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 neut ral pH. Despite the apparent similarity in function between this 'acid purple' form of bR and the native form of halorhodopsin (hR), their F TIR difference spectra measured ca. 5 ms after photolysis are signific antly different. The most striking difference is the appearance of a p ositive band at 1753 cm(-1) and a negative band at 1732 cm(-1) in the bR(acid) (purple) difference spectrum. These and other spectral featur es are similar, but not identical, to those of the bR --> O difference spectrum measured at neutral pH. The structure of the bR(acid) (purpl e) longest-lived product therefore corresponds more closely to the O p hotoproduct of the bR proton-pumping photocycle, rather than the hL ph otoproduct 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 cyc le of bR(acid) (purple). Our FTIR results deepen the distinctions betw een the hR and bR photocycles. The mechanism of chloride pumping in hR has been thought not to involve protonation or hydrogen bonding chang es of carboxylic acid groups. In bR(acid) (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 thermo dynamic or kinetic stabilization of the O intermediate.