Kinetics of Light-Induced Intramolecular Charge Transfer and Proton Release in Bacteriorhodopsin

Recent advances in understanding the intramolecular charge transfer and proton release by the light-driven proton pump bacteriorhodopsin (bR) are critically reviewed. The focus is on the time-resolved electrical methods, i.e., photocurrent and photovoltage measurements, and on transient absorption experiments with pH-sensitive dyes. Particular attention is paid to the following topics: charge translocation in the low-pH forms of bR (acid-blue and acid-purple); electrogenicity of the 13-cis cycle; results with bR mutants; surface-bound dyes to detect the proton release at specific sites on either side of the protein; the question of kinetic coupling between the deprotonation of the Schiff base and proton release; and rapid long-range migration of protons along the surface of the purple membrane.     

Molecular Dynamics Studies of Bacteriorhodopsin's Photocycles

The availability of the structure of bacteriorhodopsin from electron microscopy studies has opened up the possibility of exploring the proton pump mechanism of this protein by means of molecular dynamics simulations. In this review we summarize earlier theoretical investigations of the photocycle of bacteriorhodopsin including relevant quantum chemistry studies of retinal, structure refinement, molecular dynamics simulations, and evaluation of pK_a values. We then review a series of recent modeling efforts which refined the structure of bacteriorhodopsin adding internal water, and which studied the nature of the J intermediate and the likely geometry of the K₅₉₀ and L₅₅₀ intermediates (strongly distorted 13-cis) as well as the sequence of retinal geometry and protein conformational transitions which are conventionally summarized as the M₄₁₂ intermediate. We also review simulations of the photocycle of light-adapted bacteriorhodopsin at T=77 K and of the photocycle of dark-adapted bacteriorhodopsin, both cycles differing from the conventional photocycle through a nonfunctional (pure 13-cis) retinal geometry of the corresponding K₅₉₀ and L₅₅₀ states. The simulations demonstrate a potentially critical role of water and of minute reorientations of retinal's Schiff base nitrogen in controlling proton pumping in bR₅₆₈; the simulations also indicate the existence of heterogeneous photocycles. The results exemplify the important role of molecular dynamics simulations in extending investigations on bacteriorhodopsin to a level of detail which is presently beyond experimental resolution, but which needs to be known to resolve the pump mechanism of bacteriorhodopsin. Finally, we outline the major existing challenges in the field of bacteriorhodopsin modeling.     

Dynamic Coupling of Tyrosine 185 with the Bacteriorhodopsin Photocycle,as Revealed by Chemical Shift Assisted AF-QM/MM Calculations and Molecular Dynamic Simulations

Aromatic residues are highly conserved in microbial photoreceptors and play crucial roles in the dynamic regulation of receptor functions. However, little is known about the dynamic mechanism of the functional role of those highly conserved aromatic residues during the receptor photocycle. Tyrosine 185 (Y185) is one of the highly conserved aromatic residues within the retinal binding pocket of bacteriorhodopsin (bR). In this study, we explored the molecular mechanism of its dynamic coupling with the bR photocycle by automated fragmentation quantum mechanics/molecular mechanics (AF-QM/MM) calculations and molecular dynamic (MD) simulations based on chemical shifts obtained by 2D solid-state NMR correlation experiments. We observed that Y185 plays a significant role in regulating the retinal cis–trans thermal equilibrium, stabilizing the pentagonal H-bond network, participating in the orientation switch of Schiff Base (SB) nitrogen, and opening the F42 gate by interacting with the retinal and several key residues along the proton translocation channel. Our findings provide a detailed molecular mechanism of the dynamic couplings of Y185 and the bR photocycle from a structural perspective. The method used in this paper may be applied to the study of other microbial photoreceptors.     

Photoreactions of the Photointermediates of Bacteriorhodopsin

The photochemical reactions of the intermediates of the photochemical cycle of bacteriorhodopsin (bR) are reviewed. These reactions constitute photochemical control of the cycle and provide an independent approach for the investigation of the mechanism of light energy transduction in the purple membrane. The absorption of a light quantum by the K, L, or M intermediates converts them back to bR. These transformations interrupt the photocycle so that no proton transfer occurs after absorption of the second quantum. The action of blue light on the M intermediate causes structural changes of the chromophore, as a result of which the Schiff base is reprotonated from Asp-85, not from Asp-96 as in the usual thermal transition of M. The photoreactions of the L, M, N, and O intermediates lead to the formation of new photoproducts. Studies of the photoconversion of the intermediates can serve as an additional source of information on the nature of photoprocesses in bR: they reveal several conformers of K and bR at 90 K, different M states, two N intermediates, and provide direct evidence for the existence of a thermal back reaction from N to M. The study of the photoreactions of the J, K, L, M, N, and O intermediates is a promising method for elucidating the structures and roles of these states. Reversible photoconversions of bR and its photointermediates provide a basis for potential applications of bR in optical registration of information.     

