Trapping and spectroscopic identification of the photointermediates of bacteriorhodopsin at low temperatures

Light-driven transmembrane proton pumping by bacteriorhodopsin occurs in the photochemical cycle, which includes a number of spectroscopically identifiable intermediates. The development of methods to crystallize bacteriorhodopsin have allowed it to be studied with high-resolution X-ray diffraction, opening the possibility to advance substantially our knowledge of the structure and mechanism of this light-driven proton pump. A key step is to obtain the structures of the intermediate states formed during the photocycle of bacteriorhodopsin. One difficulty in these studies is how to trap selectively the intermediates at low temperatures and determine quantitatively their amounts in a photosteady state. In this paper we review the procedures for trapping the K, L, M and N intermediates of the bacteriorhodopsin photocycle and describe the difference absorption spectra accompanying the transformation of the all-trans-bacteriorhodopsin into each intermediate. This provides the means for quantitative analysis of the light-induced mixtures of different intermediates produced by illumination of the pigment at low temperatures.     

Charge Movements in the 13-c/s Photocycles of the Bacteriorhodopsin Mutants R82K and R82Q*

We have examined light-induced currents in oriented membranes of the bacteriorhodopsin mutants R82K and R82Q. Our results suggest that two photocurrent components found in R82K, with 30 and 300 us lifetimes, are due to the photocycle of the 13-cis rather than the all-trans form of the pigment. We investigated the pH dependence of these components and their correspondence to absorbance changes at 660 nm characteristic of pho-tointermediates of the 13-cis cycle. The presence of a D₂O effect suggests that the charge motions producing these photocurrents are related to proton or protonated amino acid movement within the molecule. The current amplitudes depend on the protonation states of at least two residues, D85 and (probably) E204. In R82Q, a 10 pis photocurrent is observed that also depends on the protonation state of D85 and is similar to the 30 us current in R82K. We attempt to explain these currents in terms of a model for interacting residues in the extracellular half of the bacteriorhodopsin channel.     

THE WHITE MEMBRANE OF CRYSTALLINE BACTERIOOPSIN IN HALOBACTERIUM HALOBIUM STRAIN R1mW AND ITS CONVERSION INTO PURPLE MEMBRANE BY EXOGENOUS RETINAL

Abstract— A new strain isolated from Halobacterium halobium designated R₁mW, contained negligible amounts of isoprenoid pigments, had a yellowish white color due to respiratory pigments and showed no proton movement in response to light. However, addition of all-trans-retinal converted R₁mW into purple cells. Formation of both halorhodopsin and bacteriorhodopsin was indicated by induction of light-dependent proton uptake and release, respectively. Both haloopsin and bacterioopsin were thus postulated to be present in R₁mW. Electron micrographs of freeze-fractured cytoplasmic membranes revealed patches in a hexagonal array of trimeric particles, comparable to the purple membrane structure. These white membrane patches were isolated by procedures similar to those for the purple membrane. The white membrane's buoyant density was about 1.18 g/m/, and its main component migrated on sodium dodecylsulfate polyacrylamide gels at the same rate as bacteriorhodopsin. The white membrane showed only a small absorption peak at ～410nm due to contaminating respiratory pigments and a strong absorption at around 275 nm and shorter wavelengths. The white membrane was thus considered to be mainly composed of bacterioopsin, which was readily converted into bacteriorhodopsin by an addition of all-trans-retinal. The absorption and CD spectra of the white membrane were measured before and after addition of retinal. The molar extinction coefficient of dark-adapted bacteriorhodopsin formed was determined to be 53000M^?1 cm^?1 at 560 nm from retinal binding studies. The CD spectrum of the white membrane was negligible in the visible region but showed several bands assigned to aromatic and backbone structures in the UV region. Retinal addition caused considerable changes in the spectrum, yielding the CD spectrum of crystalline purple membrane bacteriorhodopsin. The white membrane thus appears to be a preparation suitable for structure-function studies of bacteriorhodopsin.     

