Base Deprotonation Research Articles

The role of back-reactions and proton uptake during the N .fwdarw. O transition in bacteriorhodopsin's photocycle: a kinetic resonance Raman study

The kinetics of bacteriorhodopsin's photocycle have been analyzed at pH 5, 6, 7, 8, and 8.6 by using time-resolved resonance Raman spectroscopy. The concentrations of the various intermediates as a function of time were determined by following their resonance Raman intensities using 502-nm (L550, N550, BR568), 458-nm (M412), and 752-nm (O640) excitation. The spectral contributions to the pump + probe data from each intermediate were quantitatively separated by least-squares decomposition. These relative concentrations were then converted to absolute concentrations by using a conservation of molecules constraint. This enabled the unambiguous refinement of a variety of kinetic models to find the simplest one that accurately describes the data. The kinetic data, including the biphasic decay of L550 and M412, are best reproduced by a sequential scheme including back-reactions (BR----L----M----N----O----BR). In addition, the kinetics of the L----M and N----O steps are found to be pH-dependent. Both the forward and reverse rate constants connecting L550 and M412 increase with pH, confirming earlier proposals of catalyzed Schiff base deprotonation at alkaline pH. Below pH 7, the N550----O640 rate constant is independent of pH, but it decreases linearly with pH above 7. This indicates that the protein must pick up a proton during the N550----O640 transition and that this process becomes rate determining above pH 7. There must, therefore, be an intermediate between N550 and O640 which we denote as N+550. A molecular graphics model is presented which incorporates these observations into a mechanism for proton pumping.

Effect of genetic modification of tyrosine-185 on the proton pump and the blue-to-purple transition in bacteriorhodopsin.

The retinylidene chromophore mutant (Y185F) of bacteriorhodopsin, in which Tyr-185 is substituted by phenylalanine, is examined and compared with wild-type bacteriorhodopsin expressed in Escherichia coli; both were reinstituted similarly in vesicles. The Y185F mutant shows (at least) two distinct spectra at neutral pH. Upon light absorption, the blue species (which absorbs in the red) behaves as if "dead"--i.e., neither its tyrosine nor its protonated Schiff base undergoes deprotonation nor does its tryptophan fluorescence undergo quenching. This result is unlike either the purple species (which absorbs in the blue) or wild-type bacteriorhodopsin expressed in E. coli. As the pH increases, both the color changes and the protonated Schiff base deprotonation efficiency suggest a blue-to-purple transition of the Y185F mutant near pH 9. If this blue-to-purple transition of Y185F corresponds to the blue-to-purple transition of purple-membrane (native) bacteriorhodopsin (occurring at pH 2.6) and of wild-type bacteriorhodopsin expressed in E. coli (occurring at pH 5), the protein-conformation changes of this transition as well as the protonated Schiff base deprotonation may be controlled not by surface pH alone, but rather by the coupling between surface potential and the general protein internal structure around the active site. The results also suggest that Tyr-185 does not deprotonate during the photocycle in purple-membrane bacteriorhodopsin.

Substitution of amino acids Asp-85, Asp-212, and Arg-82 in bacteriorhodopsin affects the proton release phase of the pump and the pK of the Schiff base.

Photocycle and flash-induced proton release and uptake were investigated for bacteriorhodopsin mutants in which Asp-85 was replaced by Ala, Asn, or Glu; Asp-212 was replaced by Asn or Glu; Asp-115 was replaced by Ala, Asn, or Glu; Asp-96 was replaced by Ala, Asn, or Glu; and Arg-82 was replaced by Ala or Gln in dimyristoylphosphatidylcholine/3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate micelles at pH 7.3. In the Asp-85----Ala and Asp-85----Asn mutants, the absence of the charged carboxyl group leads to a blue chromophore at 600 and 595 nm, respectively, and lowers the pK of the Schiff base deprotonation to 8.2 and 7, respectively, suggesting a role for Asp-85 as counterion to the Schiff base. The early part of the photocycles of the Asp-85----Ala and Asp-85----Asn mutants is strongly perturbed; the formation of a weak M-like intermediate is slowed down about 100-fold over wild type. In both mutants, proton release is also slower but clearly precedes the rise of M. The amplitude of the early (less than 0.2 microseconds) reversed photovoltage component in the Asp-85----Asn mutant is very large, and the net charge displacement is close to zero, indicating proton release and uptake on the cytoplasmic side of the membrane. The data suggest an obligatory role for Asp-85 in the efficient deprotonation of the Schiff base and in the proton release phase, probably as proton acceptor. In the Asp-212----Asn mutant, the rise of the absorbance change at 410 nm is slowed down to 220 microsecond, its amplitude is small, and the release of protons is delayed to 1.9 ms. The absorbance changes at 650 nm indicate perturbations in the early time range with a slow K intermediate. Thus Asp-212 also participates in the early events of charge translocation and deprotonation of the Schiff base. In the Arg-82----Gln mutant, no net transient proton release was observed, whereas, in the Arg-82----Ala mutant, uptake and release were reversed. The pK shift of the purple-to-blue transition in the Asp-85----Glu, Arg-82----Ala, and Arg-82----Gln mutants and the similarity in the photocycle and photoelectrical signals of the Asp-85----Ala, Asp-85----Asn, and Asp-212----Asn mutants suggest the interaction between Asp-85, Arg-82, Asp-212, and the Schiff base as essential for proton release.

