Photocycle Dynamics and Vibrational Spectroscopy of the E46Q Mutant of Photoactive Yellow Protein

Abstract

The dynamics over the initial 100 ns (3 ps time resolution) of the room-temperature photocycle of the E46Q mutant of photoactive yellow protein (PYPE46Q) are measured using picosecond transient absorption (PTA) spectroscopy. Three intermediates, I0E46Q, I0⧧E46Q, and I1E46Q, are observed in the room-temperature PYPE46Q photocycle. Although their respective formation and decay rates differ, I0E46Q, I0⧧E46Q, and I1E46Q correspond to analogous intermediates (i.e., I0, I0⧧, and I1) 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 PYPE46Q. Vibrational spectra (1100−1700 cm-1) of both PYP and PYPE46Q are measured under the same experimental conditions (i.e., ω1 = 490 nm and ωs = 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 PYPE46Q 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 I0 and I0⧧ intermediates involve structural and/or electronic energy changes localized on the phenolate ring of the PYP chromophore.