Structure and Function of Halorhodopsin

A comprehensive review of the physiology, structure, and function of halorhodopsin (HR), the only known light-driven anion pump, is presented. Beside the well-studied transport function of HR in intact cells the article focuses on recent results about the molecular properties of HR. Overexpression and in vivo 2-D crystallization allowed structural investigations at a level of 7 Å resolution. The results demonstrate a very close structural relationship with the proton pump bacteriorhododopsin (BR). The retinal binding site as well as a chloride binding site near the Schiff base in HR can be modeled with the side chains placed into the corresponding positions in the BR structural model. Mechanistically the vectorial catalytic cycle of HR is similar to that of BR, as suggested before (Oesterhelt et al., J. Bioenerg. Biomembr. 1992, 24 : 181), and consists of photoisomerization of the retinal moiety which triggers chloride movement within the transport site towards the Schiff base. This is followed by an accessibility change from the extracellular (EC) channel to the cytoplasmic (CP) channel allowing chloride to be released into the cytoplasm. After reisomerization and reversion of the accessibility change, a chloride is rebound into the transport site from the extracellular surface through EC. In the absence of transported ions or the additional presence of azide, photoisomerization to 13-cis is followed directly by an accessibility change and release of a proton from the Schiff base through CP. Blue light causes photoisomerization back to trans and the accessibility change is reversed. Uptake of a proton through EC completes proton transport from the outside to the inside. Depending on relative concentrations of chloride and azide, both modes of ion translocation operate in parallel and as alternatives during individual cycles of a molecule. Future experimentation will have to fill in the many details of this molecular model of ion transport.     

蛋白质光学信息记录材料细菌视紫红质

光敏蛋白质细菌视紫红质(bR)存在于嗜盐菌紫膜中。bR分子吸收光子后,视黄醛构型发生变化,产生一系列中间态并经历一个独特的光循环过程,同时完成质子泵功能。bR具有高的空间分辨率、高的光灵敏度、高的循环次数及良好的热稳定性和非线性光学特性,是一种性能优良的生物光学材料,可广泛应用于空间光调制器、投影显示、全息记录、模式识别、光学信息存储等方面,在分子电子学、生物计算机等领域具有诱人的应用前景。     

Application of FTIR Spectroscopy to the Structural Study on the Function of Bacteriorhodopsin

Difference FTIR spectroscopy of the photointermediates of bacteriorhodopsin is informative for changes in H-bonding, the protonation states, and the bond orientation of functional residues such as C=O, N-H, and O-H of the chromophore, protein residues, peptide bonds, and internal water molecules. The vibrational bands of the chromophore are found at frequencies similar to those observed by resonance Raman spectra. Moreover, FTIR gives clear results on the N— in-plane bending vibration and C₁₄–C₁₅ stretching vibrational modes, both of which are useful for the analysis of the structure of the chromophore. The protonation states and H-bonding changes of intramembrane protonated aspartic acid residues can be revealed only by FTIR spectroscopy, in combination with proper isotope labeling and site-directed mutagenesis. The results with Asp-96 and Asp-85 in the L, M, and N photointermediates were especially useful for understanding the proton transfer mechanism. Besides amino acid groups in the protein, peptide C=O vibrations were assigned to a specific bond by the use of site-directed isotope labeling. Water molecules which undergo structural changes upon photoreaction were attributed specifically to those interacting with particular protein residues by mutational studies. On the basis of these FTIR studies, the role of the L intermediate is emphasized as the key intermediate that creates the conditions for the proton transfer reaction from the Schiff base to Asp-85 and subsequent proton uptake reactions in the cytoplasmic domain.     

