Probing the Q-proton pathway of ba3-cytochrome c oxidase by time-resolved Fourier transform infrared spectroscopy

In cytochrome c oxidase, the terminal respiratory enzyme, electron transfers are strongly coupled to proton movements within the enzyme. Two proton pathways (K and D) containing water molecules and hydrophobic amino acids have been identified and suggested to be involved in the proton translocation from the mitochondrial matrix or the bacterial cytoplasm into the active site. In addition to the K and D proton pathways, a third proton pathway (Q) has been identified only in ba3-cytochrome c oxidase from Thermus thermophilus, and consists of residues that are highly conserved in all structurally known heme-copper oxidases. The Q pathway starts from the cytoplasmic side of the membrane and leads through the axial heme a3 ligand His-384 to the propionate of the heme a3 pyrrol ring A, and then via Asn-366 and Asp-372 to the water pool. We have applied FTIR and time-resolved step-scan Fourier transform infrared (TRS2-FTIR) spectroscopies to investigate the protonation/deprotonation events in the Q-proton pathway at ambient temperature. The photolysis of CO from heme a3 and its transient binding to CuB is dynamically linked to structural changes that can be tentatively attributed to ring A propionate of heme a3 (1695/1708 cm(-1)) and to deprotonation of Asp-372 (1726 cm(-1)). The implications of these results with respect to the role of the ring A propionate of heme a3-Asp372-H2O site as a proton carrier to the exit/output proton channel (H2O pool) that is conserved among all structurally known heme-copper oxidases, and is part of the Q-proton pathway in ba3-cytochrome c oxidase, are discussed.     

ATP-independent luminal oscillations and release of Ca2+ and H+ from mast cell secretory granules: implications for signal transduction

InsP(3) is an important link in the intracellular information network. Previous observations show that activation of InsP(3)-receptor channels on the granular membrane can turn secretory granules into Ca(2+) oscillators that deliver periodic trains of Ca(2+) release to the cytosol (T. Nguyen, W. C. Chin, and P. Verdugo, 1998, Nature, 395:908-912; I. Quesada, W. C. Chin, J. Steed, P. Campos-Bedolla, and P. Verdugo, 2001, BIOPHYS: J. 80:2133-2139). Here we show that InsP(3) can also turn mast cell granules into proton oscillators. InsP(3)-induced intralumenal [H(+)] oscillations are ATP-independent, result from H(+)/K(+) exchange in the heparin matrix, and produce perigranular pH oscillations with the same frequency. These perigranular pH oscillations are in-phase with intralumenal [H(+)] but out-of-phase with the corresponding perigranular [Ca(2+)] oscillations. The low pH of the secretory compartment has critical implications in a broad range of intracellular processes. However, the association of proton release with InsP(3)-induced Ca(2+) signals, their similar periodic nature, and the sensitivity of important exocytic proteins to the joint action of Ca(2+) and pH strongly suggests that granules might encode a combined Ca(2+)/H(+) intracellular signal. A H(+)/Ca(2+) signal could significantly increase the specificity of the information sent by the granule by transmitting two frequency encoded messages targeted exclusively to proteins like calmodulin, annexins, or syncollin that are crucial for exocytosis and require specific combinations of [Ca(2+)] "and" pH for their action.     

Entrapment of human flavin-containing monooxygenase 3 in the presence of gold nanoparticles: TEM,FTIR and electrocatalysis

Background

Nanosized particles of gold are widely used as advanced materials for enzyme catalysis investigations. In some bioanalytical methods these nanoparticles can be exploited to increase the sensitivity by enhancing electron transfer to the biological component i.e. redox enzymes such as drug metabolizing enzymes.

Methods

In this work, we describe the characterization of human flavin-containing monooxygenase 3 (hFMO3) in a nanoelectrode system based on AuNPs stabilized with didodecyldimethylammonium bromide (DDAB) on glassy carbon electrodes. Once confirmed by FTIR spectroscopy that in the presence of DDAB-AuNPs the structural integrity of hFMO3 is preserved, the influence of AuNPs on the electrochemistry of the enzyme was studied by cyclic voltammetry and square wave voltammetry.

Results

Our results show that AuNPs improve the electrochemical performance of hFMO3 on glassy carbon electrodes by enhancing the electron transfer rate and the current signal-to-noise ratio. Moreover, the electrocatalytic activity of hFMO3-DDAB-AuNP electrodes which was investigated in the presence of two well known substrates, benzydamine and sulindac sulfide, resulted in K_M values of 52 μM and 27 μM, with V_max of 8 nmol min^− 1 mg^− 1 and 4 nmol min^− 1 mg^− 1, respectively, which are in agreement with data obtained with the microsomal enzyme.

