Aspartate-histidine interaction in the retinal schiff base counterion of the light-driven proton pump of Exiguobacterium sibiricum.
One of the distinctive features of eubacterial retinal-based proton pumps, proteorhodopsins, xanthorhodopsin, and others, is hydrogen bonding of the key aspartate residue, the counterion to the retinal Schiff base, to a histidine. We describe properties of the recently found eubacterium proton pump from Exiguobacterium sibiricum (named ESR) expressed in Escherichia coli, especially features that depend on Asp-His interaction, the protonation state of the key aspartate, Asp85, and its ability to accept a proton from the Schiff base during the photocycle. Proton pumping by liposomes and E. coli cells containing ESR occurs in a broad pH range above pH 4.5. Large light-induced pH changes indicate that ESR is a potent proton pump. Replacement of His57 with methionine or asparagine strongly affects the pH-dependent properties of ESR. In the H57M mutant, a dramatic decrease in the quantum yield of chromophore fluorescence emission and a 45 nm blue shift of the absorption maximum with an increase in the pH from 5 to 8 indicate deprotonation of the counterion with a pK(a) of 6.3, which is also the pK(a) at which the M intermediate is observed in the photocycle of the protein solubilized in detergent [dodecyl maltoside (DDM)]. This is in contrast with the case for the wild-type protein, for which the same experiments show that the major fraction of Asp85 is deprotonated at pH >3 and that it protonates only at low pH, with a pK(a) of 2.3. The M intermediate in the wild-type photocycle accumulates only at high pH, with an apparent pK(a) of 9, via deprotonation of a residue interacting with Asp85, presumably His57. In liposomes reconstituted with ESR, the pK(a) values for M formation and spectral shifts are 2-3 pH units lower than in DDM. The distinctively different pH dependencies of the protonation of Asp85 and the accumulation of the M intermediate in the wild-type protein versus the H57M mutant indicate that there is strong Asp-His interaction, which substantially lowers the pK(a) of Asp85 by stabilizing its deprotonated state.
Department of Physiology and Biophysics, University of California, Irvine, CA 92697, USA. balashov@uci.edu
S P
SP
Balashov
L E
LE
Petrovskaya
E P
EP
Lukashev
E S
ES
Imasheva
A K
AK
Dioumaev
J M
JM
Wang
S V
SV
Sychev
D A
DA
Dolgikh
A B
AB
Rubin
M P
MP
Kirpichnikov
J K
JK
Lanyi
D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D056449:Bacillales / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D055668:Photochemical Processes; D011522:Protons; D025604:Rhodopsins, Microbial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012545:Schiff Bases / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D001426:Bacterial Proteins; D011522:Protons; D025604:Rhodopsins, Microbial; D012545:Schiff Bases; D001224:Aspartic Acid; D006639:Histidine