Optical control of the primary step of photoisomerization of the retinal molecule in bacteriorhodopsin from the all-trans to the 13-cis state was demonstrated under weak field conditions (where only 1 of 300 retinal molecules absorbs a photon during the excitation cycle) that are relevant to understanding biological processes. By modulating the phases and amplitudes of the spectral components in the photoexcitation pulse, we showed that the absolute quantity of 13-cis retinal formed upon excitation can be enhanced or suppressed by ±20% of the yield observed using a short transform-limited pulse having the same actinic energy. The shaped pulses were shown to be phase-sensitive at intensities too low to access different higher electronic states, and so these pulses apparently steer the isomerization through constructive and destructive interference effects, a mechanism supported by observed signatures of vibrational coherence. These results show that the wave properties of matter can be observed and even manipulated in a system as large and complex as a protein.

In the light-driven proton pump bacteriorhodopsin, proton transfer from the retinal Schiff base to aspartate-85 is the crucial reaction of the transport cycle. In halorhodopsin, a light-driven chloride ion pump, the equivalent of residue 85 is threonine. When aspartate-85 was replaced with threonine, the mutated bacteriorhodopsin became a chloride ion pump when expressed in Halobacterium salinarium and, like halorhodopsin, actively transported chloride ions in the direction opposite from the proton pump. Chloride was bound to it, as revealed by large shifts of the absorption maximum of the chromophore, and its photointermediates included a red-shifted state in the millisecond time domain, with its amplitude and decay rate dependent on chloride concentration. Bacteriorhodopsin and halorhodopsin thus share a common transport mechanism, and the interaction of residue 85 with the retinal Schiff base determines the ionic specificity.

We have measured proton release into the medium after proton transfer from the retinal Schiff base to Asp⁸⁵ in the photocycle and the C=O stretch bands of carboxylic acids in wild type bacteriorhodopsin and the E204Q and E204D mutants. In E204Q, but not in E204D, the normal proton release is absent. Consistent with this, a negative band in the Fourier transform infrared difference spectra at 1700 cm⁻¹ in the wild type, which we now attribute to depletion of the protonated E204, is also absent in E204Q. In E204D, this band is shifted to 1714 cm⁻¹, as expected from the higher frequency for a protonated aspartic than for a glutamic acid. Consistent with their origin from protonated carboxyls, the depletion bands in the wild type and E204D shift in D₂O to 1690 and 1703 cm⁻¹, respectively. In the protein structure, Glu²⁰⁴ seems to be connected to the Schiff base region by a chain of hydrogen-bonded water. As with other residues closer to the Schiff base, replacement of Glu²⁰⁴ with glutamine changes the O-H stretch frequency of the bound water molecule near Asp⁸⁵ that undergoes hydrogen-bonding change in the photocycle. The results therefore identify Glu²⁰⁴ as XH, the earlier postulated residue that is the source of the released proton during the transport, and suggest that its deprotonation is triggered by the protonation of Asp⁸⁵ through a network that contains water dipoles.