Conclusions

The immobilization of hFMO3 protein in DDAB stabilized AuNP electrodes improves the bioelectrochemical performance of this important phase I drug metabolizing enzyme.

General significance

This bio-analytical method can be considered as a promising advance in the development of new techniques suitable for the screening of novel hFMO3 metabolized pharmaceuticals.     

Inter-oligomer interactions of the human prion protein are modulated by the polymorphism at codon 129

The common polymorphism at codon 129 in the human prion protein (PrP) has been shown in many studies to influence not only the pathology of prion disease but also the misfolding propensity of PrP. Here we used NMR, CD and atomic force microscopy in solution to investigate differences in β-oligomer (β^O) formation and inter-oligomer interaction depending on the polymorphism at codon 129. NMR investigations assigned the observable amide resonances to the β^O N-terminal segments, showing that it is the core region of PrP (residues 127-228) that is involved in β^O formation. Atomic force microscopy revealed distinctive 1.8 × 15 × 15-nm disk-like structures that form stacks through inter-oligomer interactions. The propensity to form stacks and the number of oligomers involved depended on the polymorphism at codon 129, with a significantly lower degree of stacking for β^O with valine at position 129. This result provides evidence for conformational differences between the β^O allelic forms, showing that the core region of the protein including position 129 is actively involved in inter-oligomer interactions, consistent with NMR observations.     

The retinal structure of channelrhodopsin-2 assessed by resonance Raman spectroscopy

Channelrhodopsin-2 mediates phototaxis in green algae by acting as a light-gated cation channel. As a result of this property, it is used as a novel optogenetic tool in neurophysiological applications. Structural information is still scant and we present here the first resonance Raman spectra of channelrhodopsin-2. Spectra of detergent solubilized and lipid-reconstituted protein were recorded under pre-resonant conditions to exclusively probe retinal in its electronic ground state. All-trans retinal was identified to be the favoured configuration of the chromophore but significant contributions of 13-cis were detected. Pre-illumination hardly changed the isomeric composition but small amounts of presumably 9-cis retinal were found in the light-adapted state. Spectral analysis suggested that the Schiff base proton is strongly hydrogen-bonded to a nearby water molecule.     

Chlorophyll f and chlorophyll d are produced in the cyanobacterium Chlorogloeopsis fritschii when cultured under natural light and near-infrared radiation

We report production of chlorophyll f and chlorophyll d in the cyanobacterium Chlorogloeopsis fritschii cultured under near-infrared and natural light conditions. C. fritschii produced chlorophyll f and chlorophyll d when cultured under natural light to a high culture density in a 20 L bubble column photobioreactor. In the laboratory, the ratio of chlorophyll f to chlorophyll a changed from 1:15 under near-infrared, to an undetectable level of chlorophyll f under artificial white light. The results provide support that chlorophylls f and d are both red-light inducible chlorophylls in C. fritschii.     

Functional cell-free synthesis of a seven helix membrane protein: in situ insertion of bacteriorhodopsin into liposomes

The expression of membrane proteins for functional and structural studies or medicinal applications is still not very well established. Membrane-spanning proteins that mediate the information flow of the extracellular side with the interior of the cell are prime targets for drug development methods that would allow screening techniques or high throughput formats are of particular interest. Here we describe a systematic approach to the liposome-assisted cell-free synthesis of functional membrane proteins. We demonstrate the synthesis of bacteriorhodopsin (bR(cf)) in presence of small unilamellar liposomes. The yield of bR(cf) per volume cell culture is comparable to that of bacteriorhodopsin in its native host. The functional analysis of bR(cf) was performed directly using the cell-free reaction mixture. Photocycle measurements reveal kinetic data similar to that determined for bR in Halobacterium salinarum cell-envelope vesicles. The liposomes can be attached directly to black lipid membranes (BLM), which allows measuring light activated photocurrents in situ. The results reveal a functional proton pump with properties identical to those established for the native protein.     