Caged mitochondrial uncouplers that are released in response to hydrogen peroxide

Caged versions of the most common mitochondrial uncouplers (proton translocators) have been prepared that sense the reactive oxygen species (ROS) hydrogen peroxide to release the uncouplers 2,4-dinitrophenol (DNP) and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) from caged states with second order rate constants of 10 (±0.8) M⁻¹ s⁻¹ and 64.8 (±0.6) M⁻¹ s⁻¹, respectively. The trigger mechanism involves conversion of an arylboronate into a phenol followed by fragmentation. Hydrogen peroxide-activated uncouplers may be useful for studying the biological process of ageing.     

Proteorhodopsin Photocycle Kinetics Between pH 5 and pH 9

The retinal protein proteorhodopsin is a homolog of the well‐characterized light‐driven proton pump bacteriorhodopsin. Basic mechanisms of proton transport seem to be conserved, but there are noticeable differences in the pH ranges of proton transport. Proton transport and protonation state of a carboxylic acid side chain, the primary proton acceptor, are correlated. In case of proteorhodopsin, the pK_a of the primary proton acceptor Asp‐97 (pK_a ≈ 7.5) is unexpectedly close to environmental pH (pH ≈ 8). A significant fraction of proteorhodopsin is possibly inactive at natural pH, in contrast to bacteriorhodopsin. We investigated photoinduced kinetics of proteorhodopsin between pH 5 and pH 9 by time resolved UV/vis absorption spectroscopy. Kinetics is inhomogeneous within that pH region and can be considered as a superposition of two fractions. These fractions are correlated with the Asp‐97 titration curve. Beside Asp‐97, protonation equilibria of other groups influence kinetics, but the observations do not point toward major differences of primary proton acceptor function in proteorhodopsin and bacteriorhodopsin. The pK_a of proteorhodopsin and some of its variants is suspected to be an example of molecular adaptation to the physiology of the original organisms.     

THE EFFECT OF CROSS-LINKING ON PHOTOCYCLING ACTIVITY OF BACTERIORHODOPSIN

Abstract— Purple membrane preparations from Halobacterium halobium were chemically modified with imidoesters. Dimethyl adipimidate (8.3 Å chain length) amidinates about five of the six free lysine residues whereas dimethyl suberimidate (11.3 Å) under the same conditions reacts with only 2–3 residues. Gel electrophoresis showed that the shorter chain length imidoesters were less effective than dimethyl suberimidate in oligomer formation. However, dimethyl adipimidate resulted in a more marked inhibition of the photoreaction activity. Monofunctional imidates, methyl acetimidate and methyl butyrimi-date, at comparable degrees of amidination, did not appreciably affect activity indicating that the presence of bulky groups on the exposed lysine residues does not cause the effects observed. Hence, the introduction of molecular mobility constraints by intramolecular cross-linking slows photocycling, and, therefore, inhibits proton pumping activity of bacteriorhodopsin. This indicates that conforma-tional changes of the protein moiety of bacteriorhodopsin occur during photocycling activity.     

A PROTON RELEASE SITE ON THE C-TERMINAL SIDE OF BACTERIORHODOPSIN

We have studied the pH dependence of the light-induced proton release and uptake by bacteriorhodopsin. The quantum efficiency of proton release in cell envelopes and proton uptake in phospholipid vesicles is high in the low pH range and begins to decline between pH 6 and 7 in cell envelopes and between pH 7–8 in phospholipid vesicles. In the cell envelope vesicles the proton release increases again above pH 8–8.5; in phospholipid vesicles a proton release is observed before proton uptake at pHs greater than 9. We suggest that the light-induced proton release observed at high pHs are due to protons released and rebound on the carboxyl terminal side of bacteriorhodopsin.     

