Bacteriorhodopsin (BR570) bathochromic band shift in an external electric field

In dry films of bacteriorhodopsin-containing purple membranes from Halobacterium halobium the external electric field (10⁴–10⁵ V · cm^?1) induces the appearance of a product spectrally close to the initial intermediate of bacteriorhodopsin (BR) photochromic cycle (batho form, K). This result and also preliminary data of the electret-thermal analysis of the preparations suggest that the dielectric polarization in chromophore-protein-lipid complexes might be an essential step of the primary stabilization of light energy in photo-bioenergetic processes.     

Electric field induced conformational changes of bacteriorhodopsin in purple membrane films

Electric field induced conformational changes of bacteriorhodopsin were studied in six types of dried film (randomly and electrically oriented membranes of purple as well as cation-depleted blue bacteriorhodopsin) by measuring the frequency dependence of the optical absorbance change and the dielectric dispersion and absorption. For the purple bacteriorhodopsin the optical absorbance change induced by alternating rectangular electric fields of ±300 kV/cm altered the sign twice in the frequency range from 0.001 Hz to 100 kHz (around 0.03 Hz and 100 kHz), indicating that the electric field induced conformational change in these samples consists of, at least, three steps. Similarly, it was found for the blue bacteriorhodopsin that at least two steps are involved. In accord with optical measurements, the dielectric behaviour due to alternating sinusoidal electric fields of±6kV/cm in the frequency range from 10 Hz to 10 MHz showed two broad dispersion/absorption regions, one below 1 kHz and the other around 10–100 kHz. This suggests that the conformational change of bacteriorhodopsin is also reflected by its dielectrical properties and that it is partially induced at 6 kV/cm. Including previous results obtained by analysis of the action of DC fields on purple membrane films, a model for a field-induced cyclic reaction for purple as well as blue bacteriorhodopsin is proposed. In addition it was found that there are electrical interactions among purple membrane fragments in dried films.     

Electric field induced conformational changes of bacteriorhodopsin in purple membrane films

Electric field-induced absorption changes of bacteriorhodopsin were studied with different samples of purple membranes which were prepared as randomly oriented and electrically oriented films of purple as well as cation-depleted blue bacteriorhodopsin. The absorption changes were proportional to the square of the field strength up to 300 kV/cm. The electric field from the intracellular side to the extracellular side of the purple bacteriorhodopsin induces a spectrum change, resulting in a spectrum similar to that of the cation-depleted blue bacteriorhodopsin. When the field was removed, the purple state was regenerated. The blue state was mainly affected by an electric field in the opposite direction, suggesting a reversible interaction with the Schiff's base bond of the retinal. Since the field-induced reaction of bacteriorhodopsin was observed in the presence of a concomitant steady ion flux, it is assumed that the generation of a local diffusion potential may play an important role in these spectral reactions. Although the fragments were fixed in the dried film, electric dichroism was observed. The dichroic contribution of the total absorbance change was about 15%. The angular displacement of the retinal transition moment was calculated to be 1.5° toward the membrane normal.     

Effects of electric field on the photocycle of bacteriorhodopsin

The photoconversion of bacteriorhodopsin and the effects of an applied electric field (5 · 10⁷ V · m^?1) were studied in dry films of purple membranes from Halobacterium halobium. The electric field was found to cause at least two different effects: (1) it blocks in part the formation of the batho-bacteriorhodopsin (K), most probably due to electrically-induced dark transition of some bacteriorhodopsin molecules into the photochemically inactive form; (2) it decreases the rate of the intermediate M decay, the rise time of the M formation being unaffected by electric field. The observed phenomena may suggest a feedback control mechanism for the regulation of the bacteriorhodopsin photocycle in purple membranes.     

Effect of water on the structure of bacteriorhodopsin and photochemical processes in purple membranes

Visible and infrared spectra of bacteriorhodopsin films under different humidities at room and low temperatures are investigated. On dehydration of purple membranes at room temperatures an additional chromophore state with the absorption band at 506 nm is revealed. The photocycle of purple membranes in the dry state is devoid of the 550 nm intermediate and involves the long-lived intermediate at 412 nm. As water is removed, the 550 nm intermediate becomes undetectable. The analysis of the infrared spectra shows that dehydration does not affect the ordering of the main network of the interpeptide hydrogen bonds which stabilizes the -helical conformation (slightly distorted in the initial humid dark- and light-adapted state); light adaptation (cis-trans isomerization) of bacteriorhodopsin results in an increase of sorbed water in purple membranes. Dehydration of purple membranes decreases the reaction rate of cis-trans isomerization.     

