Quantum efficiency of native and mutant bacteriorhodopsin obtained from blue light induced relaxation experiments

Bacteriorhodopsin was continuously excited with green background light. In this way a steady state distribution of all intermediates of the photocycle was obtained. Then a perturbation of the system was induced by a blue laser flash and the resulting absorption changes were measured. The experiments were done with native bacteriorhodopsin and with the point mutant BR Asp96→Asn, in which aspartate 96 is changed to asparagine. Blue light induced relaxation experiments revealed a rate constant belonging to the excitation of bacteriorhodopsin by the green background. With this rate constant the quantum efficiency of native bacteriorhodopsin and of BR Asp96→Asn was determined to be 0.60 ± 0.10. Signals obtained with native bacteriorhodopsin could be explained with a simple model of the photocycle consisting of three consecutive intermediates BR 568, L 550 and M 412. To describe the behavior of BR Asp96→Asn , a further photoactive intermediate after the M 412 state had to be postulated. Properties of this intermediate are similar to those of the N 550 state