Photoreactions of bacteriorhodopsin at acid pH

It has been known that bacteriorhodopsin, the retinal protein in purple membrane which functions as a light-driven proton pump, undergoes reversible spectroscopic changes at acid pH. The absorption spectra of various bacteriorhodopsin species were estimated from measured spectra of the mixtures that form at low pH, in the presence of sulfate and chloride. The dependency of these on pH and the concentration of Cl- fit a model in which progressive protonation of purple membrane produces "blue membrane", which will bind, with increasing affinity as the pH is lowered, chloride ions to produce "acid purple membrane." Transient spectroscopy with a multichannel analyzer identified the intermediates of the photocycles of these altered pigments, and described their kinetics. Blue membrane produced red-shifted KL-like and L-like products, but no other photointermediates, consistent with earlier suggestions. Unlike others, however, we found that acid purple membrane exhibited a very different photocycle: its first detected intermediate was not like KL in that it was much more red-shifted, and the only other intermediate detectable resembled the O species of the bacteriorhodopsin photocycle. An M-like intermediate, with a deprotonated Schiff base, was not found in either of these photocycles. There are remarkable similarities between the photoreactions of the acid forms of bacteriorhodopsin and the chloride transport system halorhodopsin, where the Schiff base deprotonation seems to be prevented by lack of suitable aspartate residues, rather than by low pH.

On the molecular mechanisms of solar energy storage during the photocycle of the other photosynthetic system in nature, bacteriorhodopsin

Bacteriorhodopsin (bR) is the only protein found in the purple membrane of Halobacterium halobium, a light-harvesting bacterium. bR absorbs light and undergoes a cycle involving intermediates appearing in different time domains (from fractions of picoseconds to several milliseconds). As a result, the protonated Schiff base (PSB) deprotonates, and protons are pumped from inside to the outside of the cell membrane, creating proton gradients that are energetically responsible for making ATP. Using time-resolved Raman and optical spectroscopic experiments, the following results and possible conclusions are obtained. (1) Unlike chlorophyll, bR does not use an antenna system, i.e., each absorbing molecule is a reaction center. (2) Isomerization of its retinal chromophore, which leads to the first step in energy storage by charge separation, occurs on the subpicosecond time scale. (3) The deprotonation of the PSB and a tyrosine, which occur on the 40 {mu}s time scale, is found to have activation energies comparable to H-bond energies. This, together with the fact that the temporal quenching of the tryptophan fluorescence follows the time profile of the deprotonation strongly suggests that the latter process is controlled by protein conformation changes. (4) Cations are found to be required for the deprotonation process and are believedmore » to control the protein conformation required for this process. Possible mechanisms responsible for the decrease in the pK{sub a} of the PSB from 13.3 to <2.6 during the photocycle, and are thus responsible for the deprotonation process, are discussed.« less

Nuclear magnetic resonance and infrared study of lithium coordination by phosphonates and phosphine oxides R2P(O)CH2COY and their carbanions. Comparison between ketone, ester, and amide COY groups

Lithiated carbanionic chelates α to P(O) groups are formed from four title compounds. Their infrared and 13C and 31P nuclear magnetic resonance spectra show that chelation induces peculiar structural perturbations that depend upon the nature of Y. The corresponding neutral precursors 1–4 are able to form bidentate coordinates with the Li cation, which can be observed in THF when Y = OMe or NMe2 but in benzene only when Y = Me. They are probably intermediates in the lithiated base deprotonation of neutral compounds 1–4.

