Proton magnetic resonance studies of 7 Fe ferredoxins: Lower potential of the [4 Fe–4 S] redox center than the [3 Fe–3 S] cluster

Two types of iron-sulfur clusters, [3 Fe–3 S] and [4 Fe–4 S], were identified by ¹H-NMR in ferredoxins from Thermus thermophilus, Mycobacterium smegmatis and Pseudomonas ovalis. The [4 Fe–4 S] clusters always showed the redox couples which had potentials lower than that of the [3 Fe–3 S] clusters.     

Mechanism of Fe uptake by the leaf symplast: Is Fe inactivation in leaf a cause of Fe deficiency chlorosis?

The mechanism of iron (Fe) uptake from the leaf apoplast into leaf mesophyll cells was studied to evaluate the putative Fe inactivation as a possible cause of Fe deficiency chlorosis. For this purpose, sunflower (Helianthus annuus L.) and faba bean plants (Vicia faba L.) were precultured with varied Fe and bicarbonate (HCO ₃ ^- ) supply in nutrient solution. After 2–3 weeks preculture, Fe^III reduction and ⁵⁹Fe uptake by leaf discs were measured in solutions with Fe supplied as citrate or synthetic chelates in darkness. The data clearly indicate that Fe^III reduction is a prerequisite for Fe uptake into leaf cells and that the Fe nutritional status of plants does not affect either process. In addition, varied supply of Fe and HCO ₃ ^- to the root medium during preculture had no effect on pH of the xylem sap and leaf apoplastic fluid. A varied pH of the incubation solution had no significant effect on Fe^III reduction and Fe uptake by leaf discs in the physiologically relevant pH range of 5.0–6.0 as measured in the apoplastic leaf fluid. It is concluded that Fe inactivation in the leaf apoplast is not a primary cause of Fe deficiency chlorosis induced by bicarbonate. This revised version was published online in June 2006 with corrections to the Cover Date.     

Synthesis of polypyrrole within the cell wall of yeast by redox-cycling of [Fe(CN)6]3−/[Fe(CN)6]4−

Yeast cells are often used as a model system in various experiments. Moreover, due to their high metabolic activity, yeast cells have a potential to be applied as elements in the design of biofuel cells and biosensors. However a wider application of yeast cells in electrochemical systems is limited due to high electric resistance of their cell wall. In order to reduce this problem we have polymerized conducting polymer polypyrrole (Ppy) directly in the cell wall and/or within periplasmic membrane. In this research the formation of Ppy was induced by [Fe(CN)₆]³⁻ions, which were generated from K₄[Fe(CN)₆], which was initially added to polymerization solution. The redox process was catalyzed by oxido-reductases, which are present in the plasma membrane of yeast cells. The formation of Ppy was confirmed by spectrophotometry and atomic force microscopy. It was confirmed that the conducting polymer polypyrrole was formed within periplasmic space and/or within the cell wall of yeast cells, which were incubated in solution containing pyrrole, glucose and [Fe(CN)₆]⁴⁻. After 24 h drying at room temperature we have observed that Ppy-modified yeast cell walls retained their initial spherical form. In contrast to Ppy-modified cells, the walls of unmodified yeast have wrinkled after 24 h drying. The viability of yeast cells in the presence of different pyrrole concentrations has been evaluated.     

NMR structures of the transmembrane domains of the α4β2 nAChR

The α4β2 nicotinic acetylcholine receptor (nAChR) is the predominant heteromeric subtype of nAChRs in the brain, which has been implicated in numerous neurological conditions. The structural information specifically for the α4β2 and other neuronal nAChRs is presently limited. In this study, we determined structures of the transmembrane (TM) domains of the α4 and β2 subunits in lauryldimethylamine-oxide (LDAO) micelles using solution NMR spectroscopy. NMR experiments and size exclusion chromatography-multi-angle light scattering (SEC-MALS) analysis demonstrated that the TM domains of α4 and β2 interacted with each other and spontaneously formed pentameric assemblies in the LDAO micelles. The Na(+) flux assay revealed that α4β2 formed Na(+) permeable channels in lipid vesicles. Efflux of Na(+) through the α4β2 channels reduced intra-vesicle Sodium Green? fluorescence in a time-dependent manner that was not observed in vesicles without incorporating α4β2. The study provides structural insight into the TM domains of the α4β2 nAChR. It offers a valuable structural framework for rationalizing extensive biochemical data collected previously on the α4β2 nAChR and for designing new therapeutic modulators.     

