Biology Reference
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CO 2 molecule in GFP provides a new opportunity to characterise the interactions of a
single molecule of CO 2 in a specific protein environment [ 26 ]. After photoconversion,
bound and unbound populations of photogenerated CO 2 have been observed and
characterised by X-ray crystallography and cryo-infrared crystallography in the
GFP L ,GFP M and GFP R products (at 100, 200 and 293 K). Electron density assigned
to the CO 2 molecule in the GFP L state at 100 K disappears at higher temperatures,
indicating the loss of occupancy of the binding site identified, H-bonded to Ser205.
The CO 2 vibrational signals evolve over the 100-200 K temperature range, coalescing
into a single band at 2,338 cm 1 at 200 K. The appearance of multiple overlapping
CO 2 bands suggests significant disorder for the photogenerated CO 2 already at 100 K.
The observed disorder of the CO 2 molecule underlines the unexpected mobility of the
chromophore environment at 100 K. Polarised IR data on oriented single crystals
indicated a minimum of three CO 2 environments with distinct frequencies and
orientations and additionally characterised the direction and changes of the Cys
70-Val 68 hydrogen bond during photoconversion [ 26 ].
Some conclusions can be drawn with regard to the energetics of the electron
transfer reaction from Glu 222 to the chromophore. An in-depth characterisation of
the thermodynamics of electron transfer is still not available, however [ 36 ]. Princi-
pally, the energy levels of the S1 and S2 states of the neutral and anionic chromo-
phore in intact GFP relative to the anionic carboxylate of Glu222 are not known,
from either experimental parameters or simulation and calculation [ 36 ]. This iden-
tifies an important area for future computational chemistry and theory. Considering
the S1 state of the neutral chromophore, by making the assumption that electron
transfer competes with the ESPT reaction in a parallel reaction scheme, the forward
rate constant may be estimated. The singlet excited state of the protonated species
decays with time-constants of 3 and 15 ps to form a longer lived (3 ns) deprotonated
radiative state [ 16 , 48 ]. From the apparent photoconversion quantum efficiency with
excitation at 390 nm of 1.6
10 8 s 1
is estimated [ 36 ]. For the anionic singlet state, a 3 ns lifetime would be considered,
and the quantum yield is about ten times below that for the neutral state, leading to an
estimated forward rate constant of ~5
10 3 , a forward electron transfer rate of 2.4
10 4 s 1 . This suggests that the electron
transfer rate is enhanced with increasing the free energy difference and/or reducing
the reorganisation energy. However, the free energy difference cannot be deter-
mined as the back-transfer rate is not known.
Electron transfer in redox proteins is described by Marcus theory [ 49 - 51 ]. The
non-adiabatic description of electron transfer uses “Fermi's golden rule” which
provides the transfer rate. Both classical [ 50 ] and quantum mechanical [ 49 ] treat-
ment of Marcus electron transfer theory predict that the dependence of the electron
transfer rate, k et , on the free energy
G leads to a maximum when the free energy
equals the reorganisation energy (
¼ l). Empirical expressions have been
derived for electron transfer in proteins that make predictions with regard to the,
first order, forward electron transfer rate and the free energy and reorganisation
energy of electron transfer [ 52 ]. For groups that are in van der Waals contact, the
optimised k et (when
¼ l) is expected to approach 10 13 s 1 [ 52 ]. In GFP, the
edge-to-edge distance between the Glu222 carboxylate and the phenolic ring of
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