Reaction control in bacteriorhodopsin: Impact of arg82 and asp85 on the fast retinal isomerization, studied in the second site revertant arg82ala/gly231cys and various purple and blue forms of bacteriorhodopsin

Femtosecond time-resolved optical absorption experiments reveal that the ch anges of the excited electronic state dynamics observed between bacteriorho dopsin wild type and the single mutant R82A are completely reversed in the double mutant R82A/G231C. Thus, the bacteriorhodopsin double mutant R82A/G2 31C is shown to be a second site revertant with respect to the primary ultr afast all-trans to 13-cis photoisomerization of the retinal cofactor. The r esults imply that in R82A/G231C a cofactor binding pocket is realized in wh ich, 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 photois omerization. The revertancy found for R82A/G231C and further results on the single mutants R82A, R82C, R82Q, and G231C at various pH values and ion co ncentrations confirm and broaden the range of applicability of the known co rrelation between the protonation state of aspartic acid 85 (D85) and the t ime constants of the excited electronic state decay. Among the bR mutant sy stems investigated, species with D85 deprotonated exhibit an excited electr onic state decay time constant of tau(1) = 0.52 +/- 0.05 ps whereas systems with D85 protonated show a biphasic decay with tau(1) = 1.7 +/- 0.3 and ta u(2) ranging from 6 to 12 ps. It is noted that the distribution of the tau( 2) times is much wider than that of the tau(1) times.