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the chromophore is 3.5 ˚ , which is close to van der Waals distance. The electron
transfer rate constant is estimated to be 2.4
10 8 s 1 which is five orders of
magnitude below the optimal rate. This suggests that the free energy difference is
very different in magnitude compared to the reorganisation energy. It is likely that
the reaction has a very small
G or is endoergonic, in which case it would be
expected that either a small decrease in
G or a small decrease in l will result in an
increase in the rate and in the photoconversion quantum yield [ 36 ]. This is consis-
tent with the increased photoconversion quantum yield, and the electron transfer
rate, with a shorter wavelength (254 nm UV illumination) that is resonant with a
higher order optical transition of the chromophore [ 19 ].
2.3 Light-Induced Oxidative Decarboxylation in Other
Fluorescent Proteins
GFP was the first known example showing oxidative decarboxylation of a car-
boxylic acid in a protein [ 19 ]. It has now been documented to occur also in mutants
of GFP and in related fluorescent protein. Currently, besides the fluorescent proteins
no other examples of oxidative decarboxylation are known in proteins. In GFP,
oxidative decarboxylation of Glu222 results from the light-induced formation of an
oxidative state. Similar reactions are well known in chemistry and are referred to as
the “Kolbe reaction” [ 44 ]. Thermal oxidative decarboxylation of carboxylic acids
can be catalysed by oxidants such as Mn(III) [ 43 ]. In redox proteins with strong
oxidising centres, oxidative decarboxylation of nearby ionised carboxylic groups
may also be expected to occur. Many proteins contain redox centres such as Mn(III)
or Fe(III) that are coordinated by ionised
-carboxylates. Depending on the specific
redox potentials and lifetimes of the intermediate oxidative states and the
carboxylates, and resulting quantum efficiency, oxidative decarboxylation may
limit the number of turnovers that such enzymes can make.
Since the discovery of light-induced decarboxylation in GFP, it has also been
documented in mutants of A. victoria GFP and has been found in at least two other
fluorescent proteins, DsRed and an aceGFP mutant. An A. victoria GFP mutant
called yellow fluorescent protein (YFP) (10C variant, including mutations S65G,
V68L, S72A and T203Y) undergoes photobleaching with strong CW illumination
at 514 nm into the low energy absorption band (l max ¼ 527 nm) [ 53 ]. Concomitant
with bleaching, an absorption peak at 390 nm is produced which is a photoactive
species. The report [ 53 ] mentions an additional product band at 470 nm, which
could indicate that the 390-nm band is a higher optical transition. Subsequent
excitation of this band at 390 nm causes photoactivation, recovering some of the
fluorescence lost in the bleaching process. It is reported that the photobleached
product recovers spontaneously in the dark on an hour timescale when CW green
illumination was used, but when pulsed excitation was used the photoproduct was
irreversibly bleached. Moreover, in this case a loss of 44 Da, indicating the likely
decarboxylation of a carboxylate side chain, could be demonstrated [ 53 ]. The site of
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