M. Sheves

pKa of the protonated Schiff base of bovine rhodopsin. A study with artificial pigments

Artificial bovine rhodopsin pigments derived from synthetic retinal analogues carrying electron-withdrawing substituents (fluorine and chlorine) were prepared. The effects of the electron withdrawing substituents on the pKa values of the pigments and on the corresponding Schiff bases in solution were analyzed. The data suggest that the apparent pKa of the protonated Schiff base is above 16. However, the alternative possibility that the retinal Schiff base linkage in bovine rhodopsin is not accessible for titration from the aqueous bulk medium cannot be definitely ruled out.

Resolving the primary dynamics of bacteriorhodopsin, and of a `C13C14 locked' analog, in the reactive excited state

Abstract The evolution of stimulated emission from the reactive excited state of bacteriorhodopsin is compared with that of a modified protein containing a synthetic retinal which is unable to twist around the C 13 C 14 bond. The rise of the emission is demonstrated to be structured, multistaged and wavelength dependent, appearing later in the red edge of the emission bands in both proteins. Spectral modulations associated with vibronic wavepacket motion are also observed in both. The mechanism of strong Stokes shifting of the emission resolved here, and the nature of coherences observed in the excited state are discussed.

TITRATION KINETICS OF ASP-85 IN BACTERIORHODOPSIN : EXCLUSION OF THE RETINAL POCKET AS THE COLOR-CONTROLLING CATION BINDING SITE

The spectrum (the purple↔blue transition) and function of the light‐driven proton pump bacteriorhodopsin are determined by the state of protonation of the Asp‐85 residue located in the vicinity of the retinal chromophore. The titration of Asp‐85 is controlled by the binding/unbinding of one or two divalent metal cations (Ca2+ or Mg2+). The location of such metal binding site(s) is approached by studying the kinetics of the cation‐induced titration of Asp‐85 using metal ions and large molecular cations, such as quaternary ammonium ions, R4N+ (R=Et, Pr, a divalent ‘bolaform ion’ [Et3N+‐(CH2)4‐N+Et3] and the 1:3 molecular complex formed between Fe2+ and 1,10‐phenanthroline (OP). The basic multi‐component kinetic features of the titration, extending from 10−2 to 104 s, are unaffected by the charge and size of the cation. This indicates that cation binding to bR triggers the blue→purple titration in a fast step, which is not rate‐determining. In view of the size of the cations involved, these observations indicate that the cation binding site is in an exposed location on, or close to, the membrane surface. This excludes previous models, which placed the color‐controlling Ca2+ ion in the retinal binding pocket.

Non-isomerizable artificial pigments: Implications for the primary light-induced events in bacteriorhodopsin

The primary events in the photosynthetic retinal protein bacteriorhodopsin (bR) are reviewed in light of photophysical and photochemical experiments with artificial bR in which the native retinal polyene is replaced by a variety of chromophores. Focus is on retinals in which the “critical” C13=C14 bond is locked with respect to isomerization by a rigid ring structure. Other systems include retinal oxime and non-isomerizable dyes noncovalently residing in the binding site. The early photophysical events are analyzed in view of recent pump–probe experiments with sub-picosecond time resolution comparing the behavior of bR pigments with those of model protonated Schiff bases in solution. An additional approach is based on the light-induced cleavage of the protonated Schiff base bond that links retinal to the protein by reacting with hydroxylamine. Also described are EPR experiments monitoring reduction and oxidation reactions of a spin label covalently attached to various protein sites. It is concluded that in bR the initial relaxation out of the Franck–Condon (FC) state does not involve sub-stantial C13=C14 torsional motion and is considerably catalyzed by the protein matrix. Prior to the decay of the relaxed fluorescent state (FS or I state), the protein is activated via a mechanism that does not require double bond isomerization. Most plausibly, it is a result of charge delocalization in the excited state of the polyene (or other) chromophores. More generally, it is concluded that proteins and other macromolecules may undergo structural changes (that may affect their chemical reactivity) following optical excitation of an appropriately (covalently or non-covalently) bound chromophore. Possible relations between the light-induced changes due to charge delocalization, and those associated with C13=C14 isomerization (that are at the basis of the bR photocycle), are discussed. It is suggested that the two effects may couple at a certain stage of the photocycle, and it is the combination of the two that drives the cross-membrane proton pump mechanism.