Evidence for the involvement of more than one metal cation in the Schiff base deprotonation process during the photocycle of bacteriorhodopsin

The removal of metal cations inhibits the deprotonation process of the protonated Schiff base during the photocycle of bacteriorhodopsin. To understand the nature of the involvement of these cations, a spectroscopic and kinetic study was carried out on bacteriorhodopsin samples in which the native Ca(2+) and Mg(2+) were replaced by Eu(3+), a luminescent cation. The decay of Eu(3+) emission in bacteriorhodopsin can be fitted to a minimum of three decay components, which are assigned to Eu(3+) emission from three different sites. This is supported by the response of the decay components to the presence of (2)H(2)O and to the changes in the Eu(3+)/bR molar ratio. The number of water molecules coordinated to Eu(3+) in each site is determined from the change in its emission lifetime when (2)H(2)O replaces H(2)O. Most of the emission originates from two "wet" sites of low crystal-field symmetry-e.g., surface sites. Protonated Schiff base deprotonation has no discernable effect on the emission decay of protein-bound Eu(3+), suggesting an indirect involvement of metal cations in the deprotonation process. Adding Eu(3+) to deionized bacteriorhodopsin increases the emission intensity of each Eu(3+) site linearly, but the extent of the deprotonation (and color) changes sigmoidally. This suggests that if only the emitting Eu(3+) ions cause the deprotonation and bacteriorhodopsin color change, ions in more than one site must be involved-e.g., by inducing protein conformation changes. The latter could allow deprotonation by the interaction between the protonated Schiff base and a positive field of cations either on the surface or within the protein.

Organic geochemistry of the British Kimmeridge Clay: 3. The occurrence and distribution of acyclic isoprenoid C19 alkenes in Kimmeridge Clay shale oils

The occurrence and distribution of acyclic isoprenoid C19 alkenes (pristenes) in Kimmeridge Clay shale oils has been examined. Pristenes comprise ≈ 9 to 15% of isolated alkene fractions, and ≈ 8% of total shale oil non-aromatic hydrocarbons. They are generally much more abundant than pristane, possibly because only limited hydrogenation of isoprenoid alkenes occurs during pyrolysis. Two pristene isomers are identified (mass spectrometry, retention indices, g.c. co-injection), with prist-2-ene the more abundant in several Kimmeridge shale oils. Prist-2-ene/prist-1-ene ratios show some correlation with sediment oil yield and clay mineral content. Clay-rich low oil yield sediments often give prist-2-ene dominated shale oils, whereas high oil yield sediments show prist-1-ene dominance. Results suggest the conversion of prist-1-ene to prist-2-ene during pyrolysis by a time dependent, thermodynamically favourable double bond rearrangement. Two mechanisms are suggested for the generation of prist-1-ene, the primary pyrolysis product, from C20 units CC bonded to Kimmeridge oil shale kerogen: lt]o li](i) a thermolytic, radical induced tertiary hydrogen abstraction followed by homolytic β CC bond cleavage, or li](ii) in the presence of clay minerals, a clay catalysed carbonium ion route involving Lewis acid tertiary hydrogen abstraction and heterolytic β CC bond fission. Prist-1-ene double bond rearrangement is suggested to occur by a clay-catalysed ionic pathway involving proton-donor site double bond protonation followed by collapse of the resultant tertiary carbonium ion by Lewis base deprotonation. While rearrangement is likely to be retarded in analytical flash pyrolysis, Fischer pyrolysis conditions (and laboratory thermal maturation) allow more time for secondary rearrangement. Where the rate of rearrangement is enhanced by increased clay catalyst concentration, e.g. clay rich Kimmeridge shales, prist-2-ene becomes increasingly prominent in the resultant pyrolysates.

