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

Abstract

The hypothesis was tested whether in bacteriorhodopsin (BR) the reduction of the steric interaction between the 9-methyl group of the chromophore 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 difference spectroscopy. We found that the second half of the photocycle was similarly distorted in the two modified systems: based on the amide-I band, the protonation state of D96, and the kinetics of proton uptake, four N intermediates could be identified, the last one having a lifetime of several seconds; no O intermediate could be detected; the proton uptake showed a pronounced biphasic time course; and the pKa 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 the 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 providing tight coupling between chromophore isomerization and the late proton transfer steps.

Crystal Structure of the L Intermediate of Bacteriorhodopsin: Evidence for Vertical Translocation of a Water Molecule during the Proton Pumping Cycle

“…36,37 The observations (2) and (3) are in accordance with previous observations that mutations in Leu93 and Trp182 affect the formation/decay kinetics of the L intermediate. 38,39 The observation (4) is in line with recent FTIR studies demonstrating that the indole nitrogen of Trp182 is not hydrogen bonded in the L intermediate. 40,41 The observation (6) is consistent with a recent FTIR study showing that a strong interaction between Asp85 and the OH group of Thr89 remains unchanged in the L intermediate.…”

Mechanism of Ion Transport across Membranes

“…Other mutations, nearer the retinal, also caused the slowing of the photocycle, but for these there was no correlation with the deprotonation of Asp 85 . In these cases a perturbed interaction between the 13-methyl or 9-methyl groups of the retinal and the protein interfered with reisomerization to all-trans, as suggested earlier by other kinds of evidence (32)(33)(34).…”

Structure of bacteriorhodopsin at 1.55 Å resolution

“…Site-speci®c mutagenesis of many of these residues resulted in changed absorption maxima or changed rate of thermal isomerization (e.g. see Marti et al, 1991;Sonar et al, 1993;Ihara et al, 1994;Delaney et al, 1995;Weidlich et al, 1996). The side-chain of Leu93, a residue that was found to in¯uence the rate of 13-cis-to all-trans reisomerization in the photocycle (Delaney et al, 1995), is as close as 3.6 A Ê from the 13-methyl group of the retinal.…”