Deprotonation/protonation bacteriorhodopsin

Fig. 2. Deprotonation and protonation of bacteriorhodopsin triggered by laser-excitation at 580 nm in a pulse of 100 nsec length. Methylumbilliferon was used as pH indicator, (a) deprotonation recorded at —53°C, (6) protonation recorded at -34°C.

Fig. 2. A tentative scheme of the bacteriorhodopsin pump. bR indicates the bacteriorhodopsin ground state, and L, M, N(P) and O indicate the corresponding intermediates of the photocycle. NHs, =N2 and =NH represent the protonated Schiff base of the idUtrans retinal residue, the deprotonated and the protonated Schiff bases of 13-cw retinal residues, respectively. -COOH and -COO are the protonated and the deprotonated Asp-96 carboxylic group, respectively. The outward hydrophilic H -conducting pathway (the proton well) is shaded. (From Skulachev[35].)...

The proton transfer reaction between azide and other weak bases with various pK s and the Schiff base allowed estimation of the pK of the halorhodopsin Schiff base at that time in the photocycle where the proton is exchanged. The ratio of forward and reverse proton transfer was dependent on the pKa of the weak base and reached a value of unity at a pK, of about 4.3 [127]. This is then the pK of the Schiff base at the time of proton transfer, representing a drop of about 3 pH units from its dark value. In bacteriorhodopsin it must be a similar pK, shift, but of much greater magnitude, which allows the Schiff-base deprotonation in the L — M step. 

Schiffs base, but the spectra for the M-412 intermediate indicate that this proton is lost. The deprotonation of the Schiffs base is apparently after the K intermediate [262], and proposed to be during the L to M transition [209,263,264], Reprotonation of the nitrogen is suggested to occur during the M-412 to 0-640 conversion [265], Part of the blue-shift in the formation of M-412 is, of course, explained by the fact that, in model retinal compounds, loss of the proton leads to a 440-380 nm shift [266], but other effects must also be present. Circumstantial evidence, which includes the finding of 13-cis retinal in M-like intermediates stabilized under somewhat denaturing conditions [198,267], favors the idea that the retinal is isomerized in the M intermediate, as do the more direct resonance Raman data [268,269], In fact, the K and L intermediates seem already to contain the 13-c/i isomer of retinal, as indicated by extraction of 13-c/i retinal from the L intermediate [270] and spectroscopic data on the K and L intermediates [271-274], The resonance Raman spectroscopy of bacteriorhodopsin photointermediates has been recently reviewed [275],... 

Henderson et al. [223] presented a detailed pattern of the structure of bacteriorhodopsin using high-resolution cryoelectron microscopy. Using X-ray and neutron diffraction techniques, Dencher et al. [224—227] could decode the secondary and tertiary structure of bacteriorhodopsin during the photocycle. Nevertheless, we should emphasize that the resolution still shows transitions in the active site (protonation of counterions, deprotonation of Schiff base, and reprotonation of counterions), leading to a metastable state of the protein. 

Fig. 23. (A) The halophilic bacterium H. halobium with patches containing the "purple membrane" (B) Structure of the protein bacteriorhodopsin (left) and the structural formula for the chromophore retinal (right) (C) Covalent binding of retinal with iysine-216 forming a positively-charged Schiff base (D) Illumination of the bacteriorhodopsin retinal and transformation from a trans- to a cis-configuration and releases a proton from the Schiff base to the cell exterior relaxation to ttie trans-form, with uptake of a proton from the cytoplasmic interior. The combination of deprotonation and reprotonation on opposite sides of the membrane constitutes a proton pump. See text for other details. Figures partly adapted from Becker and Deamer (1991) The World of the Cell (2nd ed) Benjamin/Cummings PubI Co. p 215.

There is now abundant evidenee that bacteriorhodopsin is a light-activated vectorial ion pump that translocates protons across the bacterial plasma membrane. The mechanism of proton translocation is unknown, although the process is linked to a complex photocycle that involves a number of intermediates [Mowery and Stoeckenius (1981) and Stoeckenius and Bogomolni (1982) see also articles in Packer (1982)] in which the Schiff base is protonated in the BR 570 state and deprotonated in the MR 412 state (Lewis et al., 1974 Aton et al., 1977 Rothschild et al., 1981). The electrochemical gradient generated in this way is used by the cell for ATP synthesis. 

Infrared spectroscopic studies of macromolecules became increasingly powerful with the development of Fourier transform techniques [44, 47, 48, 59-67]. (See Chap. 1 for a description of an FTIR spectrometer.) FTIR measurements can be used to probe changes in the bonding or interactions of individual amino acid side chains in proteins. Bacteriorhodopsin provides an illustration. When bacterio-rhodopsin is illuminated, its protonated retinylidine Schiff base chromophore isomerizes and then transfers a proton to a group in the protein. FTIR measurements showed the formation of an absorption band at 1,763 cm in addition to a set of absorption changes attributable to the chromophore [63, 68]. In bacteriorhodopsin that was enriched in [4- C]-aspartic acid the band appeared at 1,720 cm and an additional shift to 1,712 cm was obtained when the sovent was replaced by D2O. These observations indicated that the band reflected C=0 stretching of a protonated aspartic acid, leading to identification of a particular aspartic acid residue as the H" acceptor for deprotonation of the chromophore. 

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