A MODEL-INDEPENDENT APPROACH TO ASSIGNING BACTERIORHODOPSINS INTRAMOLECULAR REACTIONS TO PHOTOCYCLE INTERMEDIATES

By using factor analysis and decomposition, bacteriorhodopsin's intram olecular reactions have been assigned to photocycle intermediates. Ind ependent of specific kinetic models, the pure BR-L, BR-M, BR-N, and BR -O difference spectra were calculated by analyzing simultaneously two different measurements in the visible and infrared spectral region per formed at pH 6.5, 298 K, 1 M KCl, and pH 7.5, 288 K, 1 M KCl. Even tho ugh after M formation L, M, N, and O intermediates kinetically overlap under physiological conditions, their pure spectra have been separate d by this analysis in contrast to other approaches at which unphysiolo gical conditions or mutants have been used or specific photocycle mode ls have been assumed. The results now provide a set reference spectra for further studies. The following conclusions for physiologically rel evant reactions are drawn: (a) the catalytic proton release binding si te, asp 85, is protonated in the L to M transition and remains protona ted in the intermediates N and O; (b) the catalytic proton uptake bind ing site asp 96 is deprotonated in the M to N transition and already r eprotonated in the N to O transition; (c) proton transfer between asp 96 and the Schiff base is facilitated by backbone movements of a few p eptide carbonyl groups in the M to N transition.