Testing BR photocycle kinetics.

An improved K absorption spectrum in the visible is obtained from previous photocycle data for the D96N mutant of bacteriorhodopsin, and the previously obtained M absorption spectrum in the visible and the fraction cycling are confirmed at 25 degrees C. Data at lower temperatures are consistent with negligible temperature dependence in the spectra from 5 degrees C to 25 degrees C. Detailed analysis strongly indicates that there are two intermediates in addition to the first intermediate K and the last intermediate M. Assuming two of the intermediates have the same spectrum and using the L spectrum obtained previously, the best kinetic model with four intermediates that fits the time course of the intermediates is rather unusual, with two L's on a cul-de-sac. However, a previously proposed, more conventional model with five intermediates, including two L's with the same spectra and two M's with the same spectra, also fits the time course of the intermediates nearly as well. A new criterion that tests an individual proposed spectrum against data is also proposed.