Glutamate-194 to Cysteine Mutation Inhibits Fast Light-Induced Proton Release in Bacteriorhodopsin

Abstract

Substitution of glutamic acid-194, a residue on the extracellular surface of bacteriorhodopsin, with a cysteine inhibits the fast light-induced proton release that normally is coupled with the deprotonation of the Schiff base during the L to M transition. Proton release in this mutant occurs at the very end of the photocycle and coincides with deprotonation of the primary proton acceptor, Asp-85, during the O to bR transition. the E194C mutation also results in a slowing down of the photocycle by about 1 order of magnitude as compared to the wild type and produces a strong effect on the pH dependence of dark adaptation that is interpreted as a drastic reduction or elimination of the coupling between the primary proton acceptor Asp-85 and the proton release group. These data indicate that Glu-194 is a critical component of the proton release complex in bacteriorhodopsin.

Breaking the Carboxyl Rule

“…Following transfer of its proton to the Schiff base, its initial unprotonated state is restored. Second, the protonation state of Lys-96 does not change, but the lysine side chain supports a hydrogen-bonded network that can contain an additional proton in a similar way as the extracellular proton release network (45) is supported by Glu-194 and Glu-204 in bacteriorhodopsin (42,43).…”

“…This would explain the key differences in the sequence of events after photoexcitation. In bacteriorhodopsin, protonation of the Schiff base by Asp-96 during the M to N transition is distinctively more rapid than the proton uptake (39,42,46,55,56) leading to reprotonation of Asp-96. Asp-96 appears to be inaccessible to the bulk at the time of the proton exchange between Asp-96 and the Schiff base (33), because it does not lose the proton to the bulk up to very high pH values.…”

Amino acids with an intermolecular proton bond as proton storage site in bacteriorhodopsin

“…Earlier IR studies have assigned the highly conserved Glu-204 and/or Glu-194 as the PRG (10,11). Gerwert and coworkers carried out careful analysis of the IR spectra using the WT bR and a collection of mutants.…”

Molecular mechanisms controlling proton pumping by bacteriorhodopsin. Final report

“…Using the same approach they found that G1u204 shows all the features of the PRG X and the residue controlling pKa of Asp85, X'. [1][2][3]6,7,9].…”

“…100 AS) [9]. This correlation is observed not only in Arg82 mutants but in other mutants too: K129H [3], E194C [6], R134K [7], E204A and E204N [8]. The most likely explanation is that the initial high pKa of Asp85 indicates more hydrophobic environment that favors the protonated state of Asp85 [1].…”