Characterization of sodium and proton flows in sub-bacterial particles of Halobacterium halobium in terms of nonequilibrium thermodynamics

A thermodynamic characterization of the Na⁺-H⁺ exchange system in Halobacterium halobium was carried out by evaluating the relevant phenomenological parameters derived from potential-jump measurements. The experiments were performed with sub-bacterial particles devoid of the purple membrane, in 1 M NaCl, 2 M KCl, and at pH 6.5–7.0. Jumps in either pH or pNa were brought about in the external medium, at zero electric potential difference across the membrane, and the resulting relaxation kinetics of protons and sodium flows were measured. It was found that the relaxation kinetics of the proton flow caused by a pH-jump follow a single exponential decay, and that the relaxation kinetics of both the proton and the sodium flows caused by a pNa-jump also follow single exponential decay patterns. In addition, it was found that the decay constants for the proton flow caused by a pH-jump and a pNa-jump have the same numerical value. The physical meaning of the decay constants has been elucidated in terms of the phenomenological coefficients (mobilities) and the buffering capacities of the system. The phenomenological coefficients for the Na⁺-H⁺ flows were determined as differential quantities. The value obtained for the total proton permeability through the particle membrane via all available channels, $\text{L}{}_{\mathrm{<mtext>H</mtext>}}\text{= (}\text{?J}{}_{\mathrm{<mtext>H\; +</mtext>}}\text{?Δ}\text{pH}\text{)}{}_{\mathrm{<mtext>\Delta \psi ,\Delta </mtext><mtext>pNa</mtext>}}$, was in the range of 850–1150 nmol H⁺·(mg protein)^?1·h^?1·(pH unit)^?1 for four different preparations; for the total Na⁺ permeability, $\text{L}{}_{\mathrm{<mtext>Na</mtext>}}\text{= (}\text{?J}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{+}}\text{?Δ}\text{pNa}\text{)}{}_{\mathrm{<mtext>\Delta \psi ,\Delta </mtext><mtext>pH</mtext>}}$, it was 1620–2500 nmol Na⁺·(mg protein)^?1·h^?1·(pNa unit)^?1; and for the proton ‘cross-permeability’, $\text{L}{}_{\mathrm{<mtext>HNa</mtext>}}\text{= (}\text{?J}{}_{\mathrm{<mtext>H</mtext>}}{}^{\mathrm{+}}\text{?Δ}\text{pNa}\text{)}{}_{\mathrm{<mtext>\Delta \psi ,\Delta </mtext><mtext>pH</mtext>}}$, it was 220–580 nmol H⁺·(mg protein)^?1·h^?1·(pNa unit)^?1, for different preparations. From the above phenomenological parameters, the following quantities have been calculated: the degree of coupling (q), the maximal efficiency of Na⁺-H⁺ exchange (η_max), the flow and force efficacies (?) of the above exchange, and the admissible range for the values of the molecular stoichiometry parameter (r). We found q ? 0.4; η_max ? 5%; 0.36 ? r ? 2; ?_JNa⁺ ? 1.3 · 10⁵μmol · (RT unit)^?1 at J_Na = 1 μmolNa⁺ · (mgprotein)^?1 · h^?1; and ?_ΔpNa ? 5 · 10⁴ ΔpNa · (mg protein) · h · (RT unit)^?1 at ΔpNa = 1 unit, for different preparations.     

The absorbance spectrum of the brown holo-membrane and the comparison of pI values of bacteriorhodopsin solubilized from purple membrane and from brown holo-membrane

Brown holo-membrane was prepared by the addition of all-trans-retinal to brown apo-membrane which was isolated from Halobacterium halobium grown in the presence of nicotine. The effects of pH and NaCI concentration on the absorbance spectrum of the brown holo-membrane were investigated in comparison with those of the purple membrane. The λ_max of the dark-adapted brown holo-membrane shifted from 560 to 600 nm by lowering pH. The pK value which was determined as the mid-point pH for the spectral red-shift was 5.8 in the absence of NaCl. It was lowered to 4.5 and 3.4 in 0.1 and 1 M NaCl solutions, respectively. The pK value for the brown holo-membrane was larger than the corresponding value for the purple membrane in the NaCl solution. Bacteriorhodopsins present in the purple membrane and in the brown holo-membrane were solubilized in the nonionic detergent, lauryl ester of sucrose. For both solubilized bacteriorhodopsins, the pK value of spectral red-shift was about 3.1 in water, and the pI value, determined by chromatofocusing, was about 4.6 at 22°C.     