Desulfovibrio gigas ferredoxin II: redox structural modulation of the [3Fe–4S] cluster

Desulfovibrio gigas ferredoxin II (DgFdII) is a small protein with a polypeptide chain composed of 58 amino acids, containing one Fe₃S₄ cluster per monomer. Upon studying the redox cycle of this protein, we detected a stable intermediate (FdII_int) with four ¹H resonances at 24.1, 20.5, 20.8 and 13.7 ppm. The differences between FdII_ox and FdII_int were attributed to conformational changes resulting from the breaking/formation of an internal disulfide bridge. The same ¹H NMR methodology used to fully assign the three cysteinyl ligands of the [3Fe–4S] core in the oxidized state (DgFdII_ox) was used here for the assignment of the same three ligands in the intermediate state (DgFdII_int). The spin-coupling model used for the oxidized form of DgFdII where magnetic exchange coupling constants of around 300 cm⁻¹ and hyperfine coupling constants equal to 1 MHz for all the three iron centres were found, does not explain the isotropic shift temperature dependence for the three cysteinyl cluster ligands in DgFdII_int. This study, together with the spin delocalization mechanism proposed here for DgFdII_int, allows the detection of structural modifications at the [3Fe-4S] cluster in DgFdII_ox and DgFdII_int.     

EPR analysis of multiple forms of [4Fe–4S]3+ clusters in HiPIPs

The electron paramagnetic resonance (EPR) spectrum from the [4Fe–4S]³⁺ cluster in several high-potential iron–sulfur proteins (HiPIPs) is complex: it is not the pattern of a single, isolated S=1/2 system. Multifrequency EPR from 9 to 130 GHz reveals that the apparent peak positions (g values) are frequency-independent: the spectrum is dominated by the Zeeman interaction plus g-strain broadening. The spectra taken at frequencies above the X-band are increasingly sensitive to rapid-passage effects; therefore, the X-band data, which are slightly additionally broadened by dipolar interaction, were used for quantitative spectral analysis. For a single geometrical [4Fe–4S]³⁺ structure the (Fe–Fe)⁵⁺ mixed-valence dimer can be assigned in six different ways to a pair of iron ions, and this defines six valence isomers. Systematic multicomponent g-strain simulation shows that the [4Fe–4S]³⁺ paramagnets in seven HiPIPs from different bacteria each consist of three to four discernible species, and these are assigned to valence isomers of the clusters. This interpretation builds on previous EPR analyzes of [4Fe–4S]³⁺ model compounds, and it constitutes a high-resolution extension of the current literature model, proposed from paramagnetic NMR studies.     

Altered Localization of the δ Subunit of the GABAA Receptor in the Thalamus of α4 Subunit Knockout Mice

The α4 subunit of the GABA_A receptor (GABA_AR) is highly expressed in the thalamus where receptors containing the α4 and δ subunits are major mediators of tonic inhibition. The α4 subunit also exhibits considerable plasticity in a number of physiological and pathological conditions, raising questions about the expression of remaining GABA_AR subunits when the α4 subunit is absent. Immunohistochemical studies of an α4 subunit knockout (KO) mouse revealed a substantial decrease in δ subunit expression in the ventrobasal nucleus of the thalamus as well as other forebrain regions where the α4 subunit is normally expressed. In contrast, several subunits associated primarily with phasic inhibition, including the α1 and γ2 subunits, were moderately increased. Intracellular localization of the δ subunit was also altered. While δ subunit labeling was decreased within the neuropil, some labeling remained in the cell bodies of many neurons in the ventrobasal nucleus. Confocal microscopy demonstrated co-localization of this labeling with an endoplasmic reticulum marker, and electron microscopy demonstrated increased immunogold labeling near the endoplasmic reticulum in the α4 KO mouse. These results emphasize the strong partnership of the δ and α4 subunit in the thalamus and suggest that the α4 subunit of the GABA_AR plays a critical role in trafficking of the δ subunit to the neuronal surface. The findings also suggest that previously observed reductions in tonic inhibition in the α4 subunit KO mouse are likely to be related to alterations in δ subunit expression, in addition to loss of the α4 subunit.     

