The sequence of events triggered by light absorption that subsequently leads to proton transfer is termedphotocycle. As shown in Figure 5.2 a, the photocycle consists of several intermediate states. The intermediate states differ from each other with respect to ab-sorption maximum, structure and protonation state. In the last decade, crystallographers attempted to solve structures representing each intermediate state. These structures are categorized according to their proposed intermediate state as bR, K, L, M, N and O struc-tures. The M structures are not further subdivided into M1 and M2 structures, since the structural data, in general, does not allow a specific allocation to either M1or M2.
The proton is transported through BR along a series of protein residues. These key residues of proton transfer are explicitly shown in the structure of BR depicted in Chap-ter 1, Figure 1.7. Furthermore in Figure 5.2 b, the general position of the key residues
5.1. Proton Transfer of Bacteriorhodopsin 79
Figure 5.2. Proton Transfer of bacteriorhodopsin. a) When light is absorbed by the retinal Schiff base while BR is in the bR state, a sequence of events is triggered termed the photocycle. Consecutively, BR adopts a serial of intermediate states: bR, K, L, M1, M2, N and O. During the photocycle, a proton is transferred from the cytoplasm to the extracellular space. b)In the schematic of BR, the key residues and the proton transfer steps from the cytoplasmic to the extracellular side of the cell are indicated.
The encircled numbers refer to the sequence of the five proton transfer steps and their occurrence during the photocycle. The protonation states of the photocycle intermediate states are given in Table 5.1.
and the five proton transfer steps are indicated in a schematic of BR. The protonation of these residues has been identified experimentally for the different photocycle intermedi-ates [55, 57, 63, 207, 208]. In the transition from the bR to the K and from the K to the L intermediate state, no proton transfer takes place. The bR, K and L intermediate state differ from each other in small structural rearrangements, but not in the protonation of the key residues. The first change in protonation occurs during the transition from the L to the M1 intermediate state, when the proton is transferred from the retinal Schiff base to Asp85. During the M1 to M2 transition a proton is released to the extracellular space from the proton release group Glu194/Glu204. In the transition from the M2 to the N intermediate state, the retinal Schiff base receives a proton from Asp96. Asp96 is then protonated from the cytoplasm during the transition from the N to the O intermediate state. During the last step, the transition from the O state back to the bR state, the proton is transferred from Asp85 to the proton release group Glu194/Glu204. Although they do not change their protonation during the photocycle, Arg82, Asp115 and Asp212 are functionally important residues. Asp115 and Arg82 were experimentally shown to remain protonated, while Asp212 was shown to remain deprotonated during the photo-cycle [189, 208].
Other protonatable residues of BR are speculated to function as proton antennas. Their involvement in the proton transfer is, however, not well established. The focus of the present work lies, therefore, on the key residues of the BR proton transfer described
[bR]/[K]/[L] [M1] [M2] [N] [O]
Asp96 1 1 1 0 1
Asp115a 1 1 1 1 1
retinal Schiff base 1 0 0 1 1
Asp85 0 1 1 1 1
Asp212b 0 0 0 0 0
Arg82a 1 1 1 1 1
Glu194/Glu204 1 1 0 0 0
aAsp115 and Arg82 remain protonated during the physiological photocycle.
bAsp212 remains deprotonated during the physiological photocycle.
Table 5.1. Protonation state subsets of bacteriorhodopsin photocycle intermedi-ates. The key residues of proton transfer are listed in sequential order starting from the cytoplasmic side of BR (cf. Figure 5.2 b). [bR], [K], [L], [M1], [M2], [N] and [O] refer to the protonation state of the respective photocycle intermediate (cf. Figure 5.2 a). In principle, the table defines protonation state subsets, since residues not listed in the table may be protonated or deprotonated. The bR, K and L intermediate adopt the same protonation state. In the following, this protonation state will be referred to as [bR] state.
above. In sequential order from the cytoplasmic to the extracellular side these are: Asp96, Asp115, the retinal Schiff base, Asp85, Asp212, Arg82, Glu194 and Glu204. The pro-tonation behavior of these key residues is analyzed in detail in the results part of this chapter.
5.2 C OMPUTATIONAL D ETAILS
The Metropolis Monte Carlo calculations presented in this chapter are performed on all 20 structures of BR listed in Chapter 3, Table 3.1. The structures are grouped according to their intermediate state as bR, K, L, M and N structures. The M structures are not further subdivided into M1and M2structures, since an unambiguous classification of the structural data into M1and M2 state is, in general, not feasible. No structure is available for the O intermediate of the BR photocycle. Instead, calculations are performed on the Asp85Ser mutant structure. This structure is proposed to resemble the O intermediate and is, therefore, termed O-like intermediate structure. To simplify the differentiation between the numerous structures, the individual structures will be referred to by their intermediate state and their PDB code as given in Table 3.1: e.g., bR:1c3w or M:1f4z.
The proton release group from which the proton is released to the extracellular space is rather complex. It is the only proton donor/acceptor of BR that includes several groups.
Specifically, the proton release consists of Glu194, Glu204 and a water cluster located in between the two glutamate residues. The proton is delocalized over the proton release group [63]. For the calculation performed in this work, water molecules are not modeled explicitly but are represented by a continuum. The proton release group, thus, comprises
5.3. Protonation State Subsets 81