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Mutation of Arginine 134 to Lysine Alters the pK a s of Key Groups Involved in Proton Pumping by Bacteriorhodopsin
Author(s) -
Misra Saurav,
Martin Charles,
Kwon OhHoon,
Ebrey Thomas G.,
Chen Ning,
Crouch Rosalie K.,
Menick Donald R.
Publication year - 1997
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.1997.tb03223.x
Subject(s) - bacteriorhodopsin , protonation , chemistry , deprotonation , proton , titration , schiff base , titration curve , photoprotein , proton pump , counterion , proton transport , halobacteriaceae , weak base , crystallography , photochemistry , ion , halobacterium salinarum , inorganic chemistry , biochemistry , enzyme , organic chemistry , bioluminescence , physics , atpase , quantum mechanics , membrane
Abstract— 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 counter‐ion, D85, and of the proton release group. We calculate, in R134K, that the pK a of D85 is 4.6 in the unphotolyzed state, while the pK a of the proton release group is 8.0 in the unphotolyzed state but drops to ?5.8 in the M intermediate. The same value for the pK a 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.