Breaking the Carboxyl Rule: LYSINE 96 FACILITATES REPROTONATION OF THE SCHIFF BASE IN THE PHOTOCYCLE OF A RETINAL PROTEIN FROM EXIGUOBACTERIUM SIBIRICUM

Sergei P Balashov,Lada E Petrovskaya,Eleonora S Imasheva,Evgeniy P Lukashev,Andrei K Dioumaev,Jennifer M Wang,Sergey V Sychev,Dmitriy A Dolgikh,Andrei B Rubin,Mikhail P Kirpichnikov,Janos K Lanyi

doi:10.1074/jbc.m113.465138

Abstract

A lysine instead of the usual carboxyl group is in place of the internal proton donor to the retinal Schiff base in the light-driven proton pump of Exiguobacterium sibiricum (ESR). The involvement of this lysine in proton transfer is indicated by the finding that its substitution with alanine or other residues slows reprotonation of the Schiff base (decay of the M intermediate) by more than 2 orders of magnitude. In these mutants, the rate constant of the M decay linearly decreases with a decrease in proton concentration, as expected if reprotonation is limited by the uptake of a proton from the bulk. In wild type ESR, M decay is biphasic, and the rate constants are nearly pH-independent between pH 6 and 9. Proton uptake occurs after M formation but before M decay, which is especially evident in D2O and at high pH. Proton uptake is biphasic; the amplitude of the fast phase decreases with a pKa of 8.5 ± 0.3, which reflects the pKa of the donor during proton uptake. Similarly, the fraction of the faster component of M decay decreases and the slower one increases, with a pKa of 8.1 ± 0.2. The data therefore suggest that the reprotonation of the Schiff base in ESR is preceded by transient protonation of an initially unprotonated donor, which is probably the ε-amino group of Lys-96 or a water molecule in its vicinity, and it facilitates proton delivery from the bulk to the reaction center of the protein.

Highlights

Lysine rather than a carboxylic residue is in place of the internal proton donor in the E. sibiricum proton pump
Light-induced proton pumping by the K96A mutant in suspensions of E. coli cells and proteoliposomes was examined in same way as described previously for wild type Exiguobacterium sibiricum (ESR) [2]
Evidence is presented for a two-step mechanism for proton transfer from the bulk to the retinal Schiff base that involves an internal proton donor, which is the side chain of Lys-96 and/or interacting water molecules

Summary

Background

Lysine rather than a carboxylic residue is in place of the internal proton donor in the E. sibiricum proton pump. A lysine instead of the usual carboxyl group is in place of the internal proton donor to the retinal Schiff base in the lightdriven proton pump of Exiguobacterium sibiricum (ESR) The involvement of this lysine in proton transfer is indicated by the finding that its substitution with alanine or other residues slows reprotonation of the Schiff base (decay of the M intermediate) by more than 2 orders of magnitude. Deprotonation of the initially protonated Asp-96 was detected by FTIR during the decay of the M intermediate [16, 18, 27], which proceeds in a pH-independent manner, but the subsequent reprotonation of the carboxylate during proton uptake from the bulk is pH-dependent [28, 29] and is responsible for slowing the N to O transition and overall photocycle turnover at high pH (29 –31) These changes in protonation of the internal carboxyl residue are caused by a transient decrease of its pKa value during the photocycle from above 11 to ϳ 7.5 [28, 30, 32, 33]. That reprotonation of the Schiff base is slowed manyfold by mutations of Lys-96 suggests that Lys-96, and possibly interacting water molecules, is the site that mediates proton transfer from the bulk to the Schiff base

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION