The dynamics over the initial 100 ns (3 ps time resolution) of the room-temperature photocycle of the E46Q mutant of photoactive yellow protein (pyP(EA6Q)) measured using picosecond transient absorption (PTA) spectroscopy. Three intermediates, I-0(E46Q), I-0(not asymptotic to E46Q) and I-1(E46) , are observed in the room-temperature pyp(E46Q) photocycle. Although their respective 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(not asymptotic to), and I-1) observed by PTA in the room-temperature photocycle 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 photochemical mechanism itself. The influence of the E46Q mutation on the PYP chromophore can be independently obtained by measuring changes in the vibrational degrees of freedom of ground-state PYP and pyp(E46Q) Vibrational spectra (1100-1700 cm(-1)) of both PYP and pyp(E46a),, measured under the same experimental conditions (i.e., omega (1) = 490 nm and omega (s), 518-535 nm) using picosecond resonance coherent anti-Stokes Raman scattering (PR/CARS). 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 positions and relative intensities of vibrational bands assigned to the phenolate 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 asymptotic to) intermediates involve structural and/or electronic energy changes localized on the phenolate ring of the PYP chromophore.