Mutation of arginine 134 to lysine alters the pK(a)s of key groups involved in proton pumping by bacteriorhodopsin.
Arginine 134 is located near the extracellular surface of bacteriorhodopsin (bR) and may interact with one or more nearby glutamate residues. In the bR mutant R134K, light-induced Schiff-base deprotonation (formation of the M intermediate) exhibits several kinetic components and has a complex pH dependence. The kinetics and pH dependence of M formation were analyzed using the following general guidelines for interpreting M formation: (1) The fastest component of M formation reflects the redistribution of the Schiff-base proton to D85, the usual proton acceptor, in response to the change in the proton affinities of the Schiff base and D85 early in the photocycle; (2) Two additional components of M formation reflect transitions between spectroscopically similar substates of M. By applying these guidelines, supplemented by information about the pK(a)s of D85 and the proton release group from acid (purple-to-blue) and alkaline titrations of the absorption spectra of the unphotolyzed R134K pigment, we explain the pH dependence of M formation as being due to titration of the counterion, D85, and of the proton release group. We calculate, in R134K, that the pKa of D85 is 4.6 in the unphotolyzed state, while the pKa of the proton release group is 8.0 in the unphotolyzed state but drops to approximately 5.8 in the M intermediate. The same value for the pKa of the proton release group in the M intermediate is obtained when we use photocurrent measurements to monitor proton release. The altered values of these pK(a)s relative to the corresponding values in wild-type bR suggest that D85 and the proton release group are coupled more weakly in R134K than in the wild type.
Misra
S
S
Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign 61801, USA.
Martin
C
C
Kwon
O H
OH
Ebrey
T G
TG
Chen
N
N
Crouch
R K
RK
Menick
D R
DR
D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D001436:Bacteriorhodopsins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011522:Protons
D011522:Protons; D001436:Bacteriorhodopsins; D001120:Arginine; D008239:Lysine