Picosecond time-resolved resonance Raman spectrum of the K-590 intermediate in the room temperature bacteriorhodopsin photocycle

Abstract

The resonance Raman (RR) spectrum of the K-590 intermediate formed during the initial 3–5 ps of the bacteriorhodopsin (BR) photocycle is measured using picosecond time-resolved resonance Raman (PTR 3) spectroscopy. Room temperature samples are examined using 5–6 ps (FWHM) 565 nm pump and 590 nm, 595 nm and 608 nm probe laser pulses. Probe laser delays of 50 ps to 2 ns ensure that the only photocycle intermediate present in the reaction mixture is K-590. The relative concentrations of K-590 and BR-570 at a given time in the reaction are determined from a rate equation model which simulates picosecond transient absorption and picosecond time-resolved fluorescence measurements. The RR spectrum of K-590 is discussed with respect to RR spectra previously assigned to the K intermediate at room and low (77K) temperatures, BR-570, BR-548 (dark-adapted) and related protonated Schiff bases. Significant differences appear in the K-590 spectrum reported here relative to earlier data including: (i) a small (<2 cm −1) shift in the K-590 CC stretching frequency relative to that of BR-570, (ii) a strong 1194 cm −1 band in the CC stretching frequency region with a distinct shoulder near 1171 cm −1, and (iii) increases in the hydrogen out-of-plane band intensities throughout the 950–995 cm −1 region and near 802 cm −1.