582
Bacteriorhodopsin, Molecular Biology of
functioning of the proton pump against
large transmembrane proton gradients.
4
Properties of Single-site Mutants
Of the 248 amino acid residues in bac-
teriorhodopsin, only a handful plays an
important role in the transport. Their
replacement
with
less
functional
(e.g.
nonprotonable, wrong polarity, or size-
mismatched) residues will produce a less
effective pump, but only at a saturat-
ing light intensity, through increasing the
overall turnover time. Only one mutation,
that of Lys216, has been reported to elim-
inate transport altogether by eliminating
the covalent binding of retinal. However,
even such mutants show transport if recon-
stituted with a retinal analogue in which
the Schiff base is protonated.
The important roles played by Asp85 and
Asp96 in bacteriorhodopsin are illustrated,
nevertheless, by the altered phenotypes
of recombinant proteins in which they
are replaced by nonprotonatable residues
like asparagine.
When Asp85 is replaced, the purple
chromophore becomes blue, similar to
the color produced upon protonation of
this anionic residue at low pH (
<
3). The
p
K
a
of the retinal Schiff base is shifted
from
>
13 in the wild type to about 8. The
mutation removes the negative charge of
Asp85 that is the primary counterion to
the positively charged Schiff base and thus
the origin of the stability of the protonated
Schiff base. The mutation also removes
the proton acceptor, and the photocycle of
this mutant produces only small amounts
of the M intermediate, through loss of
the Schiff base proton in the cytoplasmic
direction. The D85N mutant shows proton
transport at a pH at which the Schiff
base is unprotonated, through a series of
photocycle steps that occur out of their
normal sequence.
Replacement of Asp96 with asparagine
greatly stabilizes the M intermediate, as
the internal proton donor to the Schiff
base is removed. The Schiff base is now
reprotonated directly from the cytoplasmic
surface, and its rate becomes slow and pH
dependent. Mutation of many residues at
the cytoplasmic surface near Asp96 does
not hinder reprotonation of the Schiff base,
but it decouples the reisomerization of the
retinal to all-trans from reprotonation of
Asp96. In these mutants, the lifetime of N
becomes very slow and pH independent.
Replacement
of
residues
implicated
in proton release, Arg82, Glu194, and
Glu204, with glutamine eliminates proton
release during the rise of the M state.
Instead, the proton is released to the
extracellular surface in the last step of
the photocycle, the O to BR reaction.
Most likely, with the proton release site
rendered nonfunctional, this proton is
directly from Asp85 that deprotonates in
the last photocycle step in any case.
5
Pump Modes, Energetics
The release and uptake of protons at
the extracellular and cytoplasmic surfaces
respectively, brought about by the in-
traprotein proton transfer reactions of the
photocycle, can be measured by absorp-
tion changes of pH indicator dyes either in
the bulk or covalently linked to a surface
residue. The pump functions well both at
pH
>
6, at which proton release to the ex-
tracellular surface precedes proton uptake
from the cytoplasmic side, and at pH
<
6
(the p
K
a
for proton release), at which the
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