It is known that the absorption maximum of halorhodopsin is red shifted by 10 nm with the uptake of a chloride ion Cl(-). According to the X-ray structure, the ion is located at the position of the counterion of the chromophore, protonated retinal Schiff base. Thus, the direction of the observed spectral change is opposite to that expected from the pi-electron redistribution (an increase in the bond alternation) induced by the counterion. The physical origin of this abnormal shift is never explained in terms of any simple chemical analogues. We successfully explain this phenomenon by a QM/MM type of excitation energy calculation. The three-dimensional structure of the protein is explicitly taken into account using the X-ray structure. We reveal that the electronic polarization of the protein environment plays an essential role in tuning the absorption maximum of halorhodopsin.

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.

The fluorescence of NO was obtained by excitation from the 184.9 nm resonance line of a low pressure mercury lamp. It was found that the β (v′=9) emission was induced by the absorption of the 184.9 nm line for [NO]=1.0 torr. Quenching of the β (v′=9) bands by He or N2 molecules was accompanied by an enhancement of the γ bands, v′=4 or 0 and 1, respectively. When CO was added to NO, intensities of the emission were weakened. These effects were explained by the following reaction scheme.

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The quenching rates of NO(B 2Π, v′=9) have been measured for He, H2, CO, CF4, N2, and CO2, relative to NO self-quenching rate, by noting the reduction in fluorescence intensity of the β (v′=9) bands. The values obtained are 1.0:0.12:0.13:0.13:0.14:0.17:0.18 for NO, He, H2, CO, CF4, N2, and CO2. Absolute quenching rates are also estimated to be close to hard-sphere collision rate constants and the quantum yield of the β (v′=9) emission to be of the order of 10−2.