Real-time spectroscopy of transition states in bacteriorhodopsin during retinal isomerization.
Real-time investigations of the rearrangement of bonds during chemical transformations require femtosecond temporal resolution, so that the atomic vibrations within the reacting molecules can be observed. Following the development of lasers capable of emitting ultrashort laser flashes on this timescale, chemical reactions involving relatively simple molecules have been monitored in detail, revealing the transient existence of intermediate species as reactants are transformed into products. Here we report the direct observation of nuclear motion in a complex biological system, the retinal chromophore of bacteriorhodopsin (bR568), as it undergoes the trans-cis photoisomerization that is fundamental to the vision process. By using visible-light pulses of less than 5 femtosecond in duration, we are able to monitor changes in the vibrational spectra of the transition state and thus show that despite photoexcitation of the anti-bonding molecular orbital involved, isomerization does not occur instantly, but involves transient formation of a so-called 'tumbling state'. Our observations thus agree with growing experimental and ab initio evidence for a three-state photoisomerization model and firmly discount the initially suggested two-state model for this process.