文摘
The hypothesis was tested whether in bacteriorhodopsin (BR) thereduction of the stericinteraction between the 9-methyl group of the chromophoreall-trans-retinal and the tryptophan atposition182 causes the same changes as observed in the photocycle of9-demethyl-BR. For this, the photocycleof the mutant W182F was investigated by time-resolved UV-vis and pHmeasurements and by static andtime-resolved FT-IR difference spectroscopy. We found that thesecond half of the photocycle was similarlydistorted in the two modified systems: based on the amide-I band, theprotonation state of D96, and thekinetics of proton uptake, four N intermediates could be identified,the last one having a lifetime ofseveral seconds; no O intermediate could be detected; the proton uptakeshowed a pronounced biphasictime course; and the pKa of group(s) on thecytoplasmic side in N was reduced from 11 in wild type BRto around 7.5. In contrast to 9-demethyl-BR, in the W182F mutantthe first part of the photocycle doesnot drastically deviate from that of wild type BR. The resultsdemonstrate the importance of the stericinteraction between W182 and the 9-methyl group of the retinal inproviding tight coupling betweenchromophore isomerization and the late proton transfersteps.