Reaction of the purple membrane with a carbodimide

Purple membrane was reacted with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at pH 4.5 and 8.0. At pH 4.5, the reaction yields cross-linked bacteriorhodopsin. The cross-linking is inhibited by pretreatment of the membrane with papain, or by the presence of carbohydrazide or glycine ethyl ester in the reaction mixture. The product of the pH 8.0 reaction is not cross-linked, but it displays altered properties. Two measures of photochemical activity (light-induced change in proton binding (Δh?) and decay of photointermediate M) show changes indicative of slowed proton uptake. The Δh? is increased by ethyl dimethylaminopropylcarbodiimide. This increase is unaffected by pretreatment of the membrane with papain, and it is not reversed by NH₂OH. However, the reaction is inhibited by millimolar concentrations of CaCl₂. The altered Δh? is not apparent in detergent-solubilized membranes. Ethyl dimethylaminopropyl-carbodiimide does not appear to cause a large alteration in the membrane surface charge, as measured by Ca²⁺ binding.We conclude that (1) at acid pH, ethyl dimethylaminopropylcarbodiimide can be used for cross-linking or for attachment of specific probes to the C-terminal region of bacteriorhodopsin, and hence to the cytoplasmic side of the purple membrane, and (2) at alkaline pH, ethyl dimethylaminopropylcarbodiimide reacts at a different type of site and appears to inhibit the proton pump.     

Stimulation of ATP synthesis in Halobacterium halobium R1 by light-induced or artificially created proton electrochemical potential gradients across the cell membrane

The relationship between proton movement and phosphorylation in Halobacterium halobium R₁ has been investigated under anaerobic conditions. The light-induced changes in the bacteriorhodopsin are accompanied by proton movements across the cell membrane which result in pH changes in the suspending medium. The initial alkaline shift is shown to be closely paralleled by (and hence correlated with) ATP synthesis. Acidification of the medium in the presence of valinomycin, under conditions of low external potassium, brings about ATP synthesis in the dark.     

Kinetics of photoconversion of protochlorophyllide 649 to chlorophyllide 676 at low temperature in etiolated cotyledons of Pharbitis nil

The kinetics of the photoconversion of protochlorophyllide 649 to chlorophyllide 676 were studied spectrophotometrically over the temperature range of ?15 – ?80°C under light-saturating conditions in etiolated cotyledons of Pharbitis nil. Photoconversion obeyed the sum of two first-order kinetics over this low temperature range. Activation energies obtained from the rate constants were about 5000 cal; this suggests that these two processes may be physical processes not chemical reactions. The results indicate that photoconversion involves two main steps. One is the step dependent on both light intensity and temperature that has been well studied. The other, which is concerned in this study, is the step dependent on temperature only, which may be the requisite for photoconversion. This latter step seems to be related to the binding mode of protochlorophyllide to a holochrome protein or to conformational changes in the protochlorophyllide-holochrome.     

Half channels mediating H transport and the mechanism of gating in the Fo sector of Escherichia coli F1Fo ATP synthase

H⁺-transporting F₁F_o ATP synthase catalyzes the synthesis of ATP via coupled rotary motors within F_o and F₁. H⁺ transport at the subunit a–c interface in trans-membranous F_o drives rotation of the c-ring within the membrane, with subunit c being bound in a complex with the γ and ε subunits extending from the membrane. Finally, the rotation of subunit γ within the α₃β₃ sector of F₁ mechanically drives ATP synthesis within the catalytic sites. In this review, we propose and provide evidence supporting the route of proton transfer via half channels from one side of the membrane to the other, and the mechanism of gating H⁺ binding to and release from Asp61 of subunit c, via conformational movements of Arg210 in subunit a. We propose that protons are gated from the inside of a four-helix bundle at the periplasmic side of subunit a to drive protonation of cAsp61, and that this gating movement is facilitated by the swiveling of trans-membrane helices (TMHs) 4 and 5 at the site of interaction with cAsp61 on the periphery of the c-ring. Proton release to the cytoplasmic half channel is facilitated by the movement of aArg210 as a consequence of this proposed helical swiveling. Finally, release from the cytoplasmic half channel is mediated by residues in a complex of interacting extra-membraneous loops formed between TMHs of both subunits a and c. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

Time-resolved single-turnover of ba3 oxidase from Thermus thermophilus

The kinetics of the oxidation of fully-reduced ba₃ cytochrome c oxidase from Thermus thermophilus by oxygen were followed by time-resolved optical spectroscopy and electrometry. Four catalytic intermediates were resolved during this reaction. The chemical nature and the spectral properties of three intermediates (compounds A, P and O) reproduce the general features of aa₃-type oxidases. However the F intermediate in ba₃ oxidase has a spectrum identical to the P state. This indicates that the proton taken up during the P → F transition does not reside in the binuclear site but is rather transferred to the covalently cross-linked tyrosine near that site. The total charge translocation associated with the F → O transition in ba₃ oxidase is close to that observed during the F → O transition in the aa₃ oxidases. However, the P_R → F transition is characterized by significantly lower charge translocation, which probably reflects the overall lower measured pumping efficiency during multiple turnovers.     

