PHOTOREACTION OF THE N-INTERMEDIATE OF BACTERIORHODOPSIN, AND ITS RELATIONSHIP TO THE DECAY KINETICS OF THE M-INTERMEDIATE

Because the M photointermediate of recombinant T46V bacteriorhodopsin decays more rapidly and the N intermediate more slowly than in wild-type, the photoreaction of N could be examined without interference from M. We found that between pH 6 and 9 the photoproducts of N included both earlier suggested M-like intermediate and red-shifted R state. However, when the photoexcitation of N was at wavelengths below 500 nm the amount of M-like product decreased with increasing pH, and at pH 9 virtually only R was produced. In the dark, T46V contains an N-like conformer, in increasing amounts with increasing pH like wild-type but in 4-5 times greater concentrations. The photoreaction of this thermally produced state is much like that of the N intermediate. It is associated with the appearance of a slowly decaying M, but we calculate that under most conditions used to follow M in the wild-type photocycle the amount of N-like conformer, and therefore the amplitude of this slow component, will not be significant. The results confirm the suggestion [Fukuda & Kouyama (1992) Biochemistry 31, 11740-11747] that an M-like state is included among the photoproducts of N, but at the same time provide support to photocycle models in which the slow component of the biphasic M decay is attributed not to this secondary photoreaction or to a separate photocycle originating from a heterogeneous initial state, but to thermal equilibration between M and N in a single photocycle.