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

Karsten Neumann ^*, Mirka-Kristin Verhoefen ^*, Ingrid Weber , Clemens Glaubitz  and Josef Wachtveitl ^*

^* Institute of Physical and Theoretical Chemistry, and Institute of Biophysical Chemistry and Center of Biomolecular Magnetic Resonance, Johann Wolfgang Goethe-University Frankfurt, Frankfurt/Main, Germany

Correspondence: Address reprint requests to Josef Wachtveitl, Institute of Physical und Theoretical Chemistry, Institute of Biophysics, Johann Wolfgang Goethe-University Frankfurt, Max-von-Laue-Str. 7, 60438 Frankfurt/Main, Germany. Tel.: 49-69-798-29351; Fax: +49 69 798 29709; E-mail: wveitl{at}theochem.uni-frankfurt.de

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 C=C (1500 cm^–1–1580 cm^–1) and C=N 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 (PR_K–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.