Photoacoustic spectroscopy of bacteriorhodopsin photocycle.

Photoacoustic spectroscopy was applied to study the energetics and the kinetics of the slow intermediates of the bacteriorhodopsin photocycle. An analysis of the modulation frequency dependence of the photoacoustic signal allowed us to estimate the enthalpy changes and the kinetic parameters associated with those intermediates. The effects of pH, salt concentration, and protein aggregation were studied. Three photoacoustic transitions were found. The two low frequency transitions were attributed to O660 and M412, respectively. The third transition was interpreted as resulting from a protein conformational change undetected spectrophotometrically. The frequency spectra were simulated between 5 and 180 Hz at pH's 5.1, 7.0, and 8.9 assuming a branching in the bacteriorhodopsin photocycle at the M412 level. The enthalpy changes associated with M412 and O660 were computed and compared with the experimental values.