Bacteriorhodopsin protonated Schiff base

Figure 2-3. Protonated Schiff-base of retinal (PSBR) and computational models used in ONIOM QM QM calculations (left). Electrostatic effects of the surrounding protein on excitation energies in bacteriorhodopsin evaluated using TD-B3LYP Amber right). (Adapted from Vreven and Morokuma [37] (Copyright American Institute of Physics) and Vreven et al. [38], Reprinted with permission.)...

The CP MAS NMR spectroscopy has been also extensively used for studies of proteins containing retinylidene chromophore like proteorhodopsin or bacteriorhodopsin. Bacteriorhodopsin is a protein component of purple membrane of Halobacterium salinarium.71 7 This protein contains 248 amino acids residues, forming a 7-helix bundle and a retinal chromophore covalently bound to Lys-216 via a Schiff base linkage. It is a light-driven proton pump that translocates protons from the inside to the outside of the cell. After photoisomerization of retinal, the reaction cycle is described by several intermediate states (J, K, L, M, N, O). Between L and M intermediate states, a proton transfer takes place from the protonated Schiff base to the anionic Asp85 at the central part of the protein. In the M and/or N intermediate states, the global conformational changes of the protein backbone take place. 

Dynamic nuclear polarisation (DNP) enhanced 15N CP MAS NMR has been exploited by Mark-Jurkauskas et al.79 in the studies of intermediates of the bacteriorhodopsin photocycle. The data for L intermediate were similar to those found for 13-ds,15-anti retylidene chloride, while those for K intermediate were similar to those of acid blue bacteriorhodopsin in which the Schiff base counterion was neutralised (Table 3). The 15N chemical shifts observed have shown that for bacteriorhodopsin, the Schiff base in K intermediate state loses contact with its counterion and establishes a new one in L intermediate state. The proton energy stored at the beginning in the electrostatic modes has been transformed to torsional modes. The transfer of energy is facilitated by the reduction of bond order alternation in the polyene chain when the counterion interaction is initially broken and is driven by the attraction of the Schiff base to a new counterion. 3D CP MAS experiments of NCOCX, NCACX, CONCA and CAN(CO)CA types have been used in studies of proteorhodopsin.71... 

Early experiments with bacteriorhodopsin (228) interpreted the Raman spectrum in terms of an unprotonated Schiff base, forming a charge-transfer complex with a protein functional group (210,212). This interpretation of the Raman data, essentially based on a comparison with the frequencies of model Schiff bases in solution, was criticized by Honig and Ebrey (48), who pointed out that consistency could also be obtained with a protonated Schiff base model. The latter hypothesis was subsequently confirmed by deuteration experiments similar to those described for rhodopsin (229,230), and by Raman spectra in denatured systems (231). In variance with the clear-cut similarity observed between the resonance-Raman spectra of rhodopsin and isorhodopsin, and those of the 11-cis and 9-cis model compounds, respectively,... 

A complex multi-chromophoric system comprises the purple membrane patches from Halobacterium salinarium. These patches are composed of about 3000 bacter-iorhodopsin proteins. The hyperpolarizability of solubilized monomeric bacterio-rhodopsin was measured by HRS and found to be 2100 x 10 esu at 1064 nm. This high value is due to the presence of a chromophore in the protein, the proto-nated Schiff base of retinal. A purple membrane patch can be treated as a two-dimensional crystal of bacteriorhodopsin proteins, and its structure is known in considerable detail. The analysis of the purple membrane tensor was performed by adding the hyperpolarizabilities of the individual proteins in the purple membrane. From (depolarized) HRS measurements on purple membrane suspensions, the structure of the purple membrane patches, and an average membrane size measured by atomic force microscopy, a fi value of 2200 x 10 esu was calculated for bacteriorhodopsin [22]. The organization of the dipolar protonated Schiff base chro-mophores in the membranes was found to be predominantly octopolar. 

