Two Groups Control Light-Induced Schiff Base Deprotonation and the Proton Affinity of Asp85 in the Arg82His Mutant of Bacteriorhodopsin

Abstract

Arg(82) is one of the four buried charged residues in the retinal binding pocket of bacteriorhodopsin (bR). Previous studies show that Arg(82) controls the pK(a)s of Asp(85) and the proton release group and is essential for fast light-induced proton release. To further investigate the role of Arg(82) in light-induced proton pumping, we replaced Arg(82) with histidine and studied the resulting pigment and its photochemical properties. The main pK(a) of the purple-to-blue transition (pK(a) of Asp(85)) is unusually low in R82H: 1.0 versus 2.6 in wild type (WT). At pH 3, the pigment is purple and shows light and dark adaptation, but almost no light-induced Schiff base deprotonation (formation of the M intermediate) is observed. As the pH is increased from 3 to 7 the M yield increases with pK(a) 4.5 to a value approximately 40% of that in the WT. A transition with a similar pK(a) is observed in the pH dependence of the rate constant of dark adaptation, k(da). These data can be explained, assuming that some group deprotonates with pK(a) 4.5, causing an increase in the pK(a) of Asp(85) and thus affecting k(da) and the yield of M. As the pH is increased from 7 to 10.5 there is a further 2.5-fold increase in the yield of M and a decrease in its rise time from 200 micros to 75 micros with pK(a) 9. 4. The chromophore absorption band undergoes a 4-nm red shift with a similar pK(a). We assume that at high pH, the proton release group deprotonates in the unphotolyzed pigment, causing a transformation of the pigment into a red-shifted "alkaline" form which has a faster rate of light-induced Schiff base deprotonation. The pH dependence of proton release shows that coupling between Asp(85) and the proton release group is weakened in R82H. The pK(a) of the proton release group in M is 7.2 (versus 5.8 in the WT). At pH < 7, most of the proton release occurs during O --> bR transition with tau approximately 45 ms. This transition is slowed in R82H, indicating that Arg(82) is important for the proton transfer from Asp(85) to the proton release group. A model describing the interaction of Asp(85) with two ionizable residues is proposed to describe the pH dependence of light-induced Schiff base deprotonation and proton release.

Unraveling the mechanism of proton translocation in the extracellular half‐channel of bacteriorhodopsin

“…Mutated protein substituting R82 with Gln, Ala or Lys delays the proton release from the EC until after proton uptake via D96 . In the R82H mutant, the t‐O to t‐bR transition is slowed, indicating that R82 is important for the proton transfer from the CC to the EC. The analysis here will focus on the wild‐type protein where the large, positively charged R82 appears to be a major component in the region between the CC and the EC.…”

“…This process is likely to be slowed by generating a high‐energy neutral Arg if following a proton‐hopping pathway, or because a lower‐energy but longer Grotthuss‐type pathway is rarely formed. Since R82 does not actively participate in a Grotthuss‐type pathway, this may help explain how the R82 mutant proteins can still pump protons …”

Crystal Structures of Acid Blue and Alkaline Purple Forms of Bacteriorhodopsin

“…53 It is worthwhile to note that this cooperativity disappears also when Arg82 is replaced with lysine or histidine. 54 Since the side-chain of this residue is shown to undergo a pronounced structural change during the purpleto-blue transition ( Figure 5), it is very likely that Arg82 plays an important role in mediating a strong coupling between the proton release complex and Asp85, as has been suggested by the structural analysis of the M intermediate. [10][11][12][13] The diffraction data from alkalized crystals show that the proton release complex undergoes a different type of structural change at around pH 10.…”

Role of Arg82 in the Early Steps of the Bacteriorhodopsin Proton-Pumping Cycle

“…22 The much slower proton transfer from Asp85 to the PRG in the O-to-bR transition of Arg82His as compared to the wild-type further supports an important functional role of Arg82. 23 Multiconformation equilibrium electrostatics computations suggested that Arg82, when oriented towards the Schiff base region, stabilises the zwitterionic state with protonated Schiff base and negatively-charged Asp85. In contrast when oriented towards the PRG, Arg82 stabilises the unprotonated Schiff base/protonated Asp85 (M-like) state, and the deprotonated state of the PRG.…”