On the heterogeneity of the M population in the photocycle of bacteriorhodopsin.

The M stage in the photocycle of bacteriorhodopsin (bR), a key step in its light-induced proton pump mechanism, is studied in water/glycerol suspensions over the temperature range between 20 and -60 degrees C. The biexponential decay of M is analyzed for wild-type (WT) bR and for its D96N, Y185F, and D115N mutants, at various pH values, according to the scheme: bR-->(hv) L-->M<-->(k1, k-1) N-->(k2) bR. The analysis leads to the conclusion that the N state is generated, with analogous rate parameters, in all cases, including the D96N mutant. Another approach involves probing the M state, generated by steady-state illumination at -60 degrees C, by fast cooling to -180 degrees C. Subsequent irradiation with blue light, followed by gradual warming up, induces the M-->(hv) M'-->bR'-->bR sequence of reactions. On the basis of characteristic difference spectra and transition temperatures observed for the M'-->bR' process, it is concluded that the initially observed M state at -60 degrees C, denoted as (M)a, is composed of three (or four) equilibrated substrates, MI, MII, MIII, and MIV. During the M-->N equilibration, which corresponds to the fast phase of the M decay, (M)a transforms into a second state, (M)b, in which MIII has been replaced by a fifth M substate, denoted as MV. MV is identified as the protein state in which an appropriate structural change allows reprotonation of the Schiff base, generating the N state. The low-temperature heterogeneity in M is discussed in terms of the two M states (M1 and M2) previously postulated [Váró, G., & Lanyi, J. K. (1990) Biochemistry 29, 2241] for the room temperature photocycle. The following conclusions are derived for both low and room temperature photocycles: (a) The M population is highly heterogeneous and pH dependent. (b) At least three transitions are observed between the initially formed M state and the M state that is equilibrated with N. These are assigned to protein conformational changes and to water molecule rearrangements. (c) In an aqueous suspension of WT bR at room temperature, the Schiff base reprotonation is controlled by D96. However, our results show that the formation and stability of the N state do not require the D96 residue. Moreover, at low temperatures, the (M)a-->(M)b protein structural transformation, which has not yet been resolved at room temperature, becomes the rate-determining step in the protonation of the Schiff base.