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Participation
of Hydrogen Bonding Residues and Water Molecules in Light-Driven Proton
Transport by Bacteriorhodopsin (bR)
Balashov, Sergei1,
Maeda, Akio1, Imasheva, Eleonora1,
Lu, Miao1, Govindjee, Rajni1,
Ebrey, Thomas2, Chen, Yumei3,
Menick, Donald3 and Crouch, Rosalie3
University of Illinois, Urbana, IL1
University of Washington, Seattle, WA2
Medical University of South Carolina, Charleston, SC3
Abstract-
Light-induced proton transport in bR is a multi-step process which is
initiated by the retinal chromophore isomerization and subsequent internal
proton transfer from the Schiff base to Asp85. A chain of hydrogen bonding
residues and water molecules connect Asp85 with the extracellular surface.
Three members of this chain, Arg82, Glu204 and Glu194 were identified
as key components of the proton release complex (PRC), which decreases
its pK upon proton transfer from the Schiff base to Asp85 and releases
a proton to the extracellular surface. Here we show that mutations of
two other members of this chain, Tyr83 and Thr205, which are conserved
in Halobacterial proton pumps produce significant effects on the rate
of light-induced proton transfer, the pK of PRC and coupling of PRC
with Asp85. Mutation of Tyr83 to Phe completely abolishes fast light-induced
proton release at neutral pH, slows deprotonation of Asp85 and recovery
of bR in the O 
bR transition. Mutation of Thr205 to Val decreases the rate of the O
bR transition
by about 30 times. These data implicate Thr205 and Tyr83 as important
participants of the proton transfer from Asp85 to the extracellular
surface. Studies of the pH dependence of the rate of light-induced proton
uptake indicate that it is mediated by transient changes of the pK and
accessibility of the proton donor, Asp96. Internal water molecules are
important participants in proton transport by bR. Comparison of the
FTIR difference spectra accompanying the photoreactions of both the
initial bR and the subsequent photocycle intermediates L and M (in L
L
and M
M transitions)
indicates that the chromophore's interactions with water molecules
undergo large changes during the photocycle. Water molecules near the
chromophore presumably act as mobile mediators connecting the Schiff
base with Asp85 and Asp96 at different stages of the photocycle.
Keywords: bacteriorhodopsin,
proton transport pathway, mutations of Thr205 and Tyr83, internal water
molecules
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