The impact of membrane lipid composition on antimicrobial function of an alpha-helical peptide

VP1, a putative alpha-helical antimicrobial peptide (alpha-AMP) inhibited growth of Bacillus subtilis and Escherichia coli at 500microM. The peptide induced stable surface pressure changes in monolayers formed from B. subtilis native lipid extract (circa 4.5mNm(-1)) but transient pressure changes in corresponding E. coli monolayers (circa 1.0mNm(-1)), which led to monolayer disintegration. Synthetic lipid monolayers mimetic of the extracts were used to generate compression isotherms. Thermodynamic analysis of B. subtilis isotherms indicated membrane stabilisation by VP1 (DeltaG(Mix)<0), via a mechanism dependent upon the phosphatidylglycerol to cardiolipin ratio. Corresponding analysis of E. coli isotherms indicated membrane destabilisation by the peptide (DeltaG(Mix)>0). Destabilisation correlated with PE levels present and appeared to involve a mechanism resembling those used by tilted peptides. These data emphasise that structure/function analysis of alpha-AMPs must consider not only their structural characteristics but also the lipid make-up of the target microbial membrane.     

Resonance Raman and FTIR spectroscopic characterization of the closed and open states of channelrhodopsin-1

Channelrhodopsin-1 from Chlamydomonas augustae (CaChR1) is a light-activated cation channel, which is a promising optogenetic tool. We show by resonance Raman spectroscopy and retinal extraction followed by high pressure liquid chromatography (HPLC) that the isomeric ratio of all-trans to 13-cis of solubilized channelrhodopsin-1 is with 70:30 identical to channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Critical frequency shifts in the retinal vibrations are identified in the Raman spectrum upon transition to the open (conductive P₂³⁸⁰) state. Fourier transform infrared spectroscopy (FTIR) spectra indicate different structures of the open states in the two channelrhodopsins as reflected by the amide I bands and the protonation pattern of acidic amino acids.     

Dual role of FMN in flavodoxin function: Electron transfer cofactor and modulation of the protein-protein interaction surface

Flavodoxin (Fld) replaces Ferredoxin (Fd) as electron carrier from Photosystem I (PSI) to Ferredoxin-NADP⁺ reductase (FNR). A number of Anabaena Fld (AnFld) variants with replacements at the interaction surface with FNR and PSI indicated that neither polar nor hydrophobic residues resulted critical for the interactions, particularly with FNR. This suggests that the solvent exposed benzenoid surface of the Fld FMN cofactor might contribute to it. FMN has been replaced with analogues in which its 7- and/or 8-methyl groups have been replaced by chlorine and/or hydrogen. The oxidised Fld variants accept electrons from reduced FNR more efficiently than Fld, as expected from their less negative midpoint potential. However, processes with PSI (including reduction of Fld semiquinone by PSI, described here for the first time) are impeded at the steps that involve complex re-arrangement and electron transfer (ET). The groups introduced, particularly chlorine, have an electron withdrawal effect on the pyrazine and pyrimidine rings of FMN. These changes are reflected in the magnitude and orientation of the molecular dipole moment of the variants, both factors appearing critical for the re-arrangement of the finely tuned PSI:Fld complex. Processes with FNR are also slightly modulated. Despite the displacements observed, the negative end of the dipole moment points towards the surface that contains the FMN, still allowing formation of complexes competent for efficient ET. This agrees with several alternative binding modes in the FNR:Fld interaction. In conclusion, the FMN in Fld not only contributes to the redox process, but also to attain the competent interaction of Fld with FNR and PSI.     

Enzymatic synthesis of novel branched sugar alcohols mediated by the transglycosylation reaction of pullulan-hydrolyzing amylase II (TVA II) cloned from Thermoactinomyces vulgaris R-47

Transglycosylation reactions are useful for preserving a specific sugar structure during the synthesis of branched oligosaccharides. We have previously reported a panosyl unit transglycosylation reaction by pullulan-hydrolyzing amylase II (TVA II) cloned from Thermoactinomyces vulgaris R-47 (Tonozuka et al., Carbohydr. Res., 1994, 261, 157–162). The acceptor specificity of the TVA II transglycosylation reaction was investigated using pullulan as the donor and sugar alcohols as the acceptor. TVA II transferred the α-panosyl unit to the C-1 hydroxyl group of meso-erythritol, C-1 and C-2 of xylitol, and C-1 and C-6 of d-sorbitol. TVA II differentiated between the sugar alcohols’ hydroxyl groups to produce five novel non-reducing branched oligosaccharides, 1-O-α-panosylerythritol, 1-O-α-panosylxylitol, 2-O-α-panosylxylitol, 1-O-α-panosylsorbitol, and 6-O-α-panosylsorbitol. The Trp³⁵⁶→Ala mutant showed similar transglycosylation reactions; however, panose production by the mutant was 4.0–4.5-fold higher than that of the wild type. This suggests that Trp³⁵⁶ is important for recognizing both water and the acceptor molecules in the transglycosylation and the hydrolysis reaction.     