Labeling of chromatophore membranes and reaction centers from the photosynthetic bacterium Rhodospirillum rubrum with the hydrophobic marker 5-[125I]iodonaphthyl-1-azide

Chromatophores of the photosynthetic bacterium Rhodospirillum rubrum and isolated reaction centers were labeled with the lipophilic membrane marker 5-[¹²⁵I]iodonaphthyl-1-azide. The two smaller reaction center proteins L and M bind more label than the larger subunit H, a fact supporting the proposed localisation of the 3 subunits obtained with hydrophilic labels. Besides these integral proteins the lipids, among them mainly the pigments and the quinones, are highly labeled suggesting a hydrophobic environment around these molecules and a preferred reactivity to iodonaphthylazide. Such a hydrophobic environment may be of great importance for the function of the photosynthetic reaction centers especially for the charge separation and the primary reactions in electron transport.     

Overexpression of miR172 suppresses the brassinosteroid signaling defects of bak1 in Arabidopsis

BRI1-Associated Receptor Kinase 1 (BAK1) is a leucine-rich repeat serine/threonine receptor-like kinase (LRR-RLK) that is involved in multiple developmental pathways, such as brassinosteroid (BR) signaling, plant immunity and cell death control in plants. Because the roundish and compact rosette leaves of bak1 mutant plants are characteristic phenotypes for deficient BR signaling, we screened genetic suppressors of bak1 according to changes in leaf shape to identify new components that may be involved in BAK1-mediated BR signaling using the activation-tagging method. Here, we report bak1-SUP1, which exhibited longer and narrower rosette leaves and an increased BR sensitivity compared with those of bak1. Analyses of the T-DNA insertional site and the gene expression that was affected by the T-DNA insertion revealed that a microRNA, namely, miR172, over-accumulates in bak1-SUP1. Detailed phenotypic analyses of bak1-SUP1 and a single mutant in which the bak1 mutation was segregated out (miR172-D) revealed that the overexpression of miR172 promotes leaf length elongation in adult plants and increases the root and hypocotyl growth during the seedling stage compared with that of wild type plants. Taken together with its increased BR sensitivity, these results suggest that miR172 regulates vegetative growth patterns by modulating BR sensitivity as well as by the previously identified developmental phase transition.     

Molecular cloning,characterization and expression analysis of ATG1 in the silkworm, Bombyx mori

Atg1 is a Serine/Threonine protein kinase that plays a pivotal role in autophagy. A complete coding sequence of ATG1 is not available for the silkworm, Bombyx mori which is a good model for studying the autophagic process.     

Leishmania mexicana: Conversion of dihydroorotate to orotate in amastigotes and promastigotes

The specific activity of dihydroorotate dehydrogenase, catalysing the conversion of l-5,6-dihydroorotate (l-DHO) to orotate, in Leishmania mexicana mexicana was found to be 22.1 ± 3.5 nmole/hr/mg protein in the amastigote, and 28.7 ± 4.6 nmole/hr/mg protein in the promastigote. The enzyme was found to be soluble and to require molecular O₂ for activity in both forms of the parasite. Oxygen utilisation was not mediated through the mitochondrial cytochrome-containing respiratory chain, and phenazine methosulphate and ferricyanide could be used as electron acceptors by the enzyme in both aerobic and anaerobic conditions. The enzyme from both amastigote and promastigote had a pH optimum of 7.0, and the K_m values for l-DHO were 11.8 ± 4.9 and 2.3 ± 0.4 μM, respectively. The pyrimidine analogs 5-methylorotate (K_i = 8.8 μM) and 5-aminoorotate (K_i = 57 μM) were shown to be competitive inhibitors of the promastigote enzyme, as was the reaction product orotate (K_i = 10 μM).     

A four-subunit cytochrome bc1 complex complements the respiratory chain of Thermus thermophilus

Several components of the respiratory chain of the eubacterium Thermus thermophilus have previously been characterized to various extent, while no conclusive evidence for a cytochrome bc₁ complex has been obtained. Here, we show that four consecutive genes encoding cytochrome bc₁ subunits are organized in an operon-like structure termed fbcCXFB. The four gene products are identified as genuine subunits of a cytochrome bc₁ complex isolated from membranes of T. thermophilus. While both the cytochrome b and the FeS subunit show typical features of canonical subunits of this respiratory complex, a further membrane-integral component (FbcX) of so far unknown function copurifies as a subunit of this complex. The cytochrome c₁ carries an extensive N-terminal hydrophilic domain, followed by a hydrophobic, presumably membrane-embedded helical region and a typical heme c binding domain. This latter sequence has been expressed in Escherichia coli, and in vitro shown to be a kinetically competent electron donor to cytochrome c₅₅₂, mediating electron transfer to the ba₃ oxidase. Identification of this cytochrome bc₁ complex bridges the gap between the previously reported NADH oxidation activities and terminal oxidases, thus, defining all components of a minimal, mitochondrial-type electron transfer chain in this evolutionary ancient thermophile.