On the molecular mechanisms of the Schiff base deprotonation during the bacteriorhodopsin photocycle

Using optical flash photolysis and time-resolved Raman methods, we examined intermediates formed during the photocycle of bacteriorhodopsin (bR), as well as the bR color change, as a function of pH (in the 7.0-1.5 region) and as a function of the number of bound Ca(2+) ions. It is found that at a pH just below 3 or with less than two bound Ca(2+) per bR, the deprotonation (the L(550) --> M(412)) step ceases, yet the K(610) and L(550) analogues are still formed as in native bR. The lack of deprotonation in the photocycle of both acid blue and deionized blue bR and the similarity of their Raman spectra as well as of their K(610) and L(550) analogues strongly suggest that both blue samples have nearly the same retinal active site. It is suggested that in both blue species, bound cations are removed via a proton-cation exchange equilibrium, either on the cation exchange column for the deionized sample or in solution for the acid blue sample. The proton-cation exchange equilibrium is found to quantitatively account for the pH dependence of the purple-to-blue color change. The different mechanisms responsible for the large reduction ( approximately 11 units) of the pK(a) value of the protonated Schiff base (PSB) during the photocycle are discussed. The absence of the L(550) --> M(412) deprotonation process in both blue species is discussed in terms of the previously proposed cation model for the deprotonation of the PSB during the photocycle of native bR. The extent of the deprotonation and the blue-to-purple color change are found to follow the same dependence on either the pH or the amount of cations added to deionized blue bR. This observed correlation is briefly discussed.

THE EFFECT OF IONIC STRENGTH AND pH ON THE PROTONATED SCHIFF BASE AND TYROSINE DEPROTONATION KINETICS DURING THE BACTERIORHODOPSIN PHOTOCYCLE

Abstract— The deprotonation kinetics of tyrosine and the protonated Schiff base during the bacteriorho‐dopsin photocycle were studied under different perturbations by transient absorption spectroscop Native purple membrane, as well as samples which were deionized (blue) then restored with Na+ or La3+ were used at pH's ranging from 7 to 10 at very low salt concentrations. The results were compared with previous studies at higher ionic strength. The important conclusions can be summarized as follows: (a) The rate constants of both the Schiff base and tyrosine deprotonation are not very sensitive to the changes of conditions. (b) An almost linear relationship is observed between the relative amplitudes of the tyrosine deprotonated during the cycle and the slow component of the Schiff base deprotonation under the different perturbations studied. This was taken to support the two site model for the protonated Schiff base, one near tyrosine and the other near its ionized form. (c) The pKa value determined from the ratio of the amplitude of the fast to the slow component of the Schiff base deprotonation is found to decrease with increasing ionic strength of the medium. At extremely low ionic strength, it was found to equal that of the tyrosine phenolic group in solution.

Deprotonation of the Schiff base of rhodopsin is obligate in the activation of the G protein.

Photolysis of rhodopsin leads to the formation of an activated intermediate that activates a G protein, thus beginning the visual cascade. This activated form of rhodopsin appears coincident in time with the spectroscopically defined intermediate, metarhodopsin II. Metarhodopsin I, the precursor of metarhodopsin II, contains a protonated Schiff base, whereas metarhodopsin II does not. The question of whether the deprotonation of the protonated Schiff base is obligate in the formation of activated rhodopsin was addressed by monomethylating the active-site lysine of permethylated rhodopsin and determining whether this pigment can activate the G protein upon photolysis. The photolysis of the new pigment, which absorbs at 520 nm, led to the formation of a relatively stable metarhodopsin I-like intermediate with a lambda max of approximately equal to 485 nm, with no apparent formation of either metarhodopsin II- or metarhodopsin III-like intermediates. The only probe available to detect formation of the active form of rhodopsin is G protein activation. Photolysis of the pigment in the presence of the G protein did not lead to measurable activation of the GTPase activity of the latter. These studies establish a functional link between Schiff base deprotonation and activation of the G protein. It is concluded that proton transfer from the protonated Schiff base of rhodopsin is obligate for the initiation of visual transduction.

Determination of retinal chromophore structure in bacteriorhodopsin with resonance Raman spectroscopy.

