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Biophys J, January 2000, p. 354-362, Vol. 78, No. 1

On the Protein Residues that Control the Yield and Kinetics of O630 in the Photocycle of Bacteriorhodopsin

Qun Li,* Smadar Bressler,* David Ovrutsky,* Michael Ottolenghi,* Noga Friedman,dagger and Mudi Shevesdagger

 *Department of Physical Chemistry, The Hebrew University, Jerusalem 91904, and  dagger Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel

The effects of pH on the yield (phi r), and on the apparent rise and decay constants (kr, kd), of the O630 intermediate are important features of the bacteriorhodopsin (bR) photocycle. The effects are associated with three titration-like transitions: 1) A drop in kr, kd, and phi r at high pH [pKa(1) ~ 8]; 2) A rise in phi r at low pH [pKa(2) ~ 4.5]; and 3) A drop in kr and kd at low pH [pKa(3) ~ 4.5]. (pKa values are for native bR in 100 mM NaCl). Clarification of these effects is approached by studying the pH dependence of phi r, kr, and kd in native and acetylated bR, and in its D96N and R82Q mutants. The D96N experiments were carried out in the presence of small amounts of the weak acids, azide, nitrite, and thiocyanate. Analysis of the mutant's data leads to the identification of the protein residue (R1) whose state of protonation controls the magnitude of phi r, kr, and kd at high pH, as Asp-96. Acetylation of bR modifies the Lys-129 residue, which is known to affect the pKa of the group (XH), which releases the proton to the membrane exterior during the photocycle. The effects of acetylation on the O630 parameters reveal that the low-pH titrations should be ascribed to two additional protein residues R2 and R3. R2 affects the rise of phi r at low pH, whereas the state of protonation of R3 affects both kr and kd. Our data confirm a previous suggestion that R3 should be identified as the proton release moiety (XH). A clear identification of R2, including its possible identity with R3, remains open.

Biophys J, January 2000, p. 354-362, Vol. 78, No. 1
© 2000 by the Biophysical Society   0006-3495/00/01/354/09  $2.00


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