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          Institute: MPI für Biophysik     Collection: Abt. Biophysikalische Chemie     Display Documents

ID: 631463.0, MPI für Biophysik / Abt. Biophysikalische Chemie
Critical Role of Asp227 in the Photocycle of Proteorhodopsin
Authors:Herz, Julia; Verhoefen, Mirka-Kristin; Weber, Ingrid; Bamann, Christian; Glaubitz, Clemens; Wachtveitl, Josef
Date of Publication (YYYY-MM-DD):2012-06-27
Title of Journal:Biochemistry
Journal Abbrev.:Biochem.
Issue / Number:28
Start Page:5589
End Page:5560
Review Status:Peer-review
Audience:Experts Only
Abstract / Description:The photocycle of the proton acceptor complex mutant D227N of the bacterial retinal protein proteorhodopsin is investigated employing steady state pH-titration experiments in the UV−visible range as well as femtosecond-pump−probe spectroscopy and flash photolysis in the visible spectral range. The evaluation of the pH-dependent spectra showed that the neutralization of the charge at position 227 has a remarkable influence on the ground state properties of the protein. Both the pKa values of the primary proton acceptor and of the Schiff base are considerably decreased. Femtosecond-time-resolved measurements demonstrate that the general S1 deactivation pathway; that is, the K-state formation is preserved in the D227N mutant. However, the pH-dependence of the reaction rate is lost by the substitution of Asp227 with an asparagine. Also no significant kinetic differences are observed upon deuteration. This is explained by the lack of a strongly hydrogen-bonded water in the vicinity of Asp97, Asp227, and the Schiff base or a change in the hydrogen bonding of it (Ikeda et al. (2007) Biochemistry 46, 5365−5373). The flash photolysis measurements prove a considerably elongated photocycle with pronounced pHdependence. Interestingly, at pH 9 the M-state is visible until the end of the reaction cycle, leading to the conclusion that the mutation does not only lower the pKa of the Schiff base in the unphotolyzed ground state but also prevents an efficient reprotonation reaction
External Publication Status:published
Document Type:Article
Communicated by:N. N.
Affiliations:MPI für Biophysik/Abteilung Biophysikalische Chemie
External Affiliations:Institute of Physical and Theoretical Chemistry, Johann Wolfgang Goethe-University, Max von Laue-Straße 7, 60438 Frankfurt am Main, Germany.
Institute of Biophysical Chemistry and Center of Biomolecular Magnetic Resonance, Johann Wolfgang Goethe-University, Max von Laue-Straße 9, 60438 Frankfurt am Main, Germany.
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