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Elimination of proton donor strongly affects directionality and efficiency of proton transport in ESR, a light-driven proton pump from Exiguobacterium sibiricum

Author:
Siletsky, Sergey A., Mamedov, Mahir D., Lukashev, Evgeniy P., Balashov, Sergei P., Dolgikh, Dmitriy A., Rubin, Andrei B., Kirpichnikov, Mikhail P., Petrovskaya, Lada E.
Source:
Biochimica et biophysica acta 2019 v.1860 no.1 pp. 1-11
ISSN:
0005-2728
Subject:
lysine, probability, sodium azide, schiff bases, alanine, pH, mutants, protons, proton pump, Exiguobacterium
Abstract:
ESR from Exiguobacterium sibiricum is a retinal protein which functions as a proton pump. Unusual feature of ESR is that a lysine residue is present at a site for the internal proton donor, which in other proton pumps is a carboxylic residue. Replacement of Lys96 with alanine slows reprotonation of the Schiff base by two orders of magnitude, indicating that Lys96 and interacting water molecules function as internal proton donor to the Schiff base. In this work we examined time resolved generation of light-induced electric potential ΔΨ by the K96A mutant reconstituted into proteoliposomes. We found that the ΔΨ component, which accompanied reprotonation of the Schiff base in wild type ESR, was not only slowed but also decreased greatly in the mutant, and negative phase appeared at high pH. This indicates a higher probability of back reactions in ESR than in bacteriorhodopsin since no negative components have been observed in homologous mutants of BR, D96N and D96A. The higher rate of back reactions in ESR is probably caused by different arrangement of the proton acceptor site compared to that in BR and different sequence of proton release and uptake. Addition of sodium azide, which substitutes for the internal proton donor, restores both the rate and amplitude of the ΔΨ components related to the Schiff base reprotonation in the K96A mutant. This indicates that overall proton transport results from competition of forward and reverse reactions, and emphasizes the importance of internal donor for high efficiency and directionality of H+ transfer.
Agid:
6142958