Critical role of Asp227 in the photocycle of proteorhodopsin.
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 pK(a) values of the primary proton acceptor and of the Schiff base are considerably decreased. Femtosecond-time-resolved measurements demonstrate that the general S(1) 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 pH-dependence. 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 pK(a) of the Schiff base in the unphotolyzed ground state but also prevents an efficient reprotonation reaction.
Institute of Physical and Theoretical Chemistry, Johann Wolfgang Goethe-University, Max von Laue-Strasse 7, 60438 Frankfurt am Main, Germany.
Julia
J
Herz
Mirka-Kristin
MK
Verhoefen
Ingrid
I
Weber
Christian
C
Bamann
Clemens
C
Glaubitz
Josef
J
Wachtveitl
D001224:Aspartic Acid / Q000737:chemistry*; D017666:Deuterium Oxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D008958:Models, Molecular; D009154:Mutation; D055668:Photochemical Processes; D012172:Retinaldehyde / Q000737:chemistry; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics; D025604:Rhodopsins, Microbial; D013053:Spectrophotometry; D014867:Water / Q000737:chemistry
D025604:Rhodopsins, Microbial; C413548:proteorhodopsin; D014867:Water; D001224:Aspartic Acid; D012243:Rhodopsin; D017666:Deuterium Oxide; D012172:Retinaldehyde