Picosecond time-resolved fluorescence spectroscopy of K-590 in the bacteriorhodopsin photocycle

Abstract

The fluorescence spectrum of a distinct isometric and conformational intermediate formed on the 10(-11) s time scale during the bacteriorhodopsin (BR) photocycle is observed at room temperature using a two laser, pump-probe technique with picosecond time resolution. The BR photocycle is initiated by pulsed (8 ps) excitation at 565 nm, whereas the fluorescence is generated by 4-ps laser pulses at 590 nm. The unstructured fluorescence extends from 650 to 880 nm and appears in the same general spectral region as the fluorescence spectrum assigned to BR-570. The transient fluorescence spectrum can be distinguished from that assigned to BR-570 by a larger emission quantum yield (approximately twice that of BR-570) and by a maximum intensity near 731 nm (shifted 17 nm to higher energy from the maximum of the BR-570 fluorescence spectrum). The fluorescence spectrum of BR-570 only is measured with low energy, picosecond pulsed excitation at 590 nm and is in good agreement with recent data in the literature. The assignment of the transient fluorescence spectrum to the K-590 intermediate is based on its appearance at time delays longer than 40 ps. The K-590 fluorescence spectrum remains unchanged over the entire 40-100-ps interval. The relevance of these fluorescence data with respect to the molecular mechanism used to model the primary processes in the BR photocycle also is discussed.

Fluorescence spectra of bacteriorhodopsin and the intermediates O and Q at room temperature

“…4). The spectra reported by Kouyama et al [7] and by Atkinson et al [8] are similar to that in the present work but the spectra are slightly blue-shifted. Atkinson et al eliminated the fluorescent intermediates O and Q by using the sample-jet-flow system but a small amount of K with a 2ma x at 731 nm might be accumulated within the duration of excitation light (8-ps full width at half maximum).…”

“…It grounds on the structure of the retinoid protein in the excited state. Since Lewis et al's first reports [4][5][6][7][8], many authors have challenged the measurement of the fluorescence spectrum of bR568 at room temperature. However, their results have been inconsistent with one another.…”

Primary picosecond molecular events in the photoreaction of the BR5.12 artificial bacteriorhodopsin pigment.

“…Timeresolved measurements reveal that fluorescence from K*-590 appears with the same -3.5-ps time constant found via absorption (9,13). The maximum of the K*-590 fluorescence spectrum is blue-shifted by "17 nm relative to BR*-570 and K*-590 has a 2-fold higher fluorescence yield (13,14). No fluorescence has been associated with J-625 (9,13).…”

“…The fluorescence spectrum is measured by using a scanning 1-m spectrometer [1.2-nm band pass (14)]. RR scattering is detected with a triple monochromator and an intensified multichannel detector (19).…”

“…Fluorescence spectroscopy has been utilized to characterize the initial BR photocycle intermediates (9,13,14). Timeresolved measurements reveal that fluorescence from K*-590 appears with the same -3.5-ps time constant found via absorption (9,13).…”