Heyne, K; Herbst, J; Dominguez-Herradon, B; Alexiev, U; Diller, R
JOURNAL OF PHYSICAL CHEMISTRY B 2000, 104, 6053-6058
Femtosecond time-resolved optical absorption experiments reveal that the changes of the excited electronic state dynamics observed between bacteriorhodopsin wild type and the single mutant R82A are completely reversed in the double mutant R82A/G231C. Thus, the bacteriorhodopsin double mutant R82A/G231C is shown to be a second site revertant with respect to the primary ultrafast all-trans to 13-cis photoisomerization of the retinal cofactor. The results imply that in R82A/G231C a cofactor binding pocket is realized in which, at physiological pH, the arginine residue in position 82 (R82) is not, but a deprotonated D85 is needed for a wild-type-like fast retinal photoisomerization. The revertancy found for R82A/G231C and further results on the single mutants R82A, R82C, R82Q, and G231C at various pH values and ion concentrations confirm and broaden the range of applicability of the known correlation between the protonation state of aspartic acid 85 (D85) and the time constants of the excited electronic state decay. Among the bR mutant systems investigated, species with D85 deprotonated exhibit an excited electronic state decay time constant of t1= 0.52 ± 0.05 ps whereas systems with D85 protonated show a biphasic decay with t1= 1.7 ± 0.3 and t2 ranging from 6 to 12 ps. It is noted that the distribution of the t2 times is much wider than that of the t1times.