Unlike visual rhodopsins that bleach upon illumination, archaeal rhodopsins exhibit photocycle. This is highly advantageous in ultrafast spectroscopic studies and these techniques have been extensively applied in addition to low-temperature spectroscopy [2,12,13]. In particular, bacteriorhodopsin has been regarded historically as the model system to test new spectroscopic methods. As in visual rhodop-sins, the light absorption of archaeal rhodopsins causes formation of red-shifted primary intermediates . The primary K intermediate can be stabilized at 77 K. Time-resolved visible spectroscopy of bacteriorhodopsin reveals the presence of the precursor, called the J intermediate [12,13]. The J intermediate is more red-shifted (kmax ~625 nm) than the K intermediate (kmax ~590 nm). The excited state of bacteriorhodopsin possesses blue-shifted absorption, which decays nonexpo-nentially. The two components of the stimulated emission decay at about 200 and 500 fs . The J intermediate is formed in <500 fs, and converted to the K intermediate within 3 ps [12,69].
So when does isomerization take place from the all-trans to 13-cis form? To answer this question, as for visual rhodopsin, all-trans-locked 5-membered retinal was incorporated into bacteriorhodopsin [70-72]. In experiments with a picosecond time resolution, an intermediate was found with properties similar to those of the J intermediate . Together with the ultrafast pump probe  and coherent anti-Stokes Raman  spectroscopic results, it was concluded that isomerization around C13=C14 is not a prerequisite for producing the J intermediate. More importantly, since the J intermediate is a ground-state species, isomerization does not take place in the excited state of bacteriorhodopsin according to their interpretation [70-72]. However, other experimental data favor a common mechanism between visual and archaeal rhodopsins; namely, isomerization taking place in the excited state. Femtosecond visible-pump and infrared-probe spectroscopy showed the 13-cis characteristic vibrational band at 1190 cm-1 appearing with a time constant of ~0.5 ps, indicating that the all-trans to 13-cis isomerization takes place in femtoseconds . This time scale is coincident with formation of the J intermediate. Fourier transform of the transient absorption data with <5 fs resolution also showed the appearance of the 13-cis form in <1 ps, supporting the suggestion that the all-trans to 13-cis isomerization takes place in femtoseconds . Previous anti-Stokes resonance Raman spectroscopy proposed that the J intermediate is a vibrational hot state of the K intermediate . Thus, many experimental results are consistent with the isomerization model in the excited state.
Comparative investigation of mutant proteins of bacteriorhodopsin, other archaeal rhodopsins, and the protonated Schiff base of all-trans-retinal in solution is useful for better understanding of the primary photoisomerization mechanism. Ultrafast spectroscopy of various bacteriorhodopsin mutants revealed that only the replacements of the charged residues reduced the photoisomerization rate, leading to less efficient photoisomerization . This observation explains an important role of the electrostatic interaction of the counterion complex in the primary photoisomerization mechanism (Fig. 4.8A). The excited state is more long lived in the chloride pump halorhodopsin [77-79] and light-sensor N. pharaonis phoborhodopsin , and hence less efficient for photoisomerization. These observations suggest that bacteriorhodopsin possesses the optimized structure for the primary photoisomerization mechanism, though the structures in Fig. 4.8 look essentially similar
As for visual rhodopsins, spectroscopic studies of the protonated Schiff base of all-trans-retinal in solution are important for understanding the isomerization mechanism. We first reported the excited state dynamics of the protonated Schiff base of all-trans-retinal in methanol solution , and found that the kinetics is very similar to that of the 11-cis form (Fig. 4.6B). The only difference was that the lifetimes are 1.2-1.4 times longer in the all-trans form than in the 11-cis form [53,81]. Slightly faster decay of the 11-cis form may be reflected by their molecular structures, namely the initial steric hindrance between C10-H and C13-CH3 in the 11-cis form (Fig. 4.3) that accelerates the fluorescence decay. Interestingly, it was found that the all-trans-locked 5-membered system, which prohibits both C11=C12 and C13=C14 isomerizations, exhibits similar kinetics to those of the all-trans form in solution . These results are entirely different from those of the 11-cis-locked 5-membered system, in which the excited-state lifetime is 5-times longer (Fig. 4.6B,C) . This suggests more complex excited-state dynamics for the all-trans form. Observation of the J-like state in protein [70-72] might be correlated with such properties of the protonated Schiff base of the all-trans form.
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