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Fig. 11 Photoinduced
backbond cleavage and
b
-elimination in “green-
to-red” photoconvertable
fluorescent proteins.
Absorbance spectra of green
and red forms of KikGRX
(a) and Kaede (b) after
denaturation (reproduced
with permission [
71
])
b
His62 in EosFP, followed by a
-elimination step in which Glu212 acts as a proton
acceptor [
66
]. Glu212 is known to be essential for photoconversion of EosFP, since
the E212Q mutant does not photoconvert [
66
]. Hayashi et al. [
73
] suggested a
“water-assisted” mechanism to explain the loss of a water molecule in the red
photoproduct of Kaede [
73
]. Tsutsui et al. [
71
] discuss one specific detail of the
b
-elimination, which can follow either a E2-type elimination which is bimolecular,
or an E1-type elimination which is unimolecular. Interestingly, the red photoproduct
in KikGR(X) was found to be structurally different compared to Kaede and EosFP,
having the C
in a
cis
rather than a
trans
configuration [
71
]. This shows that free bond rotation occurs in a reaction
intermediate that favours the “E1” mechanism of
¼
C double bond between His62-C
a
and His62-C
b
-elimination.
Lelimousin et al. [
74
] proposed that intersystem crossing occurs in the reaction
pathways, arguing that this explains the low quantum yield of photoconversion
[
74
]. Hybrid QM/MM calculations were used to calculate the relative energies of
b
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