CeO2纳米晶修饰的细菌视紫红质薄膜M态寿命的研究

紫膜中的蛋白质细菌视紫红质（bR)具有独特的光循环和光致变色特性，在分子电子学和生物电子技术领域具有广泛的潜在应用价值。本文采用稀土纳米晶CeO2对细菌视紫红质－聚乙烯醇（bR-PVA)薄膜进行化学修饰。研究了纳米晶对bR光循环中的重要中间态M态的寿命的影响。发现上述稀土纳米晶可延长M态寿命，且晶粒尺寸越小对M态寿命的影响越大。纳米晶周围的羟基有助于阻碍席夫碱获得质子，从而使M态寿命延长。     

Application of Raman Spectroscopy to Retinal Proteins

Raman spectroscopic studies on photoreactive retinal proteins are comprehensively described, including the basic physics of Raman scattering and illustrative examples of the types of information on the structure and function of the retinal chromophore and its environment which can be obtained from the vibrational Raman spectra. In addition, practical advice and recipes are given which should enable the reader to plan and eventually perform a Raman experiment in a photolabile retinal protein. A dominant role is played by the resonance Raman (RR) experiment with visible laser excitation which selectively probes the retinal chromophore. Much discussion is devoted to bacteriorhodopsin (bR) and its photocycle as a paradigm for a light-induced reaction of a retinal protein. Various time-resolved techniques are described to study the temporal evolution of the bR chromophore by probing RR spectra of intermediate states. Vibrational Raman spectra are interpreted in terms of structure and structural changes of the chromophore. RR spectroscopic studies on halorhodopsin, sensory rhodopsin, and visual pigments are reported, as well as on modified proteins in which retinal analogues are incorporated, and on site-specific mutants. Results of ultraviolet RR experiments which selectively probe the aromatic side chains in the protein backbone are reported. In addition, a promising new technique of near-infrared Raman excitation is discussed. Finally, application of coherent anti-Stokes Raman spectroscopy (CARS) to retinal proteins is reported.     

pH-Dependent Protonation of Surface Carboxylate Groups in PsbO Enables Local Buffering and Triggers Structural Changes

Photosystem II (PSII) catalyzes the splitting of water, releasing protons and dioxygen. Its highly conserved subunit PsbO extends from the oxygen-evolving center (OEC) into the thylakoid lumen and stabilizes the catalytic Mn₄CaO₅ cluster. The high degree of conservation of accessible negatively charged surface residues in PsbO suggests additional functions, as local pH buffer or by affecting the flow of protons. For this discussion, we provide an experimental basis, through the determination of pK_a values of water-accessible aspartate and glutamate side-chain carboxylate groups by means of NMR. Their distribution is strikingly uneven, with high pK_a values around 4.9 clustered on the luminal PsbO side and values below 3.5 on the side facing PSII. pH-dependent changes in backbone chemical shifts in the area of the lumen-exposed loops are observed, indicating conformational changes. In conclusion, we present a site-specific analysis of carboxylate group proton affinities in PsbO, providing a basis for further understanding of proton transport in photosynthesis.     

Dynamics of the KB Proton Pathway in Cytochrome ba3 from Thermus thermophilus

The ba₃ cytochrome c oxidase from Thermus thermophilus is a B-type oxygen-reducing heme-copper oxidase and a proton pump. It uses only one proton pathway for transfer of protons to the catalytic site, the K^B pathway. It was previously shown that the ba₃ oxidase has an overall similar reaction sequence to that in mitochondrial-like A-type oxidases. However, the timing of loading the pump site, and formation and decay of catalytic intermediates is different in the two types of oxidases. In the present study, we have investigated variants in which two amino acids of the K^B proton pathway leading to the catalytic site were exchanged; Tyr-248 (located ∼23 Å below the active site towards the cytoplasm) in subunit I (Y248T) and Glu-15 (∼26 Å below the active site, ∼16 Å from Tyr-248) in subunit II (E15^IIQ). Even though the overall catalytic turnover in these two variants is similar and very low (<1 % of wildtype), the substitutions had distinctly different effects on the kinetics of proton transfer to the catalytic site. The results indicate that the Glu-15^II is the only essentially crucial residue of the K^B pathway, but that the Tyr-248 also plays a distinct role in defining an internal proton donor and controlling the dynamics of proton transfer to the pump site and the catalytic site.     