Bacteriorhodopsin as an Example of a Light-Driven Proton Pump

Apart from the long known visual pigments, another retinal protein complex exists in nature, viz. bacteriorhodopsin from halobacteria. In contrast to the visual pigments such as the rhodopsins, which act as light sensors in the eye, bacteriorhodopsin actually transforms light energy. This energy conversion is connected with the asymmetric incorporation of bacteriorhodopsin in the lattice structure of the purple membrane which forms patches on the cell surface of halobacteria. Alongside the chlorophyll system, the purple membrane system represents the second light energy conversion principle to be discovered in living nature. Bacteriorhodopsin acts as a light-driven proton pump or as the main component of such a pump system. Absorption of light triggers off a cycle of reactions coupled with the spatially oriented uptake and release of a proton. In the intact cell an electrochemical gradient is thus built up across the cell membrane of the bacterium in which part of the absorbed light energy is stored and which is not dependent upon redox processes as in the case of respiration or photosynthesis. This electrochemical gradient can supply the energy required for ATP synthesis in the cell; a reversible proton-translocating ATPase serves as catalyst system.     

ISOMER COMPOSITION and SPECTRA OF THE DARK and LIGHT ADAPTED FORMS OF ARTIFICIAL BACTERIORHODOPSINS*

Abstract– The isomer composition and spectral properties of 15 artificial bacteriorhodopsin (bR) pigments, based on a series of retinal analogs with polyene residue modified below C₉ are determined for both dark-adapted (DA) and light-adapted (LA) forms. Similarly to native bR, in all cases only two isomers, C₁₃=C₁₄cis (13-cis) and M-trans, are observed. However, the artificial DA pigments have a lower 13-d.s content than native DA bR (? 66%) while the corresponding LA pigments have a much higher 13-cis content (11-69%) than native LA bR (<2%). Thus, in variance with the native pigment, in all of the artificial systems light also induced the reversed all-trans13-cis process. The data are accounted for in terms of specific steric interactions between the polyene and the protein binding site which allow a (C₁₅-anti)(C_ls-syn) isomerization during the photocycle of the artificial pigments, but not in the case of native bR. This accounts for the high proton pumping efficiency of the natural pigment. The nature of a highly red shifted light-adapted form of two of the artificial pigments is investigated and discussed. It is also shown that, in variance with native bR, several artificial pigments exhibit identical absorption spectra for their 13-cis and all-trans isomers. It is concluded that the spectral data for the above species of artificial pigments do not lead to a clear molecular model for the origin of the spectral shift between 13-cis and all-trans bR.     

Membrane potential is involved in regulation of photosynthetic reactions in the marine diatom Thalassiosira weissflogii

High-intensity Chl fluorescence transients (OJIP transients) and light-induced kinetics of the delayed light emission were measured in diatom microalga Thalassiosira weissflogii in the presence of various uncouplers and photosynthetic inhibitors. The I step in the OJIP transients in T. weissflogii was essentially reduced or completely absent but was restored in the presence of uncouplers valinomycin, FCCP, and nigericin. Moreover, valinomycin enhanced ΔpH-dependent non-photochemical fluorescence quenching following the OJIP rise. In the presence of valinomycin, the transthylakoid membrane potential was significantly inhibited as evaluated by measurements of the delayed light emission. The results suggest a membrane potential control of the fluorescence yield in T. weissflogii. Possible mechanisms underlying the observed effects of uncouplers are discussed.     

PHOTOTACTIC ORIENTATION BY THE CILIATE, STENTOR COERULEUS

Stentor coeruleus exhibits negative phototaxis to visible light, in addition to a step-up photophobic response. The negative phototaxis was established by demonstrating the swimming of Stentor toward a focused beam away from the light source. The action spectrum showed a maximum at 610–620 nm and is essentially identical to that of the step-up photophobic response. Proton uncouplers such as micromolar concentrations of FCCP and TPMP⁺ inhibited the negative phototaxis.     

The mechanism of bacteriorhodopsin functioning. II. Ejection and injection of proton

Transmission of vibrational excitation energy conserved in cis-conformation of retinal to the outlet proton channel is considered from the perspective of quantum theory. A distribution of vibrational excitations in the channel is found; it allowed to calculate the magnitude of the directed drift proton current. The differences between velocities of proton movement in active and passive channels are considered. A transition of retinal from cis- to all -trans- conformation with the subsequent capture of proton by Schiff base out of the inlet channel is described. The lack of proton in this channel, i.e., in the H-bonded chain, is eliminated at the expense of the capture of a proton out of the cytoplasmic water enviroment. The correspondence between theoretically established states and spectroscopically identified forms of bacteriorhodopsin (inter-mediates L, M, N, and O) is proposed.     

