Photocycle of Bacteriorhodopsin

After the absorption of a photon by retinal, this molecule transmits the stimulation to the whole protein. The isomerization of the retinal after the stimulus, besides the contacts with the residues of the RBP, transduces the signal, through the different helices, from the middle of the protein to the cytoplasmic and extracellular side of the protein. The final result of this process yields the transport of a proton from the cytoplasmic to the extracellular side of the protein. At room temperature and in the light adapted form, the photocycle lasts for about 10ms. For an easy identification of the ongoing events occurring in the photocycle, they were grouped chronologically in photo intermediates. from the basal state or BR going to J, K, L, M, N and the late O. Some conformational steps pass by in each of these photo intermediates. As an example, there are available several crystallographic structures about photo intermediates. Some of them are derived from WT BR crystals analyzed at low temperature, trapping almost a pure intermediate. As well, some single or multiple mutations yield what should be considered an intermediate-like structure. Besides, the existence of some different methods for crystallizing BR has added more controversy to the structure field because there are discrepancies among structures of the same intermediate.

The important characteristics of the intermediate states are:

BR: Resting state just before beginning the photocycle. Retinal is in all-trans configuration, because the protein has to be light adapted to initiate the photocycle. In this step, Schiff’s Base is protonated.

J: In the first 500 fs, after the photon is being absorbed by the retinal, J occurs. It is hardly detectable because it is very fast. Retinal remains in all-trans conformation and SB remains protonated. A reorganization of the electronic density of the retinal between the bonds between C=C and C-C occurs in this step. During this stage there is certain torsion of the polyenic chain of the retinal.

K: The retinal isomerization from all-trans to 13-cis, 15-anti occurs in the transition from J to K. This 13-cis, 15-anti form, is different from the 13-cis, 15-syn form occurring in DA BR. This photo intermediate is easily detectable compared to J. SB is still protonated here.

L: The Asp96 becomes the most relevant residue in this photointermediate. Some waters enclosed between Asp96 and SB, moves after retinal isomerization, producing alteration in the Asp96 environment. These movements yield a relaxation of the helices directed to surround the new conformation of the retinal. Recently, some structural studies point to certain movements in helix C during this intermediate state.

M: This is the most well-known photo intermediate. SB deprotonation is the most remarkable feature in this intermediate. The proton released from the SB, moves to the Asp85, which becomes protonated. M intermediate consists of two photo intermediates, which are known M1 and M2.

  • M1: The first movement of the proton occurs in this intermediate. In this intermediate, SB deprotonates, and the proton is taken by Asp85. This transfer aids in release of a proton to the extracellular side by a residue or a complex known as X or proton release group.
  • M2: This is also referred to as the late M. Here, the accessibility of the retinal changes from the extracellular to the cytoplasmic part, in fact to promote the reprotonation of the SB in the N intermediates. Besides, some conformational changes occur in this step too. The helices F and G open such in the way that the cytoplasmic half of the protein becomes an open channel to facilitate the entry of water molecules.

N: From M2 to N, SB befalls protonated from Asp96, which deprotonates. The opening of the helices in the late M, favors the entrance of water molecules, decreasing the pKa of Asp96, thus facilitating the deprotonation of this residue in favor of the SB. Between SB and the Asp96 exists a hydrogen bonded network, that allows the transit of the proton to reprotonate the SB.

O: From M to O, Asp96 reprotonates, and the retinal reisomerizes to all-trans conformation. From which residue gets Asp96 the proton still remains unclear. Nevertheless, some hypothesis points to an antenna complex configured by Asp36, 38, 102 and 104, which would capture the proton from the bulk, to yield it to Asp96. During this intermediate, Asp85 is still protonated, but in the O to BR transition, this residue deprotonates and proton releasing group protonates.

All these events described as the photocycle, should be considered in order to determine the path of the proton through the protein. Nonetheless, the crystal structures obtained in the last years, allow us to figure out more precisely not only the way of the proton, but also the structure of some intermediates. These structures of intermediates are obtained at low temperatures and in a non-natural environment for the protein. However, some general ideas can be inferred of each intermediate considering each structure. The consensus of some conformations contrasts with the controversy generated about substantial differences among the structure of one intermediate (L-intermediate) published by different groups.