On the protein residues that control the yield and kinetics of O630 in the photocycle of bacteriorhodopsin

The effects of pH on the yield (phi(r)), and on the apparent rise and decay constants (k(r), k(d)), of the O-630 intermediate are important features of the bacteriorhodopsin (bR) photocycle. The effects are associated with three titration-like transitions: 1) A drop in k(r), k(d), and phi(r) at high pH [pK(a)(1) similar to 8]; 2) A rise in phi(r) at low pH [pK(a)(2) similar to 4.5]; and 3) A drop in k(r) and k(d) at low pH [pK(a)(3) similar to 4.5]. (pK(a) values are for native bR in 100 mM NaCl). Clarification of these effects is approached by studying the pH dependence of phi(r), k(r), and k(d) 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 (R-1) whose state of protonation controls the magnitude of phi(r), k(r), and k(d) at high pH, as Asp-96. Acetylation of bR modifies the Lys-129 residue, which is known to affect the pK(a) of the group (XH), which releases the proton to the membrane exterior during the photocycle. The effects of acetylation on the O-630 parameters reveal that the low-pH titrations should be ascribed to two additional protein residues R-2 and R-3. R-2 affects the rise of phi(r) at low pH, whereas the state of protonation of R-3 affects both k(r) and k(d). Our data confirm a previous suggestion that R-3 should be identified as the proton release moiety (XH). A clear identification of R-2, including its possible identity with R-3, remains open.