QM/MM techniques will be used to determine structures and IR spectra of the bR photo-cycle intermediates. To achieve this, we will use DFT methods as well as a re-parametrized SCC-DFTB method and determine the infrared spectra via calculation of the Hessian matrix as well as via Fourier transform of the velocity- and dipole-autocorrelation functions. These calculations may help resolving the assignment problem of atomic (X-ray) structures to experimental IR spectra and in the determination of the later structures of the bR photocycle. Of particular interest is the identity and structure of the so-called proton release group (PRG). We will test the suggestion that this group consists of a protonated water cluster at the extracellular side, a hypothesis which explains the experimental IR spectra. The determination of the PRG opens the way for the investigation of the second and fifth proton transfer step, the proton release from the PRG to the bulk in the M state and the last proton transfer step between Asp85 and the PRG between the O and bR ground state. Here, minimum pathway search algorithms as well as umbrella sampling techniques will be applied.

Investigation of the Proton Transfer Pathways from the O to bR-ground state of Bacteriorhodopsin. In bacteriorhodopsin, absorption of light by the retinal chromophore initiates a photo-cycle resulting in a net transfer of proton from the cytoplasmic side to the extracellular side of the protein. The last proton transfer takes place from the  O intermediate on a millisecond timescale to recover the bR ground state. This proton transfer is one of slowest during the photo-cycle and occurs over 11 Angstrom distance and is therefore challenging to investigate. Several features are unknown for this transfer for example the orientation and the role of the Arg82 side-chain or number and location of the waters.  We are currently performing minimum energy QM/MM path calculations to investigate the energy-barriers for this proton transfer reaction.

Proton Transfer at the extracellular side of Bacteriorhodopsin. In bacteriorhodopsin, the isomerization of the retinal chromophore results in a net proton transfer from the cytoplasmic to the extracellular side of the membrane. During the last proton transfer step from O-to-bR, a proton is transferred from the Asp85 to the extracellular proton release group. The FTIR studies have indicated a possibility of a transient [O] state characterized by anionic Asp85 and neutral Asp212. Our minimum-energy path analysis indicate that the proton transfer from Asp85 to Asp212 is possible and depends crucially upon the retinal geometry and the number and orientations of active-site waters.

Investigation of the Proton Release Group at the extracellular side of Bacteriorhodopsin. In bacteriorhodopsin, absorption of light by the retinal chromophore initiates the proton transfer from the Schiff-base to  the nearby aspartic acid. Concomitant with this first proton transfer, a proton is released to the surface of the protein from the so-called Proton Release Group(PRG) located at the extracellular side of the protein. The identity of this group has been controversial. The original idea of Glu204 being the PRG was revised to combination of Glu204 and Glu194 forming  PRG. The FTIR experiments indicated that an IR continuum absorption band present in the ground state dissapears during the formation of the M state. Protonated water clusters are known to generate such continuum bands. Based on this argument, in 2001, it was proposed the PRG is composed of protonated water cluster such as zundel ion.  We are currently simulating such water clusters with M and L states with the Infra-red(IR) analysis of the simulation to follow. We are also investigating the role of the Schiff-base deprotonation and orientation of Arg82 side-chain on the PRG.