Determination of the pKa of the Aci-Nitro Intermediate in o-Nitrobenzyl Systems

The structural volume changes following photoexcitation of o-nitrobenzyl systems are used to estimate the excited state pK_a of the aci-nitro intermediate in aqueous solutions. The rather large contractions induced in solution by UV excitation are due to the rapid deprotonation of the short-lived aci-nitro intermediate, which leads to the formation of two charged species. The magnitude of the measured contraction as a function of the pH in acidic solutions follows a sigmoidal curve, from which it is possible to extract the pK_a of the aci-nitro intermediate. This method is generally applicable to short-lived intermediates with stronger acid character than the parent compound, provided they undergo irreversible chemical transformations to a product that cannot rebind the photodetached proton. The reaction volume for water formation from its ionic constituents at basic pH allows the determination of the deprotonation quantum yields.     

The Deuterium Isotope Effect as a Tool to Investigate Enzyme Catalysis: Proton-Transfer Control Mechanisms in Cytochrome c Oxidase

Cytochrome c oxidase is a membrane-bound redox-driven proton pump. The coupling of the exergonic electron-transfer reactions from cytochrome c to oxygen to proton translocation across the membrane requires control of internal electron- and proton-transfer reactions. In this work, we focus on the kinetics of electron and proton transfer during those reaction steps that are coupled to proton pumping in cytochrome c oxidase. The results show that during the first pumping step (peroxy → oxoferryl transition), proton transfer regulates intramolecular electron transfer. The proton transfer takes place in two steps: (1) Internal proton transfer from a protonatable group, proposed to be Glu(I-286), in the so-called D-pathway, to an oxygen intermediate at the binuclear center (τ ≅ 100 μs); (2) Rapid re-protonation of Glu(I-286) from the bulk solution (τ < 100 μs). Only after proton uptake from solution the last (fourth) electron is transferred “one step closer” towards the binuclear center (from Cu_A to heme a). During the second proton-pumping step (oxoferryl → oxidized), this electron is transferred to the binuclear center, linked to the uptake of a proton through the D-pathway. The electron-transfer rate displays a kinetic-isotope effect (k_H/k_D) of 6 ± 1 (in a pH range in which the pH dependence of the rate is small), which indicates that the electron transfer is rate-limited by the proton transfer. The entry into the D-pathway (around Asp(I-132)) is composed of a cluster of negatively-charged amino acid residues together with a number of histidines, forming a so-called proton-collecting antenna designed to allow rapid protonation of groups within the proton-transfer pathway.     

Experimental pKa Value Determination of All Ionizable Groups of a Hyperstable Protein

Electrostatic interactions significantly contribute to the stability and function of proteins. The stabilizing or destabilizing effect of local charge is reflected in the perturbation of the pK_a value of an ionizable group from the intrinsic pK_a value. Herein, the charge network of a hyperstable dimeric protein (ribbon–helix–helix (rhh) protein from plasmid pRN1 from Sulfolobus islandicus) is studied through experimental determination of the pK_a values of all ionizable groups. Transitions were monitored by multiple NMR signals per ionizable group between pH 0 and 12.5, prior to a global analysis, which accounted for the effects of neighboring residues. It is found that for several residues involved in salt bridges (four Asp and one Lys) the pK_a values are shifted in favor of the charged state. Furthermore, the pK_a values of residues C40 and Y47, both located in the hydrophobic dimer interface, are shifted beyond 13.7. The necessary energy for such a shift is about two-thirds of the total stability of the protein, which confirms the importance of the hydrophobic core to the overall stability of the rhh protein.     

Ionic end‐capping of (semi)telechelic polymers by mesogens

Two methods have been used to end‐cap linear polymer chains at one end or at both ends by a mesogen through ionic bonding. These polymers are designated as liquid‐crystalline halato(semi)telechelic polymers (LC H(S)TPs). The first method relies on the ion exchange reaction between the metal counterion of halato(semi)telechelic polymers and an ionic mesogen. The second method is based on the proton transfer from a sulfonic or carboxylic acid end‐group to a tertiary aliphatic amine, this approach being controlled by the relative pK_a values of the acidic and basic groups. If the pK_a difference is not large enough, strong hydrogen‐bonding is observed by Fourier‐transform infrared (FTIR) spectroscopy rather than proton transfer. The resulting materials have been characterized by differential scanning calorimetry (DSC), polarized optical microscopy (POM) and small‐angle X‐ray scattering (SAXS). © 2000 Society of Chemical Industry     