Binding of α-synuclein with Fe(III) and with Fe(II) and biological implications of the resultant complexes

Parkinson’s disease (PD) is hallmarked by the abnormal intracellular inclusions (Lewy bodies or LBs) in dopaminergic cells. Amyloidogenic protein α-synuclein (α-syn) and iron (including both Fe(III) and Fe(II)) are both found to be present in LBs. The interaction between iron and α-syn might have important biological relevance to PD etiology. Previously, a moderate binding affinity between α-syn and Fe(II) (5.8 × 10³ M⁻¹) has been measured, but studies on the binding between α-syn and Fe(III) have not been reported. In this work, electrospray mass spectrometry (ES-MS), cyclic voltammetry (CV), and fluorescence spectroscopy were used to study the binding between α-syn and Fe(II) and the redox property of the resultant α-syn-Fe(II) complex. The complex is of a 1:1 stoichiometry and can be readily oxidized electrochemically and chemically (by O₂) to the putative α-syn-Fe(III) complex, with H₂O₂ as a co-product. The reduction potential was estimated to be 0.025 V vs. Ag/AgCl, which represents a shift by −0.550 V vs. the standard reduction potential of the free Fe(III)/Fe(II) couple. Such a shift allows a binding constant between α-syn and Fe(III), 1.2 × 10¹³ M⁻¹, to be deduced. Despite the relatively high binding affinity, α-syn-Fe(III) generated from the oxidation of α-syn-Fe(II) still dissociates due to the stronger tendency of Fe(III) to hydrolyze to Fe(OH)₃ and/or ferrihydrite gel. The roles of α-syn and its interaction with Fe(III) and/or Fe(II) are discussed in the context of oxidative stress, metal-catalyzed α-syn aggregation, and iron transfer processes.     

XANES of carboxy and cyanomet-myoglobin The role of the distal histidine in the bent Fe−C−O configuration

The ligand bonding geometry of carboxy-and cyanomet-myoglobin (MbCO and MbCN) has been measured by the XANES method (X-ray Absorption Near Edge Structure). A comparison between the ligand bonding geometry of carboxy- and cyanomet-myoglobin and of chelated protoheme methyl ester shows that the bent Fe–C–O configuration is the same in both systems. Therefore, we suggest that this configuration is not associated with any steric contraint imposed by the side chains of the aminoacid residues at the distal side of the heme pocket.     

Modification at the 5-Position of 4-Deoxypyridoxol and α4-Norpyridoxol

In an attempt to relate structure to anticoccidial activity, a number of 5-modified analogs of 4-deoxypyridoxol (4-DOP) and α⁴-norpyridoxol have been synthesized and their biological activities examined. The compounds prepared include the 5-(3-hydroxypropyl), 5-(2-hydroxyethyl), 5-(1-hydroxyethyl), formyl and acetyl analogs of 4-DOP, and 5-(3-hydroxypropyl), formyl, ethoxycarbonyl, carbamoyl and hydroxyl analogs of α⁴-norpyridoxol. Among these compounds, 4-deoxyisopyridoxal and α⁴-norisopyridoxal were found to exhibit anticoccidal activity.     

Structural and dynamic studies of the γ-M4 trans-membrane domain of the nicotinic acetylcholine receptor

A structural characterization of a synthetic peptide corresponding to the fourth transmembrane domain (M4-TMD) of the γ-subunit of the nicotinic acetylcholine receptor from Torpedo californica has been undertaken. Solid-state NMR and CD spectroscopy studies indicate that upon reconstitution into lipid vesicles or magnetically aligned lipid bilayers, the synthetic M4-TMD adopts a linear α-helical conformation with the helix aligned within 15° of the membrane normal. Furthermore, analysis of the motional averaging of anisotropic interactions present in the solid-state NMR spectra of the reconstituted peptide, indicate that the dynamics of the peptide within the bilayer are highly sensitive to the phase adopted by the lipid bilayer, providing an insight into how the interaction of lipids with this domain may play a important role in the modulation of this receptor by its lipid environment.     

