Initial Reaction Dynamics of Proteorhodopsin Observed by Femtosecond Infrared and Visible Spectroscopy

We present a comparative study using femtosecond pump/probe spectroscopy in the visible and infrared of the early photodynamics of solubilized proteorhodopsin (green absorbing variant) in D2O with deprotonated (pD 9.2) and protonated (pD 6.4) primary proton acceptor Asp-97. The vis-pump/vis-probe experiments show a kinetic isotope effect that is more pronounced for alkaline conditions, thus decreasing the previously reported pH-dependence of the primary reaction of proteorhodopsin in H2O. This points to a pH dependent H-bonding network in the binding pocket of proteorhodopsin, that directly influences the primary photo-induced dynamics. The vis-pump/IR-probe experiments were carried out in two different spectral regions and allowed to monitor the retinal CC (1500 cm−1–1580 cm−1) and CN stretching vibration as well as the amide I mode of the protein (1590 cm−1–1680 cm−1). Like the FTIR spectra of the K intermediate (PRK–PR difference spectra) in this spectral range, the kinetic parameters and also the quantum efficiency of photo-intermediate formation are found to be virtually independent of the pD value.