Effective Light-Induced Hydroxylamine Reactions Occur with C13 = C14 Nonisomerizable Bacteriorhodopsin Pigments

The light-driven proton pump bacteriorhodopsin (bR) undergoes a bleaching reaction with hydroxylamine in the dark, which is markedly catalyzed by light. The reaction involves cleavage of the (protonated) Schiff base bond, which links the retinyl chromophore to the protein. The catalytic light effect is currently attributed to the conformational changes associated with the photocycle of all-trans bR, which is responsible for its proton pump mechanism and is initiated by the all-trans --> 13-cis isomerization. This hypothesis is now being tested in a series of experiments, at various temperatures, using three artificial bR molecules in which the essential C13==C14 bond is locked by a rigid ring structure into an all-trans or 13-cis configuration. In all three cases we observe an enhancement of the reaction by light despite the fact that, because of locking of the C13==C14 bond, these molecules do not exhibit a photocycle, or any proton-pump activity. An analysis of the rate parameters excludes the possibility that the light-catalyzed reaction takes place during the approximately 20-ps excited state lifetimes of the locked pigments. It is concluded that the reaction is associated with a relatively long-lived (micros-ms) light-induced conformational change that is not reflected by changes in the optical spectrum of the retinyl chromophore. It is plausible that analogous changes (coupled to those of the photocycle) are also operative in the cases of native bR and visual pigments. These conclusions are discussed in view of the light-induced conformational changes recently detected in native and artificial bR with an atomic force sensor.

On the protein residues that control the yield and kinetics of O(630) in the photocycle of bacteriorhodopsin.

The effects of pH on the yield (phi(r)), and on the apparent rise and decay constants (k(r), k(d)), of the O(630) intermediate are important features of the bacteriorhodopsin (bR) photocycle. The effects are associated with three titration-like transitions: 1) A drop in k(r), k(d), and phi(r) at high pH [pK(a)(1) approximately 8]; 2) A rise in phi(r) at low pH [pK(a)(2) approximately 4.5]; and 3) A drop in k(r) and k(d) at low pH [pK(a)(3) approximately 4. 5]. (pK(a) values are for native bR in 100 mM NaCl). Clarification of these effects is approached by studying the pH dependence of phi(r), k(r), and k(d) in native and acetylated bR, and in its D96N and R82Q mutants. The D96N experiments were carried out in the presence of small amounts of the weak acids, azide, nitrite, and thiocyanate. Analysis of the mutants data leads to the identification of the protein residue (R(1)) whose state of protonation controls the magnitude of phi(r), k(r), and k(d) at high pH, as Asp-96. Acetylation of bR modifies the Lys-129 residue, which is known to affect the pK(a) of the group (XH), which releases the proton to the membrane exterior during the photocycle. The effects of acetylation on the O(630) parameters reveal that the low-pH titrations should be ascribed to two additional protein residues R(2) and R(3). R(2) affects the rise of phi(r) at low pH, whereas the state of protonation of R(3) affects both k(r) and k(d). Our data confirm a previous suggestion that R(3) should be identified as the proton release moiety (XH). A clear identification of R(2), including its possible identity with R(3), remains open.

Low temperature FTIR study of the Schiff base reprotonation during the M-to-bR backphotoreaction: Asp 85 reprotonates two distinct types of Schiff base species at different temperatures

We have applied low temperature difference FTIR spectroscopy to investigate intermediates produced from the M intermediate upon blue light excitation (<480 nm). In agreement with an earlier report by Balashov and Litvin (1981), who studied these intermediates with low temperature visible absorption spectrophotometry, we have observed at least three stages in this backphotoreaction. The initial photoproduct is stable at 100 K, and two products of subsequent thermal reactions are observed upon raising the temperature to 130 and 160 K, respectively.The alterations in the C=N stretching mode of the Schiff base have been identified by isotopically labeling the retinal chromophore, and changes in C=O stretching modes of amino acid residues with acidic side chains have been investigated. Analysis of the C=N stretching mode shows that the Schiff base remains unprotonated after the photochemical reaction at 100 K. Moreover, there are two types of Schiff bases, presumably associated with different bR species, that become thermally reprotonated at 130 and 160 K, respectively. Bands associated with the C=O stretching modes suggest that Asp 85 rather than Asp 96 reprotonates the Schiff base during the M to bR backphotoreaction. This conclusion is consistent with earlier observations that the polarity of electrical signals during this photochemical back reaction is reversed as compared to the thermal regeneration of bR from M.

Resolving the primary dynamics of bacteriorhodopsin, and Locked analogs in the reactive excited state.

Stimulated emission from excited bacteriorhodopsin is compared with that from isomerize. Ultrafast Stokes shifting of emission, and coherent vibrations in the excited state are observed in both for the first time. Biological relevance of coherent motions is tested by comparing to similar motions in free protonated Sciffbase (PSB) of retinal.

A Comparative Study of the Initial Photoinduced Event in Bacteriorhodopsin: Can It Be Isomerization?

Ultrafast excitation of native and artificial bacteriorhodopsin leads to a revision of the currently accepted photophysical models based on a ≤ 100 fs C13–C14 isomerization of the retinal chromophore.

Following Photoinduced Dynamics in Bacteriorhodopsin with 7 fsec Impulsive Vibrational Spectroscopy

Spectral modulations induced by 6fsec photoexcitation of bacteriorhodopsin are Fourier analyzed. Long lived undulations are assigned to ground state vibrational coherences, while possible excited state contributions are very short lived consisting mainly of HOOP motions.