The Initial Reaction Dynamics of Proteorhodopsin observed by Femtosecond Infrared and Visible Spectroscopy
Karsten Neumann 1, Mirka-Kristin Verhoefen 1, Ingrid Weber 2, Clemens Glaubitz 2 and Josef Wachtveitl 3*
1 Institute of Physical and Theoretical Chemistry, Johann Wolfgang Goethe-University Frankfurt, German
2 Institute of Biophysical Chemistry and Center of Biomolecular Magnetic Resonance, Johann Wolfgang Go
3 Goethe Universität Frankfurt am Main
* To whom correspondence should be addressed. E-mail: wveitl{at}theochem.uni-frankfurt.de.
Submitted on November 8, 2007
Revised on December 10, 2007
Accepted on 8 February 2008
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Abstract |
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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 (Lenz, M.O., R. Huber, B. Schmidt, P. Gilch, R. Kalmbach, M. Engelhard, and J. Wachtveitl. 2006. Biophys. J. 91:255-262). This points to a pH dependent H-bonding network in the binding pocket of proteorhodopsin, that directly influences the primary photoinduced 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 (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.
Key Words:
femtosecond spectroscopy, photocycle, proteorhodopsin, retinal