The Effect of Different Metal Cation Binding on the Proton Pumping in Bacteriorhodopsin

The first section of this paper is a detailed summary of studies made by us and others on metal cations binding to deionized bacteriorhodopsin (dIbR) and its variants. Our studies include the luminescence experiments of Eu³⁺ binding to dIbR and potentiometric studies of Ca²⁺ binding to dIbR, to deionized bR mutants, to bacterioopsin, and to dIbR with its C-terminus removed. The results suggest the presence of two classes of binding sites, one class has two high-affinity constants, and one has one low-affinity constant. For Ca²⁺ binding, there is one metal cation in each of the two high-affinity sites which are coupled to the charged aspartates 85 and 212 (known to be in the retinal cavity) but not coupled to each other. The low-affinity class can accommodate 0–6 Ca²⁺ ions and most of them are bound to the surface. Mg²⁺ has a slightly smaller value for its binding constant to the highest-affinity site. Thus, one expects more Ca²⁺ than Mg²⁺ bound to the two high-affinity sites. In the second section, we summarize our recent study on the effect of metal cation charge density (Ca²⁺, Mg²⁺, Eu³⁺, Tb³⁺, Ho³⁺, Dy³⁺) on the kinetics of both Schiff base deprotonation and proton transport to the extracellular surface. For all metal cations, the apparent rate constant of the slow components of the deprotonation process is the same as that for the transport process at 22 °C. The temperature studies, however, show this apparent equality to be fortuitous and to result from cancellation of the contribution of the energy and entropy of activation. Thus, while the entropy of activation is positive for the deprotonation process, it is negative for the proton transport process. These kinetic parameters depend weakly on the charge density, but in an opposite sense for the two processes. These results suggest that the deprotonation is not the rate-limiting step for the proton transport process. A possible mechanism is proposed in which a hydrated metal cation is used to induce the deprotonation of the protonated Schiff base and to dissociate one of its H₂O molecules to donate the proton in the L → M process.     

Cucurbit[n]uril Host-Guest Complexes of Acids,Photoacids, and Super Photoacids

The supramolecular chemistry of host-guest complexes of cucurbit[n]urils (CB[n]) with acidic guests in the ground (HG⁺) and excited states (HG⁺*) are reviewed. The effects of CB[n] complexation on the guests’ pK_a and/or pK_a* values are related to relative binding constants and host-guest structures of the acid form of the guest and its conjugate base. Included are carbon acids, guests of biological and medicinal interest, dyes and related polyaromatic guests, and other organic and organometallic guests. The applications of the pK_a shifts to the solubility, stability, and bioavailabilty of drug molecules, the stability and enhanced spectral properties of dyes, and in pH-induced self-sorting, micelle formation, host-guest shuttling, and controlled guest release, are discussed.     

Synthesis and Characterization of a Novel Series of Amphiphilic Mercapto-1,2,4-Triazole Schiff Base Ligands: Investigation of their Behavior in Hydro-Organic Solutions

A novel series of four Schiff base amphiphiles derived from 3-mercapto-1,2,4-triazole and different alkyl chains were successfully prepared by a new synthetic three-step method. The chemical structures of the different ligands were characterized by elemental analysis, FT-IR spectroscopy, ¹H-NMR and ¹³C-NMR spectra. The effect of the chain length on the solution behavior of the amphiphilic ligands were studied, in both homogeneous and heterogeneous systems, using pH-metric and spectrophotometric methods. Based on the electronic spectroscopy data, some parameters governing their surfactant properties, such as the critical micelle concentration (CMC), the micellization free energy (ΔG _mic) and the hydrophilic-lipophilic balance (HLB) were evaluated in chloroform and discussed. The behavior of the four Schiff bases in the heterogeneous chloroform-water mixture was then explored through the establishment of their acidity (pK_A) and distribution (Log K _d) constants in 1 M chloride medium and the acidity constants in aqueous medium (pK_a) were deduced. Results showed that an increase of the alkyl chain decreases the distribution of the ligands and increases their acidity. The extractive performance of the Schiff base amphiphiles were investigated towards Ni(II) from a chloride medium at 30 °C. The analysis of extraction data revealed that the synthesized Schiff bases exhibit a better and faster extractability than many ligands reported in the literature.