The important role of stratum corneum lipids for the cutaneous barrier function

The skin protects the body from unwanted influences from the environment as well as excessive water loss. The barrier function of the skin is located in the stratum corneum (SC). The SC consists of corneocytes embedded in a lipid matrix. This lipid matrix is crucial for the lipid skin barrier function. This paper provides an overview of the reported SC lipid composition and organization mainly focusing on healthy and diseased human skin. In addition, an overview is provided on the data describing the relation between lipid modulations and the impaired skin barrier function. Finally, the use of in vitro lipid models for a better understanding of the relation between the lipid composition, lipid organization and skin lipid barrier is discussed. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.     

Plastoquinones are effectively reduced by ferredoxin:NADP+ oxidoreductase in the presence of sodium cholate micelles. Significance for cyclic electron transport and chlororespiration

The effect of sodium cholate and other detergents (Triton X-100, sodium dodecyl sulphate, octyl glucoside, myristyltrimethylammonium bromide) on the reduction of plastoquinones (PQ) with a different length of the side-chain by spinach ferredoxin:NADP(+) oxidoreductase (FNR) in the presence of NADPH has been studied. Both NADPH oxidation and oxygen uptake due to plastosemiquinone autoxidation were highly stimulated only in the presence of sodium cholate among the used detergents. Sodium cholate at the concentration of 20 mM was found to be the most effective on both PQ-4 and PQ-9-mediated oxygen uptake. The FNR-dependent reduction of plastoquinones incorporated into sodium cholate micelles was stimulated by spinach ferredoxin but inhibited by Mg(2+) ions. It was concluded that the structure of sodium cholate micelles facilitates contact of plastoquinone molecules with the enzyme and creates favourable conditions for the reaction similar to those found in thylakoid membranes for PQ-9 reduction. The obtained results were discussed in terms of the function of FNR as a ferredoxin:plastoquinone reductase both in cyclic electron transport and chlororespiration.     

Isolation of the first toxin from the scorpion Buthus occitanus israelis showing preference for Shaker potassium channels

We have purified BoiTx1, the first toxin from the venom of the Israeli scorpion, Buthus occitanus israelis, and studied its activity and genomic organization. BoiTx1 is a 37 amino acid-long peptide contained six conserved cysteines, and is classified as an alpha-KTx3.10 toxin. The pharmacological effects of BoiTx1 were studied on various cloned K(+) channels expressed in Xenopus laevis oocytes. BoiTx1 inhibited currents through Drosophila Shaker channels with an IC(50) value of 3.5+/-0.5nM, yet had much lesser effect on its mammalian orthologs. Thus, BoiTx1 is the first member of the alpha-KTx3 family that preferentially affects insect potassium channels.     

Interactions between the active metabolite of tryptophan pyrolysate mutagen, N-hydroxy-Trp-P-2, and lipids: the role of lipid peroxides in the conversion of N-hydroxy-Trp-P-2 to non-reactive forms

The interactions between lipids and the mutagenic active metabolite of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (N-hydroxy-Trp-P-2), were studied. Oleic acid showed an inhibitory effect on the formation of this active metabolite mainly by inhibition of hepatic microsomal oxidation systems. On the other hand, microsomal lipids from rat liver and commercial pig liver lecithin diminished the amount of N-hydroxy-Trp-P-2 without inhibiting the metabolism of Trp-P-2. The direct reaction of these lipids with N-hydroxy-Trp-P-2 was disclosed by experiments using N-hydroxy-Trp-P-2 and lipids without microsomes. Furthermore, the participation of lipid peroxides in this reaction was suggested by a linear relationship between the concentrations of the conjugated diene of lipids and the disappearance of N-hydroxy-Trp-P-2. When [3H]N-hydroxy-Trp-P-2 was incubated in the presence of pig liver lecithin, the polar products which were not formed in the incubation without lipids were newly detected by thin-layer chromatography (TLC) analysis.     