N-glycosylation of ß4 integrin controls the adhesion and motility of keratinocytes

α6?4 integrin is an essential component of hemidesmosomes and modulates cell migration in wound healing and cancer invasion. To elucidate the role of N-glycosylation on ?4 integrin, we investigated keratinocyte adhesion and migration through the re-expression of wild-type or N-glycosylation-defective ?4 integrin (ΔN?4) in ?4 integrin null keratinocytes. N-glycosylation of ?4 integrin was not essential for the heterodimer formation of ?4 integrin with α6 integrin and its expression on a cell surface, but N-glycosylation was required for integrin-mediated cell adhesion and migration. Concomitantly with the reduction of ?4 integrin in the membrane microdomain, the intracellular signals of Akt and ERK activation were decreased in cells expressing ΔN?4 integrin. Forced cross-linking of ?4 integrin rescued the decreased ERK activation in ΔN?4 integrin-expressing cells to a similar extent in wild-type ?4 integrin-expressing cells. Surprisingly, compared with cells expressing wild-type ?4 integrin, an alternation in N-glycan structures expressed on epidermal growth factor receptor (EGFR), and the induction of a stronger association between EGFR and ?4 integrin were observed in ΔN?4 integrin-expressing cells. These results clearly demonstrated that N-glycosylation on ?4 integrin plays an essential role in keratinocyte cellular function by allowing the appropriate complex formation on cell surfaces.     

Studies on the cyanophytes of Cuba 4–6

The article comprises three subsequently published studies of the taxonomy of Cuban cyanophyte/cyanobacterial flora: (4)Lyngbyopsis willei: This oscillatorialean genus and species, described byGardner (1927) from mountain creeks in Puerto Rico, was found more than 50 years later in similar localities in Cuba. The morphological variability of the Cuban populations is described and similarity with the genusSchizothrix (sect.Inactis) discussed. —(5)Cylindrospermum-species: The morphological variability of twenty-oneCylindrospermum-populations collected in Cuba was studied, documented by graphical methods and compared with the published data. Four new taxa were recognized (C. minutissimum v.rinoi, C. zonatum, C. bourrellyi, andC. desikacharyi). The other populations belong to the variation ranges ofC. breve, C. minutissimum, C. michailovskoense, andC. muscicola v.kashmiriense.— (6)Gomphosphaerioideae-species: Fifty eight populations (9 species) of the subfamilyGomphosphaerioideae (Microcystaceae, Chroococcales) from freshwater biotopes of Cuba were evaluated: Four planktic species with a probable cosmopolitan distribution were found (Coelosphaerium kuetzinginianum, C. minutissimum, Snowella lacustris andCoelomoron pusillus), and from the genusCoelomoron Buell two new species,C. microcystoides andC. vestitus were described. The tropical planktic speciesWoronichinia fremyi forms occasionally water blooms in larger reservoirs. Two tropical species from the genusGomphosphaeria Kütz, were recognized,G. multiplex (Nyg.) c. n. andG. semen-vitis sp. n.     

Structure–Function Analysis of the DNA Translocating Portal of the Bacteriophage T4 Packaging Machine

Tailed bacteriophages and herpesviruses consist of a structurally well conserved dodecameric portal at a special 5-fold vertex of the capsid. The portal plays critical roles in head assembly, genome packaging, neck/tail attachment, and genome ejection. Although the structures of portals from phages φ29, SPP1, and P22 have been determined, their mechanistic roles have not been well understood. Structural analysis of phage T4 portal (gp20) has been hampered because of its unusual interaction with the Escherichia coli inner membrane. Here, we predict atomic models for the T4 portal monomer and dodecamer, and we fit the dodecamer into the cryo-electron microscopy density of the phage portal vertex. The core structure, like that from other phages, is cone shaped with the wider end containing the “wing” and “crown” domains inside the phage head. A long “stem” encloses a central channel, and a narrow “stalk” protrudes outside the capsid. A biochemical approach was developed to analyze portal function by incorporating plasmid-expressed portal protein into phage heads and determining the effect of mutations on head assembly, DNA translocation, and virion production. We found that the protruding loops of the stalk domain are involved in assembling the DNA packaging motor. A loop that connects the stalk to the channel might be required for communication between the motor and the portal. The “tunnel” loops that project into the channel are essential for sealing the packaged head. These studies established that the portal is required throughout the DNA packaging process, with different domains participating at different stages of genome packaging.