The two pKa's of aspartate-85 and control of thermal isomerization and proton release in the arginine-82 to lysine mutant of bacteriorhodopsin

Abstract

To explore the role of Arg82 in the catalysis of proton transfer in bacteriorhodopsin, we replaced Arg82 with Lys, which is also positively charged at neutral pH but has an intrinsic pKa of about 1.7 pH units lower than that of Arg. In the R82K mutant expressed in Halobacterium salinarium, we found the following: (1) The pKa of the purple-to-blue transition at low pH (which reflects the pKa of Asp85) is 3.6 +/- 0.1. At high pH a second inflection in the blue-to-purple transition with pKa = 8.0 is found. The complex titration behavior of Asp85 indicates that the pKa of Asp85 depends on the protonation state of another amino acid residue, X', which has a pKa = 8.0 in R82K. The fit of the experimental data to a model of two interacting residues shows that deprotonation of X' at high pH causes a shift in the pKa of Asp85 from 3.7 to 6.0. In turn, protonation of Asp85 decreases the pKa of X' by 2.3 pH units. This suggests that X' can release a proton upon formation of the M intermediate and the concomitant protonation of Asp85 in the photocycle. (2) The rate constant of dark adaptation, kda, is proportional to the fraction of blue membrane between pH 2 and 10, indicating that thermal isomerization proceeds through the transient protonation of Asp85. The pH dependence of kda shows that two groups with pKal = 3.9 and pKa2 = 8.0 control the rate of dark adaptation in R82K. The 1.7 pH unit shift in pKa2 in R82K compared to the wild type (WT) (pKa2 = 9.7) supports the hypothesis that X' is Arg82 in WT and Lys82 in R82K (or at least that these groups are the principal part of a cluster of residues that constitute X'). (3) Under steady state illumination, the efficiency of proton transport in R82K incorporated in phosphatidylcholine vesicles is at least 40% of that in the WT. A flash-induced transient signal of the pH-sensitive dye pyranine is similar to that in the WT (proton release precedes uptake), but the amplitude is small in R82K (about 15% of that found in the WT), indicating that only a small fraction of protons is released fast in R82K. This supports the suggestions that Arg82 is associated with the proton release pathway (acts as a proton release group or part of a proton release complex) and that Lys cannot efficiently substitute for Arg in this process.(ABSTRACT TRUNCATED AT 400 WORDS)

The Influence of a Transmembrane pH Gradient on Protonation Probabilities of Bacteriorhodopsin: The Structural Basis of the Back-Pressure Effect

“…These nontitrating residues are tyrosine residues (always protonated), R82 (always protonated), and D212 (always deprotonated). Their constant protonation over large pH ranges are consistent with experimental data 62,66,68,69 and previous theoretical calculations. 33 The protonation probability of D85 depends on the pH at the two sides of the membrane.…”

“…In contrast, the titration curves of D85 and D115 clearly deviate from a standard sigmoidal curve. Such irregular titration curves have also been observed in previous experimental 66,67 and theoretical studies 27,29,33,34 without the presence of a transmembrane proton gradient. They occur as a result of the strong electrostatic interactions between the residues that titrate at the same pH range.…”

Spectroscopic and Photochemical Characterization of a Deep Ocean Proteorhodopsin

“…This unique behavior among microbial rhodopsins indicates that an additional titratable group modulates the environment of the chromophore in BPR. Note that the diprotic titration behavior of BPR differs from the two pK a spectral transitions reported for bacteriorhodopsin (20) and sensory rhodopsin I (21). The diprotic behavior in those cases are attributable to the dependence of the pK a of the retinylidene Schiff base counterion aspartyl carboxylate (Asp-85 in BR and Asp-76 in SRI, which correspond to Asp-97 in BPR) on the titration of another residue in the protein.…”

Proton Affinity Changes Driving Unidirectional Proton Transport in the Bacteriorhodopsin Photocycle

“…Moreover, the experimental pH-dependence of the purple-to-blue transition is complex, apparently involving couplings to elements of the extracellular proton-release system. 39,40 The photocycle is known to occur via the same stages 41 in an extremely wide range of pH, from at least 5 to 9, which is larger than the typical uncertainties associated with pK a predictions based on the methodologies 32,34 used here. The present work focuses on relative changes of proton affinities during the cycle, which are less sensitive to methodological uncertainties.…”

“…The importance of Arg82 for the proton release mechanism was noted in earlier computational and experimental studies. 39,44 A proton transfer from the SB to Asp85 (p.t. 1), and the release of a proton (p.t.…”