Photosynthetic electron transport activity in heat-treated barley leaves: The role of internal alternative electron donors to photosystem II

Electron transport processes were investigated in barley leaves in which the oxygen-evolution was fully inhibited by a heat pulse (48 °C, 40 s). Under these circumstances, the K peak (∼ F_400 μs) appears in the chl a fluorescence (OJIP) transient reflecting partial Q_A reduction, which is due to a stable charge separation resulting from the donation of one electron by tyrozine Z. Following the K peak additional fluorescence increase (indicating Q_A⁻ accumulation) occurs in the 0.2-2 s time range. Using simultaneous chl a fluorescence and 820 nm transmission measurements it is demonstrated that this Q_A⁻ accumulation is due to naturally occurring alternative electron sources that donate electrons to the donor side of photosystem II. Chl a fluorescence data obtained with 5-ms light pulses (double flashes spaced 2.3-500 ms apart, and trains of several hundred flashes spaced by 100 or 200 ms) show that the electron donation occurs from a large pool with t_1/2 ∼ 30 ms. This alternative electron donor is most probably ascorbate.     

No role of DT-diaphorase (NQO1) in the protection against oxidized quercetin

Quercetin is one of the most studied alimentary antioxidants. During its antioxidant activity, quercetin becomes oxidized into its ortho-quinone/quinone methide, denoted as QQ. QQ is toxic since it is highly reactive towards thiols. DT-diaphorase (NQO1) might protect against QQ toxicity by reducing QQ to quercetin. However, conflicting data have been reported. The aim of the present study is to elucidate the role of DT-diaphorase in the protection against QQ-mediated thiol reactivity. It was found that QQ is indeed a substrate for DT-diaphorase. However, QQ reacted much faster with glutathione or protein thiols than with DT-diaphorase in experiments with isolated compounds as well as with human liver cytosol or blood plasma. This indicates that DT-diaphorase has no role in the protection against QQ.     

Spectral effects of supplementary lighting on the secondary metabolites in roses,chrysanthemums, and campanulas

To investigate the effect of the light spectrum on photosynthesis, growth, and secondary metabolites Rosa hybrida ‘Scarlet’, Chrysanthemum morifolium ‘Coral Charm’, and Campanula portenschlagiana ‘BluOne’ were grown at 24/18 °C day/night temperature under purpose-built LED arrays yielding approximately 200 μmol m⁻² s⁻¹ at plant height for 16 h per day. The four light treatments were (1) 40% Blue/60% Red, (2) 20% Blue/80% Red, (3) 100% Red, and (4) 100% White (Control). The plant height was smallest in 40% Blue/60% Red in roses and chrysanthemums, while the biomass was smallest in the white control in roses and in 100% Red in chrysanthemums. The total biomass was unaffected by the spectrum in campanulas, while the leaf area was smallest in the 40% Blue/60% Red treatment. In 100% Red curled leaves and other morphological abnormalities were observed. Increasing the blue to red ratio increased the stomatal conductance though net photosynthesis was unaffected, indicating excess stomatal conductance in some treatments. With higher blue light ratio all phenolic acids and flavonoids increased. In view of the roles of these secondary metabolites as antioxidants, anti-pathogens, and light protectants, we hypothesize that blue light may predispose plants to better cope with stress.     

Cross-linking of transmembrane helices in proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli: implications for the structure and function of the membrane domain

Proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains an α and a β subunit of 54 and 49 kDa, respectively, and is made up of three domains. Domain I (dI) and III (dIII) are hydrophilic and contain the NAD(H)- and NADP(H)-binding sites, respectively, whereas the hydrophobic domain II (dII) contains 13 transmembrane α-helices and harbours the proton channel. Using a cysteine-free transhydrogenase, the organization of dII and helix-helix distances were investigated by the introduction of one or two cysteines in helix-helix loops on the periplasmic side. Mutants were subsequently cross-linked in the absence and presence of diamide and the bifunctional maleimide cross-linker o-PDM (6 Å), and visualized by SDS-PAGE.In the α₂β₂ tetramer, αβ cross-links were obtained with the αG476C-βS2C, αG476C-βT54C and αG476C-βS183C double mutants. Significant αα cross-links were obtained with the αG476C single mutant in the loop connecting helix 3 and 4, whereas ββ cross-links were obtained with the βS2C, βT54C and βS183C single mutants in the beginning of helix 6, the loop between helix 7 and 8 and the loop connecting helix 11 and 12, respectively. In a model based on 13 mutants, the interface between the α and β subunits in the dimer is lined along an axis formed by helices 3 and 4 from the α subunit and helices 6, 7 and 8 from the β subunit. In addition, helices 2 and 4 in the α subunit together with helices 6 and 12 in the β subunit interact with their counterparts in the α₂β₂ tetramer. Each β subunit in the α₂β₂ tetramer was concluded to contain a proton channel composed of the highly conserved helices 9, 10, 13 and 14.