A PROTON PATHWAY WITH LARGE PROTON POLARIZABILITY AND THE PROTON PUMPING MECHANISM IN BACTERIORHODOPSIN - FOURIER-TRANSFORM DIFFERENCE SPECTRA OF PHOTOPRODUCTS OF BACTERIORHODOPSIN AND OF ITS PENTADEMETHYL ANALOG

The first part of the photocycle of bacteriorhodopsin (BR) (BR570, K630, L550 and M412) is studied by difference Fourier transform-infrared (FT-IR) spectroscopy. These results are compared with the respective difference spectra of the pentademethyl analogue (BR(a)). With the BR intermediates, the same bands are observed as in previous studies. This is especially true in the carbonyl region. With BR(a), however, the behaviour of the intermediates is different from the respective intermediates of BR. The photocycle is interrupted before the L state and returns to BR(a). In the case of BR, in the K intermediate, a very broad band is observed in the region 2800-2200 cm-1. In the L intermediate, a continuous absorption is observed beginning at 2800 cm-1 and extending toward smaller wavenumbers over the whole region studied. This continuum vanishes with the formation of the M412 intermediate and, instead, two very broad bands are found in the region 2700-2200 cm-1. The broad band in the K state indicates that at least one strong hydrogen bond is formed in which the proton is not well localized. From the continuum observed in the L intermediate, it is concluded that a hydrogen-bonded chain is formed showing large proton polarizability caused by collective proton motion. This chain is discussed on the basis of a structural model based on literature data. The Schiff base-Asp 85 and Tyr 185-Asp 212 bonds show proton polarizability. With the transition to the intermediate M412, within both hydrogen bonds, the protons are shifted within these hydrogen bonds to Asp 85 and Asp 212, due to changes of the local electrical fields and to specific interactions arising due to conformational changes of the protein. In this way the negative charges in the neighbourhood of Arg 82 are neutralized and hence the proton potential at Arg 82 raised. Thus, the positive charge is shifted to the outside of the proton channel and released to the bulk water phase. The continuum is no longer observed in M412 since the proton potentials in the chain are now asymmetrical. Hence, in the M412 intermediate only broad bands are found, indicating strong but asymmetrical hydrogen bonds present in the active centre.