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 pKa of the protonated Schiff base in bacteriorhodopsin is shifted from around 7 in model compounds in solution to well above 10 [86,87]. In contrast, the halorhodopsin Schiff-base pK is not raised and a pH-dependent equilibrium at 7.4 between the... 

Hendrickx et al. [41 2] have reported the first hyperpolarizabilities of retinal, retinal Schiff base and retinal protonated Schiff base at 1064 nm excitation wavelength. Retinal protonated Schiff base is responsible for the linear and NLO properties of bacteriorhodopsin protein. Their measured hyperpolarizabilities are 3600 X 10 ° esu for retinal protonated Schiff base and 470 x IO" esu for retinal Schiff base. They also investigated theoretical understanding of the first hyperpolarizabilities of retinal derivatives. Results are shown in Table 5. 

HPLC analysis also revealed that the protonated Schiff base of all-traws-retinal in solution is isomerized predominantly into the 11-cis form (82% 11-cis, 12% 9-cis, and 6% 13-ds in methanol) [23]. The 11-cis form as a photoproduct is the nature of retinochrome, not those of archaeal rhodopsins. This suggests that the protein environment of retinochrome serves as the intrinsic property of the photoisomerization of the retinal chromophore. In contrast, it seems that the protein environment of archaeal rhodopsins forces the reaction pathway of the isomerization to change into the 13-cis form. In this regard, it is interesting that the quantum yield of bacteriorhodopsin (0.64) is 4—5 times higher than that in solution (-0.15) [21,23], The altered excited state reaction pathways in archaeal rhodopsins never reduce the efficiency. Rather, archaeal rhodopsins discover the reaction pathway from the all-trans to 13-cis form efficiently. Consequently, the system of efficient isomerization reaction is achieved as well as in visual rhodopsins. Structural and spectroscopic studies on archaeal rhodopsins are also reviewed in Section 4.3. 

Halobacteria contain four rhodopsins bacteriorhodopsin, halorhodopsin, sensor-yrhodopsin, and phoborhodopsin (Fig. 4.2A) [11-17]. Bacteriorhodopsin and halorhodopsin are light-driven ion pumps, which act as an outward proton pump and an inward Ch pump, respectively. Sensoryrhodopsin and phoborhodopsin are photoreceptors that act to produce attractant and repellent responses in phototaxis, respectively. These four archaeal rhodopsins have similar structures seven helices constitute the transmembrane portion of the protein, and a retinal chromophore is bound to a lysine residue of the seventh helix via a protonated SchifF base linkage (Fig. 4.1). A negatively charged counterion is present to stabilize the positive charge inside the protein the counterion is an aspartate except for in halorhodopsin, which possesses a chloride ion. In sensoryrhodopsin, interaction with a transmembrane transducer protein raises the pKa of the aspartate, so that the aspartate is protonated at neutral pH. 

Fig. 3. Resonance Raman spectrum of the all-trani-retinal protonated Schiff base chromo-phore in light-adapted bacteriorhodopsin. The assignments are of the various enhanced vibrational normal modes are indicated. (From Smith el al. )...

Bacteriorhodopsin is found in differentiated regions of the cytoplasmic membrane of several Halobacteria. These so-called purple membranes are composed of two major lipid species (25% by weight) and one protein component which contains retinal (the aldehyde form of vitamin A) as a covalently bound prosthetic group. The retinal is bound via a protonated Schiff-base linkage to a lysine residue of the protein moiety of bacteriorhodopsin and absorbs light with a maximum absorbance at 570 nm [18]. 

Figure 19.7. Example of a time course in the bacteriorhodopsin photocycle at a single wavenumber (1188cm ). Data were recorded simultaneously with a 200-MHz and a 200-kHz transient recorder, so no inconsistency is seen in the overlap region near 20 ps. The 1188-cm band is assigned to a C-C stretching vibration of the l3-cis protonated Schiff base retinal. (Reproduced from [7], by permission of the Society for Applied Spectroscopy copyright 1997.)...

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