In this study, integrated (MOZYME + DFT) method (Ohno et al. Chem. Phys. Lett. 2001, 341, 387.) is applied to elucidate how the pK(a)'s of retinal Schiff base (RSB) and Asp85 in bacteriorhodopisn (bR) are controlled by the surrounding protein matrix, especially a hydrogen bonding network involving RSB. The whole protein is divided into two layers. Layer 1 contains only the hydrogen bonding network and is treated at the DFT level of theory. The rest of the protein is calculated using a linear-scaling molecular orbital method called MOZYME that can explicitly take into account the protein three-dimensional structure. Here we focus our attention on the pK(a) changes of RSB and Asp85 on going from the ground state to the M intermediate, because they are key factors of the proton translocation mechanism in bR. The three-dimensional structures of both states are taken from corresponding X-ray data. The calculation successfully reproduces the experimental fact that RSB and Asp85 form the zwitterions in the ground state. On the other hand, the fact that these residues are in the neutral form in the M intermediate is reproduced only when the side chain of Thr89 takes a special orientation capable of forming hydrogen bond(s) with Asp85. It is shown that such hydrogen bond formation and the disappearance of water 402 are the major factors stabilizing the neutral state of the (RSB + Asp85) system in the M intermediate. Finally, we discuss a role of Thr89 in the proton translocation process.