Two forms of N intermediate (N(open) and N(closed)) in the bacteriorhodopsin photocycle

Abstract

Glutaraldehyde, aluminum ions and glycerol (that inhibit the M intermediate decay in the wild-type bacteriorhodopsin and azide-induced M decay in the D96N mutant by stabilization of the M(closed)) accelerate the N decay in the D96N mutant. The aluminum ions, the most potent activator of the N decay, induce a blue shift of the N difference spectrum by approximately 10 nm. Protonated azide as well as acetate and formate inhibit the N decay in both the D96N mutant and the wild-type protein. It is concluded that the N intermediate represents, in fact, an equilibrium mixture of the two ('open' and 'closed') forms. These two forms, like M(closed) and M(open), come to an equilibrium in the microseconds range. The absorption spectrum of the N(open) is slightly shifted to red in comparison to that of the N(closed). Again, this resembles the M forms. 13-cis-all-trans re-isomerization is assumed to occur in the N(closed) form only. Binding of 1-2 molecules of protonated azide stabilizes the N(open) form. Existence of the 'open' and 'closed' forms of the M and N intermediates provides the appropriate explanation of the cooperative phenomenon as well as some other effects on the bacteriorhodopsin photocycle. Summarizing the available data, we suggest that M(open) is identical to the M(N) form, whereas M1 and M2 are different substates of M(closed).