Reaction Pathways of Photoexcited Retinal in Proteorhodopsin Studied by Pump−Dump−Probe Spectroscopy

Abstract

Proteorhodopsin (pR) is a membrane-embedded proton pump from the microbial rhodopsin family. Light absorption by its retinal chromophore initiates a photocycle, driven by trans/cis isomerization on the femtosecond to picosecond time scales. Here, we report a study on the photoisomerization dynamics of the retinal chromophore of pR, using dispersed ultrafast pump-dump-probe spectroscopy. The application of a pump pulse initiates the photocycle, and with an appropriately tuned dump pulse applied at a time delay after the dump, the molecules in the initial stages of the photochemical process can be de-excited and driven back to the ground state. In this way, we were able to resolve an intermediate on the electronic ground state that represents chromophores that are unsuccessful in isomerization. In particular, the fractions of molecules that undergo slow isomerization (20 ps) have a high probability to enter this state rather than the isomerized K-state. On the ground state reaction surface, return to the stable ground state conformation via a structural or vibrational relaxation occurs in 2-3 ps. Inclusion of this intermediate in the kinetic scheme led to more consistent spectra of the retinal-excited state, and to a more accurate estimation of the quantum yield of isomerization (Φ ) 0.4 at pH 6).

Reaction dynamics of the chimeric channelrhodopsin C1C2

“…3d ), indicating this species is red-shifted with respect to the steady-state ground state absorption of C1C2. Such GSI properties are similar to those observed previously in proteorhodopsin and PYP 26 , 30 . It is uncertain whether the somewhat oscillatory spectral shape of the GSI is real or spurious.…”

“… To estimate the relative amplitude of the SADS (and thereby the photoproduct quantum yield), we assume that the ground-state bleach amplitude of the three excited states and of the photoproduct state should be roughly the same. Dumping the excited state in rhodopsins is expected to give rise to intermediates on the ground state potential energy surface 30 , which need to be included in the model. …”

“…Dumping the excited state in rhodopsins is expected to give rise to intermediates on the ground state potential energy surface 30 , which need to be included in the model.…”

Second-Chance Forward Isomerization Dynamics of the Red/Green Cyanobacteriochrome NpR6012g4 from Nostoc punctiforme

“…GSIs have been observed in other PDP experiments 25-28 and for isomerizing systems; they are typically interpreted as twisted or partially isomerized ground-state populations. 25 The decay of the GSI in NpR6012g4 is multi-phasic on an apparent 10-ps time scale (Figs. 3B and 4B), with full decay occurring by 50 ps (Fig.…”

A QM/MM study of the initial excited state dynamics of green-absorbing proteorhodopsin

“…To study such ultrafast dynamics, time-resolved absorption and infrared spectroscopy measurements have been conducted mainly on GPR. [20][21][22][23][24][25][26] The ultrafast dynamics of native GPR at pH 9 was found to be best described by an excited state relaxation and a biexponential decay with time constants of 140 fs, 280 fs and 9.5 ps, respectively. 21 However, the underlying molecular mechanism of the isomerization has not been explored by computational methods, mainly due to the lack of a crystal structure of GPR.…”