Y. Zhou et al., Photocycle dynamics and vibrational spectroscopy of the E46Q mutant of photoactive yellow protein, J PHYS CH A, 105(23), 2001, pp. 5719-5726
The dynamics over the initial 100 ns (3 ps time resolution) of the room-tem
perature photocycle of the E46Q mutant of photoactive yellow protein (pyP(E
A6Q)) measured using picosecond transient absorption (PTA) spectroscopy. Th
ree intermediates, I-0(E46Q), I-0(not asymptotic to E46Q) and I-1(E46) , ar
e observed in the room-temperature pyp(E46Q) photocycle. Although their res
pective formation and decay rates differ, I-0(E46Q), I-0(not asymptotic to
E46Q) and I-1(E46a) correspond to analogous intermediates (i.e., I-0, I-0(n
ot asymptotic to), and I-1) observed by PTA in the room-temperature photocy
cle of the wild type (WT) photoactive yellow protein (PYP). These PTA data
show that the replacement of glutamic acid 46 with glutamine influences the
kinetic properties of the PYP photocycle, but does not alter the general p
hotochemical mechanism itself. The influence of the E46Q mutation on the PY
P chromophore can be independently obtained by measuring changes in the vib
rational degrees of freedom of ground-state PYP and pyp(E46Q) Vibrational s
pectra (1100-1700 cm(-1)) of both PYP and pyp(E46a),, measured under the sa
me experimental conditions (i.e., omega (1) = 490 nm and omega (s), 518-535
nm) using picosecond resonance coherent anti-Stokes Raman scattering (PR/C
ARS). Although the 14 vibrational bands observed in the PR/CARS spectrum of
PYPE46Q, are generally analogous to those found in the PR/CARS spectrum of
PYP, detailed comparisons reveal significant differences in both the posit
ions and relative intensities of vibrational bands assigned to the phenolat
e part of the cinnamyl chromophore. These PR/CARS results demonstrate that
while the chromophore within both PYP and PYPE46Qw have similar vibrational
degrees of freedom, the E46Q mutation selectively alters the structure of
the phenolate ring, apparently through differences in the hydrogen bonding
network involving glutamic acid 46 and the negatively charged oxygen in the
phenolate ring. When considered together, the changes in the kinetic rate
constants for the photocycle (PTA data) and in the vibrational spectra (PR/
CARS data) caused by the E46Q mutation suggest that the I-0 and I-0(not asy
mptotic to) intermediates involve structural and/or electronic energy chang
es localized on the phenolate ring of the PYP chromophore.