The analysis of the vibrational spectrum of the retinal chromophore in bacteriorhodopsin with isotopic derivatives provides a powerful "structural dictionary" for the translation of vibrational frequencies and intensities into structural information. Of importance for the proton-pumping mechanism is the unambiguous determination of the configuration about the C13=C14 and C=N bonds, and the protonation state of the Schiff base nitrogen. Vibrational studies have shown that in light-adapted BR568 the Schiff base nitrogen is protonated and both the C13=C14 and C=N bonds are in a trans geometry. The formation of K625 involves the photochemical isomerization about only the C13=C14 bond which displaces the Schiff base proton into a different protein environment. Subsequent Schiff base deprotonation produces the M412 intermediate. Thermal reisomerization of the C13=C14 bond and reprotonation of the Schiff base occur in the M412------O640 transition, resetting the proton-pumping mechanism. The vibrational spectra can also be used to examine the conformation about the C--C single bonds. The frequency of the C14--C15 stretching vibration in BR568, K625, L550 and O640 argues that the C14--C15 conformation in these intermediates is s-trans. Conformational distortions of the chromophore have been identified in K625 and O640 through the observation of intense hydrogen out-of-plane wagging vibrations in the Raman spectra (see Fig. 2). These two intermediates are the direct products of chromophore isomerization. Thus it appears that following isomerization in a tight protein binding pocket, the chromophore cannot easily relax to a planar geometry. The analogous observation of intense hydrogen out-of-plane modes in the primary photoproduct in vision (Eyring et al., 1982) suggests that this may be a general phenomenon in protein-bound isomerizations. Future resonance Raman studies should provide even more details on how bacterio-opsin and retinal act in concert to produce an efficient light-energy convertor. Important unresolved questions involve the mechanism by which the protein catalyzes deprotonation of the L550 intermediate and the mechanism of the thermal conversion of M412 back to BR568. Also, it has been shown that under conditions of high ionic strength and/or low light intensity two protons are pumped per photocycle (Kuschmitz & Hess, 1981). How might this be accomplished?(ABSTRACT TRUNCATED AT 400 WORDS)

ON THE TYROSINATE INVOLVEMENT IN THE SCHIFF BASE DEPROTONATION IN THE PROTON PUMP CYCLE OF BACTERIORHODOPSIN

Abstract— The ultraviolet transient absorption assigned to the tyrosinate species in bacteriorhodopsin is followed in time and as a function of pH. Both its rise time and titration curve closely resemble those observed for the production of the M412 intermediate. These results may support a recently proposed mechanism that couples tyrosinate production to the Schiff base deprotonation in the proton pump of bacteriorhodopsin.

Effect of iodination on the pK of Schiff base deprotonation and M 412 production in purple membrane

Previously, kinetic resonance Raman measurements as a function of pH have been used to demonstrate that, microseconds after light absorption, the pK of Schiff base deprotonation during the bacteriorhodopsin photocycle is 10.2 ± 0.3, whereas before the light event, the pK is > 12 (2). In this investigation, we have iodinated purple membrane suspensions and have found that the pK of Schiff base deprotonation in the photocycle has been lowered to between 7 and 8 for iodinated bacteriorhodopsin. These results, together with our previous data on the pK of Schiff base deprotonation, suggest that the amino acid tyrosine could be a critical component in the deprotonation mechanism.

Experimental evidence for secondary protein-chromophore interactions at the Schiff base linkage in bacteriorhodopsin: Molecular mechanism for proton pumping

Resonance Raman spectroscopy of the retinylidene chromophore in various isotopically labeled membrane environments together with spectra of isotopically labeled model compounds demonstrates that a secondary protein interaction is present at the protonated Schiff base linkage in bacteriorhodopsin. The data indicate that although the interaction is present in all protonated bacteriorhodopsin species it is absent in unprotonated intermediates. Furthermore, kinetic resonance Raman spectroscopy has been used to monitor the dynamics of Schiff base deprotonation as a function of pH. All our results are consistent with lysine as the interacting group. A structure for the interaction is proposed in which the interacting protein group in an unprotonated configuration is complexed through the Schiff base proton to the Schiff base nitrogen. These data suggest a molecular mechanism for proton pumping and ion gate molecular regulation. In this mechanism, light causes electron redistribution in the retinylidene chromophore, which results in the deprotonation of an amino acid side chain with pK >10.2 +/- 0.3 (e.g., arginine). This induces subsequent retinal and protein conformational transitions which eventually lower the pK of the Schiff base complex from >12 before light absorption to 10.2 +/- 0.3 in microseconds after photon absorption. Finally, in this low pK state the complex can reprotonate the proton-deficient high pK group generated by light, and the complex is then reprotonated from the opposite side of the membrane.

The pK of Schiff base deprotonation in bacteriorhodopsin

Kinetic resonance Raman spectroscopy as a function of pH has been utilized to determine the pK of Schiff base deprotonation during the bacteriorhodopsin photochemical cycle. It is shown that the pK of Schiff base deprotonation is between 9.9 and 10.3, microseconds after light absorption and is >12 before photon initiation of photochemical cycling associated with proton pumping.