A ROLE FOR MENADIONE IN THE PURPLE MEMBRANE PROTON PUMP?

Abstract— It has been assumed that proton pumps such as purple membrane lack redox loops. However, purple membrane does contain an electron carrier. Kates et al. (Meth. Enzymol. 88,98–1 111, 1982) reported the presence of 1 mole of vitaminMK–8 to 6 mol of bacteriorhodopsin among the nonpolar lipids. Is this quinone functionally important in the proton pump mechanism? Proton pumping rates were measured with lipid-free bacteriorhodopsin reconstituted in vesicles to which varying amounts of vitamin K₁ were added. With soybean lipids, in the presence of tetraphenyl boron, the pump quantum yield was 0.04H⁺/photon. This result was independent of the amount of vitamin K, added over a range of 0 to a 100-fold mole ratio to bacteriorhodopsin. A similar result was obtained with H. halobium lipids. The pump quantum yield in vesicles is much less than reported for membrane sheets and whole cells. The results support the conclusion that a vitamin K Q-cycle is not involved in the purple membrane proton pump.     

LIGHT-INDUCED PROTON DISSOCIATION AND ASSOCIATION IN BACTERIORHODOPSIN

Abstract— Light-induced proton release and uptake by acetylated and unmodified bacteriorhodopsin were measured. Bacteriorhodopsin, when illuminated, shows a net proton release at neutral and alkaline pH's, but in acidic pH, it shows an uptake of protons. In the presence of high concentrations of guanidine hydrochloride, light caused only proton release even in acidic pH and the maximum extent of the release was one proton per bacteriorhodopsin molecule around pH 8.
Acetylation of bacteriorhodopsin caused no alteration in the absorption spectrum of purple complex (bR₅₇₀) and M₄₁₂-intermediate, but decreased the decay rate of the M₄₁₂-intermediate. Light-induced release of protons was not observed even in neutral pH values, and only the proton uptake was noticed by acetylated purple membrane fragments. In high concentrations of guanidine hydrochloride, no proton uptake or release by illumination was observed. Vesicles were reconstituted from acetylated purple membrane. These vesicles had almost no ability for light-induced proton transport. The role of amino group(s) in light-induced proton release and transport through the purple membrane is discussed.     

Controlling Light-Induced Proton Transfer from the GFP Chromophore

Green Fluorescent Protein (GFP) is known to undergo excited-state proton transfer (ESPT). Formation of a short H-bond favors ultrafast ESPT in GFP-like proteins, such as the GFP S65T/H148D mutant, but the detailed mechanism and its quantum nature remain to be resolved. Here we study in vacuo, light-induced proton transfer from the GFP chromophore in hydrogen-bonded complexes with two anionic proton acceptors, I⁻ and deprotonated trichloroacetic acid (TCA⁻). We address the role of the strong H-bond and the quantum mechanical proton-density distribution in the excited state, which determines the proton-transfer probability. Our study shows that chemical modifications to the molecular network drastically change the proton-transfer probability and it can become strongly wavelength dependent. The proton-transfer branching ratio is found to be 60 % for the TCA complex and 10 % for the iodide complex, being highly dependent on the photon energy in the latter case. Using high-level ab initio calculations, we show that light-induced proton transfer takes place in S₁, revealing intrinsic photoacid properties of the isolated GFP chromophore in strongly bound H-bonded complexes. ESPT is found to be very sensitive to the topography of the highly anharmonic potential in S₁, depending on the quantum-density distribution upon vibrational excitation. We also show that the S₁ potential-energy surface, and hence excited-state proton transfer, can be controlled by altering the chromophore microenvironment.     

