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

An unequivocal answer is given to the question of why the reported fluorescence spectra of bacteriorhodopsin (bR568) have been different from one another. The inconsistency is shown to arise from the accumulation of the fluorescent intermediates O and Q (KN) by cw excitation light. Their fractions in the photo-stationary states depend on the excitation power and the suspension pH. We report the intermediate-free fluorescence spectrum of bR568 obtained with a weak excitation source (632.8 nm, 5.3 x 10(15)-1.9 x 10(16) photons cm-2.s-1) and a near-IR sensitive intensified photodiode array system. The fluorescence maxima of the spectra, F(lambda) and f(nu), are located at 755 +/- 10 nm and 12700 +/- 200 cm-1, respectively. The spectrum of O is identical to that of the deionized purple membrane bR605 (Fmax = 750 +/- 5 nm, fmax = 13,000 +/- 100 cm-1). Q (KN) exhibits a blue-shifted spectrum more than that of bR568 (Fmax < 720 nm, fmax > 13,400 cm-1).