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a
b
His
148
Glu
222
Leu
220
Thr
203
c
d
Arg
96
Arg
96
Gln
94
Gln
94
His
148
His
148
Thr
203
Thr
203
Ser
205
Ser
205
Glu
222
Glu
222
Fig. 9 “Photoinduced decarboxylation and conformational changes in the chromophore vicinity.
Coordinates of the wild-type structure (PDB 1gfl) are shown with carbon atoms in
green
, whereas
the structure of the photoproduct GFP
R
is shown with carbon atoms in
yellow
. Electron density
shown is the refined 2Fo-Fc map contoured at 1.6
s
for the 1.8
˚
resolution structure.
(a) Conformational changes of Thr 203 and His 148. (b) Absence of electron density for the
O
e
1, O
e
2 and Cd atoms of Glu 222 in GFP
R
.(c) Proposed hydrogen bonds in the chromophore
vicinity of chain A in the GFP
483
structure include solvent molecules 173[Z], 175[Z] and 203[Z].
(d) Chromophore structure in GFP
A
. The hydrogen-bonding network includes solvent atoms 25
and 63” (reproduced with permission [
19
])
transfer of charge between the phenolic- and imidazolidinone rings of the chromo-
phore will compete (efficiently) with electron transfer from Glu 222 to the chromo-
phore. If an electron is transferred, the g-carboxyl radical of Glu 222 will
decarboxylate via the “Kolbe” mechanism [
19
]. This then leads to retro-transfer of
an electron and a proton, or alternatively a hydrogen radical (Fig.
10
).An experi-
mental test for the accumulation of a radical intermediate was negative, which shows
that at least at 200K, the lifetime of the carboxylate radical intermediate as well as
the predicted chromophore radical intermediate is less than ~7 s. Considering the
distances involved, these processes can proceed in the ns time domain, which is also
in agreement with the low quantum efficiency of the reaction. Together with the low-
and room-temperature photoconversion experiments, a two-step mechanism of the
photoconversion of GFP
A
to GFP
R
was proposed.
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