Substitution of amino acids Asp-85, Asp-212, and Arg-82 in bacteriorhodopsin affects the proton release phase of the pump and the pK of the Schiff base.

Abstract

Photocycle and flash-induced proton release and uptake were investigated for bacteriorhodopsin mutants in which Asp-85 was replaced by Ala, Asn, or Glu; Asp-212 was replaced by Asn or Glu; Asp-115 was replaced by Ala, Asn, or Glu; Asp-96 was replaced by Ala, Asn, or Glu; and Arg-82 was replaced by Ala or Gln in dimyristoylphosphatidylcholine/3- [(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate miceiles at pH 7.3. In the Asp-85 -Ala and Asp-85 --Asn mutants, the absence of the charged carboxyl group leads to a blue chromophore at 600 and 595 nm, respectively, and lowers the pK of the Schiff base deprotonation to 8.2 and 7, respectively, suggesting a role for Asp-85 as counterion to the Schiff base. The early part of the photocycles of the Asp-85 -* Ala and Asp-85 -* Asn mutants is strongly perturbed; the formation of a weak M-like intermediate is slowed down about 100-fold over wild type. In both mutants, proton release is also slower but dearly precedes the rise of M. The amplitude of the early (<0.2 ps) reversed photovoltage component in the Asp-85 -* Asn mutant is very large, and the net charge displacement is close to zero, indicating proton release and uptake on the cytoplasmic side of the membrane. The data suggest an obligatory role for Asp-85 in the efficient deprotonation of the Schiff base and in the proton release phase, probably as proton acceptor. In the Asp-212 -* Asn mutant, the rise of the absorbance change at 410 nm is slowed down to 220 Ls, its amplitude is small, and the release of protons is delayed to 1.9 ms. The absorbance changes at 650 nm indicate perturbations in the early time range with a slow K intermediate. Thus Asp-212 also participates in the early events of charge translocation and deprotonation of the Schiff base. In the Arg-82 -Gln mutant, no net transient proton release was observed, whereas, in the Arg-82 Ala mutant,, uptake and release were reversed. The pK shift of the purple-to-blue transition in the Asp-85 --Glu, Arg-82-Ala, and Arg-82 --Gin mutants and the similarity in the photocycle and photoelectrical signals of the Asp-85 -Ala, Asp-85 --Asn, and Asp-212 --Asn mutants suggest the interaction between Asp-85, Arg-82, Asp-212, and the Schiff base as essential for proton release.Site-directed mutagenesis has shown and Asp-212 to be essential for proton translocation by bacteriorhodopsin (bR) (1). The very low activity in mutants Asp-96 -+ Ala (D96A) and Asp-96 -* Asn (D96N) at pH 7 is due to a markedly slowed-down decay of the photocycle intermediate M and the associated charge movement (2-5). was concluded to be the internal proton donor for the reprotonation of the Schiff base (SB) leading to the decay of M (3, 4). Fourier-transform infrared spectroscopy has revealed the protonation states of Asp-85, Asp-96, Asp-115, and Asp-212 in the K, L, and M intermediates and provided clues to the time course of proton transfer (6, 7). The data suggest that Asp-85 and Asp-212 are deprotonated in the ground state and become protonated in the L -* M transition (6). A...

PURPLE MEMBRANE: SURFACE CHARGE DENSITY and THE MULTIPLE EFFECT OF pH and CATIONS

“…Studies of the bR mutants have indicated that certain amino acids such as Asp-85, Asp-212, and Tyr-185, which are located in the proton channel near the Schiff base and involved in proton pumping, are also closely related to the regulation of the spectral transition of the purple membrane to the blue membrane (Mogi et af., 1988;Butt et al, 1989;Soppa et al, 1989;Otto et al, 1990;Subramaniam et al, 1990). As noted above, it appears that the color of bR is primarily regulated by a negative charge near the positively charged, protonated Schiff base (e.g.…”

“…This lysine, Lys-216, is therefore neutralized in the membrane, although the protonated Schiff base which it forms with the retinal is positively charged. Asp-85 has been suggested to act as a counterion to the protonated Schiff base Otto et al, 1990;Subramaniam et al, 1990), and its protonation probably leads to the formation of the blue membrane (see below). Jonas and Ebrey (1990) have proposed that the removal of a divalent cation from the bR active site Figure 1.…”

Effects of genetic replacements of charged and H-bonding residues in the retinal pocket on Ca2+ binding to deionized bacteriorhodopsin.

“…The absorption maximum of the retinal chromophore is believed to be primarily regulated by the electrostatic interactions between the charged or polar side chains of amino acids and the PSB, as well as certain carbon atoms along the retinal electronic system (10-14). From the structure (15) and genetic modification studies of bR (16,17), it has been found that Asp-85, Asp-212, and Arg-82 are charged groups in the retinal pocket. They are suggested to make a major contribution to the regulation of color of bR (17).…”

The early development and application of FTIR difference spectroscopy to membrane proteins: A personal perspective

“…Proton release involves a proton transfer from the Schiff base (SB) to Asp-85 (L → M) [42] (see step 2 of Fig. 13) and an almost simultaneous ejection of a proton into the outer medium [178]. This involves the initial movement of Arg-82 away from the active site due to proton transfer from the Schiff base to counterion Asp-85, and towards an extracellular proton release group [42] subsequently identified as Glu-194 and Glu-204 [48,49,68].…”