PHOTOCHROMIC SYNERGISM OF BACTERIORHODOPSIN- and HALORHODOPSIN-MEDIATED PHOTOPHOSPHORYLATION IN Halobacterium halobium*

Quantitative action spectroscopy was performed in Halobacterium halobium. using four suited pigment mutants, namely the bacteriorhodopsin and halorhodopsin positive mutant strain M-l (BR⁺, HR⁺), the bacteriorhodopsin positive but halorhodopsin negative strain M-18 (BR⁺, HR^-), the bacteriorhodopsin negative but halorhodopsin positive strain L-33 (BR^-, HR⁺), and the bacteriorhodopsin and halorhodopsin negative strain L-07 (BR^-, HR⁺). The approached questions were: First, photoenergetic synergism of halorhodopsin and bacteriorhodopsin in intact cells; second, photochromism and cellular function of the blue light-absorbing intermediates, i.e. M-412 and HR-410 in bacteriorhodopsin and in halorhodopsin, respectively. Dark-adapted cells of mutant strain M-l show wavelength-dependency of quantum yield of photo-phosphorylation, φ_ATP. An 1.4-fold enhancement was found at 575 nm wavelength where the long wavelength absorbance bands of bacteriorhodopsin and halorhodopsin intersect. The enhancement vanished after a 30 min pulse of orange light (600 Wm^-2 bandpass from 495 to 750 nm), but was restored after a 30 min pulse of blue light (100 Wm^-2 bandpass from 325 to 480 nm). Photoreversibility of this enhancement probably reflects phototransformation of halorhodopsin from its ground state into its inactive intermediate, HR-410, and vice versa. The halorhodopsin-mediated enhancement with maximum quantum yield of photophosphorylation, φ_ATP= 0.06, i.e. a quantum requirement of = 17 photons/ATP, is partly substituted by a rise in phosphate potential and explained in terms of a voltage-regulated gating effect on the H⁺-driven ATP-synthase, superimposed on the chemiosmotic mechanism of energy coupling. The blue-absorbing photochromic intermediate, M-412 of bacteriorhodopsin, dissipates light energy upon photoexcitation that is reflected by a spectral decline in quantum yield of photophosphorylation to a minimum value of = 0.01 at 415 nm, i.e. a quantum requirement of = 100 photons/ATP.     

Polyampholytic Graft Copolymers as Matrix for TiO2/Eosin Y/[Mo3S13]2− Hybrid Materials and Light-Driven Catalysis

An effective strategy to enhance the performance of inorganic semiconductors is moving towards organic-inorganic hybrid materials. Here, we report the design of core–shell hybrid materials based on a TiO₂ core functionalized with a polyampholytic (poly(dehydroalanine)-graft-(n-propyl phosphonic acid acrylamide) shell (PDha-g-PAA@TiO₂). The PDha-g-PAA shell facilitates the efficient immobilization of the photosensitizer Eosin Y (EY) and enables electronic interactions between EY and the TiO₂ core. This resulted in high visible-light-driven H₂ generation. The enhanced light-driven catalytic activity is attributed to the unique core–shell design with the graft copolymer acting as bridge and facilitating electron and proton transfer, thereby also preventing the degradation of EY. Further catalytic enhancement of PDha-g-PAA@TiO₂ was possible by introducing [Mo₃S₁₃]²⁻ cluster anions as hydrogen-evolution cocatalyst. This novel design approach is an example for a multi-component system in which reactivity can in future be independently tuned by selection of the desired molecular or polymeric species.     

Spectral characteristics of the photoreceptor pigment of phototaxis in Dictyostelium discoideum

Abstract —A photoresponsive pigment, described previously as the photoreceptor pigment for phototaxis in Dictyostelium discoideum, was examined by low-temperature spectroscopy. The absorption spectrum of the purified pigment frozen in darkness indicates the reduced form of a high-spin heme protein, and the spectrum of the pigment frozen during irradiation (to freeze in the photoproduct) indicates the oxidized form of the heme protein. The light-induced absorbance changes measured at room temperature also indicate photooxidation of a heme pigment. The action spectrum for the light-induced absorbance change shows a primary peak at about 430 nm and a broad, less active maximum in the 550–580 nm region. The absorption spectrum of the reduced pigment and the action spectrum for its photooxidation are both similar to the action spectrum for phototaxis of the pseudoplasmodia of D. discoideum.