Two forms of N intermediate (Nopen and Nclosed) in the bacteriorhodopsin photocycle

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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 Mclosed) 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 ∽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 Mclosed and Mopen, come to an equilibrium in the microseconds range. The absorption spectrum of the Nopen is slightly shifted to red in comparison to that of the Nclosed. Again, this resembles the M forms. 13-cis-all-trans re-isomerization is assumed to occur in the Nclosed form only. Binding of 1–2 molecules of protonated azide stabilizes the Nopen 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 Mopen is identical to the MN form, whereas M1 and M2 are different substates of Mclosed.