Time-resolved titrations of the Schiff base and of the Asp85 residue in artificial bacteriorhodopsins.
Deprotonation/protonation processes involving the retinal Schiff base and the Asp85 residue play dominant roles in the light-induced proton pump of bacteriorhodopsin (bR). Although the pKa values of these two moieties in unphotolyzed bR are well established, the kinetics of the respective titrations in the native pigment are difficult to interpret, primarily due to the extreme (nonphysiological) pKa values of the two moieties (12.2 +/- 0.2 and 2.7, in 0.1 M NaCl, for the Schiff base and for Asp85, respectively). These difficulties are circumvented by applying stopped-flow techniques, time resolving the titrations of several artificial bRs in which the pKa values of the above two residues are substantially modified: 13-CF3 bR, pKa (Schiff base) = 8.2 +/- 0.2; 13-demethyl-11,14-epoxy bR, pKa (Schiff base) = 8.2 +/- 0.1 (in 0.1 M NaCl); aromatic bR, pKa (Asp85) = 5.2 +/- 0.1 (in water). The R82Q bR mutant, pKa (Asp85) congruent to 7.2 was also employed. A major objective was to verify whether the basic relationships of homogeneous kinetics obeyed by elementary acid/base systems in solution (primarily, the possibility to express the equilibrium constant as the ratio of the forward and back rate constants) are also obeyed by the Schiff base and Asp85 moieties. We found that this is the case for the Schiff base in the pH range between 7 and 9 but not at lower pH. These observations led to the conclusion that the Schiff base is titrable from the outside medium via a proton channel, which becomes saturated, and thus rate determining, below pH approximately equal to 7.(ABSTRACT TRUNCATED AT 250 WORDS)
Druckmann
S
S
Department of Physical Chemistry, Hebrew University, Jerusalem, Israel.
Ottolenghi
M
M
Rousso
I
I
Friedman
N
N
Sheves
M
M
D001224:Aspartic Acid / Q000737:chemistry; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D017494:Proton Pumps; D011522:Protons; D012545:Schiff Bases / Q000737:chemistry
D017494:Proton Pumps; D011522:Protons; D012545:Schiff Bases; D001224:Aspartic Acid; D001436:Bacteriorhodopsins