Protonation Probability of the Proton Release Group

The proton release group consists of Glu194 and Glu204. The water molecules located in between these two glutamate residues are represented by a continuum with a high permittivity. The two glutamate residues are considered as one group, Glu194/Glu204, that can bind two protons. The protonation probability of these two glutamate residues is considered together. So far the protonation probability of single residues is considered.

For residues that can bind one proton, the protonation probability equals the expected value for the number of protons bound. Glu194/Glu204 can bind up to two protons.

Therefore, the expected value for the number of protons bound to the group takes values between 0 and 2. This expected value is given by the sum of the individual protonation probabilities of Glu194 and Glu204. For instance, an expected value of 2 indicates that both Glu194 and Glu204 are protonated.

In the [bR] and [M1] state, Glu194/Glu204 bind one proton that is released to the ex-tracellular side during the M1 to M2transition. Glu194/Glu204 are deprotonated in the [M2], [N] and [O] protonation state. The protonation of Glu194/Glu204 takes place during the last step of the photocycle after which the protein has returned to the ground state.

The bR and K structures have a doubly protonated Glu194/Glu204 at low pH values as shown in Figure 5.15 a and b, respectively. At physiological pH values, exactly one proton is bound to Glu194/Glu204. At high pH values, in most bR and K structures still one proton is bound to Glu194/Glu204. Only the bR structure bR:1f50 and the K structure K:1qko show a tendency for a completely deprotonated proton release group at higher pH values.

Two L structures, L:1o0a and L:1vjm.a, bind one proton to Glu194/Glu204 over the whole pH range as shown in Figure 5.15 c. The other two L structures, L:1e0p and L:1vjm.b, are the only BR structures with a completely deprotonated Glu194/Glu204 at physiological pH values. A deprotonated Glu194/Glu204 is characteristic for the [M2], the [N] and the [O] protonation state. For L:1vjm.b, a high probability for both the [M2] and the [O] state can indeed be seen (cf. Figure 5.5 c). In contrast, L:1e0p shows unusual protonation behavior for Asp212 and has, thus, no probability for any of the later photo-cycle intermediate protonation states at physiological pH values values (cf. Figure 5.13 c).

At higher pH values, however, a probability for the [M2] state can be observed reflecting the fact that Asp212 has deprotonated and Glu194/Glu204 are also deprotonated (cf.

Figure 5.5 d).

In the M structures, Glu194/Glu204 binds in general one proton in the pH range between 0 and 8 as can be seen in Figure 5.15 d. A deprotonation of Glu194/Glu204 can only be seen at higher pH values. Thus, for these M structures, a probability for the [M2] can only be observed at high pH values (cf. Figure 5.6). As discussed above at physiological

5.4. Protonation Probabilities of the Key Residues of Proton Transfer 99

Figure 5.15.Sum of protonation probabilities (expected value) of Glu194/Glu204.

The two glutamate residues of the proton release group, Glu194 and Glu204, are con-sidered as one group that can bind two protons. The sum of protonation probabilities ranges, therefore, from 0 to 2. It is plotted against pH for the different intermediate structures:a)bR,b)K,c)L, andd)M structures,e)N structure, andf)O-like interme-diate structure. The curves are assigned to the individual structures by their respective intermediate state and PDB code (cf. Table 3.1).

pH values, a probability for the [M2] state can only be seen for the L structure L:1vjm.b.

With respect to the protonation of Glu194/Glu204, the N structure behaves like the M structures as can be seen by comparing Figure 5.15 e and 5.15 d. Since Glu194/ Glu204 are deprotonated in the later photocycle intermediate protonation states, i.e., the [M2], [N] and [O] state, the N structure shows no probability for these states at physiological pH. At high pH values, however, this structure has a low probability for both the [M2] and the [N] state (cf. Figure 5.7).

0 2 4 6 8 10 12 14

Figure 5.16. Individual protonation probabilities of Glu194 and Glu204.The pro-tonation probabilities of Glu194 and Glu204 are plotted against pH for: a)bR:1c3w,b) bR:1qhj,c)M:1f4z andd)M:1m0m. The examples are chosen to give a general impres-sion of the individual behavior of Glu194 and Glu204. The structures are named by their intermediate state and PDB code (cf. Table 3.1).

In the O-like structure, Glu194/Glu204 bind two protons at low pH values. With in-creasing pH, the protonation probability decreases slowly, but remains above 1 as can be seen in Figure 5.15 f. As discussed above, Arg82 is deprotonated in this structure (Figure 5.14 f). Since this structure was resolved from the Asp85Ser mutant of BR, non-physiological protonation behavior may have been expected for the mutated Asp85 and the protonatable residues closest to it,i.e., the retinal Schiff base and Asp212. Instead, the protonatable residue that bridge the distance between Asp85 and the extracellular space,i.e., Arg82 and Glu194/Glu204, show a tendency to assume their neutral state.

Figure 5.16 depicts individual protonation probabilities for Glu194 and Glu204. Exem-plary four structures were chosen to give an overview of the protonation behavior of Glu194 and Glu204. As can be seen, the protonation probabilities of Glu194 and Glu204 add up to 1 at physiological pH values. Interestingly, the distribution of the proton varies significantly between the structures. For bR:1c3w, in general Glu194 is protonated while Glu204 is deprotonated. In contrast for M:1f4z, Glu194 is deprotonated while Glu204 is protonated. For bR:1qhj and M:1m0m, both Glu194 and Glu204 are partly protonated.