STERIC INTERACTION BETWEEN THE 9-METHYL GROUP OF THE RETINAL AND TRYPTOPHAN-182 CONTROLS 13-CIS TO ALL-TRANS REISOMERIZATION AND PROTON UPTAKE IN THE BACTERIORHODOPSIN PHOTOCYCLE

The hypothesis was tested whether in bacteriorhodopsin (BR) the reduct ion of the steric interaction between the 9-methyl group of the chromo phore all-trans-retinal and the tryptophan at position 182 causes the same changes as observed in the photocycle of 9-demethyl-BR. For this, the photocycle of the mutant W182F was investigated by time-resolved UV-vis and pH measurements and by static and time-resolved FT-IR diffe rence spectroscopy. We found that the second half of the photocycle wa s similarly distorted in the two modified systems: based on the amide- I band, the protonation state of D96, and the kinetics of proton uptak e, four N intermediates could be identified, the last one having a lif etime of several seconds; no O intermediate could be detected; the pro ton uptake showed a pronounced biphasic time course; and the pK(a) of group(s) on the cytoplasmic side in N was reduced from 11 in wild type BR to around 7.5. In contrast to 9-demethyl-BR, in the W182F mutant t he first part of the photocycle does not drastically deviate from that of wild type BR. The results demonstrate the importance of the steric interaction between W182 and the 9-methyl group of the retinal in pro viding tight coupling between chromophore isomerization and the late p roton transfer steps.