Replacement effects of neutral amino acid residues of different molecular volumes in the retinal binding cavity of bacteriorhodopsin on the dynamics of its primary process

Abstract

We have determined the rate and quantum yield of retinal photoisomerization, the spectra of the primary transients, and the energy stored in the K intermediate in the photocycle of some bacteriorhodopsin mutants (V49A, A53G, and W182F) in which residue replacements are found to change the Schiff base deprotonation kinetics (and thus the protein-retinal interaction). Because of their change in the local volume resulting from these individual replacements, these substitutions perturb the proton donor-acceptor relative orientation change and thus the Schiff base deprotonation kinetics. These replacements are thus expected to change the charge distribution around the retinal, which controls its photoisomerization dynamics. Subpicosecond transient spectroscopy as well as photoacoustic technique are used to determine the retinal photoisomerization rate, quantum yield, and the energy stored in the K-intermediate for these mutants. The results are compared with those obtained for wild-type bacteriorhodopsin and other mutants in which charged residues in the cavity are replaced by neutral ones. In some of the mutants the rate of photoisomerization is changed, but in none is the quantum yield or the energy stored in the K intermediate altered from that in the wild type. These results are discussed in terms of the shapes of the potential energy surfaces of the excited and ground states of retinal in the perpendicular configuration within the protein and the stabilization of the positive charge in the ground and the excited state of the electronic system of retinal.

The photoisomerization of retinal in bacteriorhodopsin: Experimental evidence for a three-state model

“…However, this perturbation need not influence the quantum efficiency for photoisomerization. Indeed, mutations that alter the excited state lifetime by more than an order of magnitude were found to have a negligible impact on the quantum efficiency for photoisomerization (14,22). The role of the protein in mediating the all-trans to 13-cis photoisomerization appears be largely as a catalyst along the C 13 AC 14 bond and as an inhibitor along all other double bonds (21).…”

“…An implication that emerged from some of the earlier work is that such a bias might facilitate rapid conversion to the 13-cis form and may even be necessary to achieve a high quantum yield for photoisomerization. On the other hand, recent studies of bR mutants revealed quantum efficiencies for photoisomerization that were remarkably constant and similar to the efficiency for native bR, despite excited-state lifetimes that varied by an order of magnitude (14,(20)(21)(22). This suggests that rapid conversion to the 13-cis form might not be necessary to attain a high quantum efficiency for photoisomerization.…”

Redetermination of the Quantum Yield of Photoisomerization and Energy Content in the K-Intermediate of Bacteriorhodopsin Photocycle and Its Mutants by the Photoacoustic Technique

“…The experimental approach we used previously [13][14][15] to determine these values was a combination of subpicosecond transient absorption and the photoacoustic technique. Transient absorption was used to determine the quantum yield of photoisomerization, and the photoacoustic signal was used to verify this number and estimate the energy content in the K-intermediate.…”

The effect of the protein environment on the structure and charge distribution of the retinal Schiff base in bacteriorhodopsin

“…However, considering the results obtained from the present study, it may be concluded that these amino acids primarily play their role by the construction of the binding pocket for the retinal chromophore and by coupling the protein and chromophore conformational changes through the steric interaction with the chromophore structure. These structural eects, added to the dominant steric and electronic restrictions of the binding pocket [32,33], would explain the discrimination exhibited by the protein binding site for dierent analogs during incubation studies [34]. With regard to this, the location of the methyl groups on the polyene side chain is of utmost importance in determining the overall shape of the retinal ligands [34].…”

“…With regard to this, the location of the methyl groups on the polyene side chain is of utmost importance in determining the overall shape of the retinal ligands [34]. These eects can also in¯uence the rate of the photoisomerization and dynamics of the ground and excited states of the retinal Schi base [32,33,35,36]. The importance of the methyl groups has also been discussed during the study of the excited-state potential energy surface of isomerization in dierent isomers of the retinal Schi base [37,38].…”