 1, bacteriorhodopsin (bR) is thought to function as a halide ion pump, in contrast to its biological function as a proton pump at neutral pH. Despite the apparent similarity in function between this 'acid purple' form of bR and the native form of halorhodopsin (hR), their FTIR difference spectra measured ca. 5 ms after photolysis are significantly different. The most striking difference is the appearance of a positive band at 1753 cm-1 and a negative band at 1732 cm-1 in the bRacid purple difference spectrum. These and other spectral features are similar, but not identical, to those of the bR-->O difference spectrum measured at neutral pH. The structure of the bRacid purple longest-lived product therefore corresponds more closely to the O photoproduct of the bR proton-pumping photocycle, rather than the hL photoproduct seen on a similar time scale in the hR photocycle. The 1753- and 1732-cm-1 bands are largely unaffected by the D212N mutation, but both appear to lose a portion of their intensities with either the D85N or D96N mutation. Thus Asp-85 and -96 likely undergo substantial changes in hydrogen-bonding environment during the halide-pumping cycle of bRacid purple. Our FTIR results deepen the distinctions between the hR and bR photocycles. The mechanism of chloride pumping in hR has been thought not to involve protonation or hydrogen bonding changes of carboxylic acid groups. In bRacid purple, however, it seems likely that at least one carboxylic acid might play an important role in the mechanism of chloride pumping, leading to an increase in thermodynamic or kinetic stabilization of the O intermediate.
Mitrovich
Q M
QM
Biochemistry Department, University of Virginia, Charlottesville 22908, USA.
Victor
K G
KG
Braiman
M S
MS
D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism*; D006217:Halobacterium / Q000378:metabolism; D025602:Halorhodopsins; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010777:Photochemistry; D011487:Protein Conformation; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D013997:Time Factors
D025602:Halorhodopsins; D001436:Bacteriorhodopsins