Patterning of diverse mammalian cell types in serum free medium with photoablation

Integration of living cells with novel microdevices requires the development of innovative technologies for manipulating cells. Chemical surface patterning has been proven as an effective method to control the attachment and growth of diverse cell populations. Patterning polyelectrolyte multilayers through the combination of layer‐by‐layer self‐assembly technique and photolithography offer a simple, versatile, and silicon compatible approach that overcomes chemical surface patterning limitations, such as short‐term stability and low‐protein adsorption resistance. In this study, direct photolithographic patterning of two types of multilayers, PAA (poly acrylic acid)/PAAm (poly acryl amide) and PAA/PAH (poly allyl amine hydrochloride), were developed to pattern mammalian neuronal, skeletal, and cardiac muscle cells. For all studied cell types, PAA/PAAm multilayers behaved as a cytophobic surface, completely preventing cell attachment. In contrast, PAA/PAH multilayers have shown a cell‐selective behavior, promoting the attachment and growth of neuronal cells (embryonic rat hippocampal and NG108‐15 cells) to a greater extent, while providing little attachment for neonatal rat cardiac and skeletal muscle cells (C2C12 cell line). PAA/PAAm multilayer cellular patterns have also shown a remarkable protein adsorption resistance. Protein adsorption protocols commonly used for surface treatment in cell culture did not compromise the cell attachment inhibiting feature of the PAA/PAAm multilayer patterns. The combination of polyelectrolyte multilayer patterns with different adsorbed proteins could expand the applicability of this technology to cell types that require specific proteins either on the surface or in the medium for attachment or differentiation, and could not be patterned using the traditional methods. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Ionic events following GABA receptor activation in an identified insect motor neuron

The ionic events underlying gamma-aminobutyric acid (GABA) receptor activation on the cell body of a cockroach identified motor neuron were investigated by using current-clamp and voltage-clamp techniques. The reversal potential for GABA-induced hyperpolarization was -77.0 +/- 2.4 mV (mean +/- s.e.m.; n = 22). The reversal potential for GABA was highly sensitive to changes in external chloride, only weakly affected by changes in external potassium, and independent of changes in either sodium or calcium ion concentration. Intracellular ion-sensitive microelectrodes confirmed that an influx of chloride ions mediated the GABA response. Intracellular injection of acetate, citrate, sulphate, fluoride or ammonium caused no change in the reversal potential for GABA. Intracellular injection of chloride, bromide, chlorate, bromate, or methyl sulphate shifted the reversal potential for GABA to values more positive than resting membrane potential. Evidence for chloride accumulating and for extrusion mechanisms was examined by using putative inhibitors. However, internal application of ammonium ions, and external application of 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS), 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), acetazolamide, furosemide, ammonium, zinc and copper ions, were all without effect on the reversal potential for GABA.     

Peristaltic transport of multilayered power-law fluids with distinct viscosities: a mathematical model for intestinal flows

This paper is concerned with the theoretical study of two-dimensional peristaltic flow of power-law fluids in three layers with different viscosities. The analysis is carried out under low Reynolds number and long wavelength approximations. The shapes of the interfaces are described by a system of non-linear algebraic equations which are solved numerically as streamlines. It is found that the non-uniformity in the intermediate and peripheral layers diminishes when the viscosity of the intermediate layer is increased and that of the outermost layer is kept unaltered for both the pseudo-plastic and dilatant fluids. Similar are the observations when the viscosity of the outermost layer is increased and that of the intermediate layer is kept fixed. The flow rate increases with the viscosities of the peripheral and the intermediate layers but the viscosity of the outermost layer is more effective. However, the knowledge of the effect of the viscosity of the intermediate layer facilitates us to achieve the required flow rate without disturbing the outermost layer. An increase in the flow behaviour index too favours larger flow rates. The trapping limits increase with the viscosity of the intermediate layer but decrease with the viscosity of the outermost layer and the flow behaviour index. Thus, a medicinal intervention that creates a more viscous intermediate layer and reduces pseudo plasticity may reduce constipation.     

Fatty acid elongase is required for shoot development in rice

Organisms are covered extracellularly with cuticular waxes that consist of various fatty acids. In higher plants, extracellular waxes act as indispensable barriers to protect the plants from physical and biological stresses such as drought and pathogen attacks. However, the effect of fatty acid composition on plant development under normal growth conditions is not well understood. Here we show that the ONION1 (ONI1) gene, which encodes a fatty acid elongase (β-ketoacyl CoA synthase) involved in the synthesis of very-long-chain fatty acids, is required for correct fatty acid composition and normal shoot development in rice. oni1 mutants containing a reduced amount of very-long-chain fatty acids produced very small shoots, with an aberrant outermost epidermal cell layer, and ceased to grow soon after germination. These mutants also showed abnormal expression of a KNOX family homeobox gene. ONI1 was specifically expressed in the outermost cell layer of the shoot apical meristem and developing lateral organs. These results show that fatty acid elongase is required for formation of the outermost cell layer, and this layer is indispensable for entire shoot development in rice.     

Generation, characterization, and docking studies of DNA-hydrolyzing recombinant F(ab) antibodies

Previously we established a series of catalytic antibodies (catAbs) capable of hydrolyzing DNA prepared by hybridoma technology. A group of these catAbs exhibited high reactivity and substrate specificity. To determine the molecular basis for these catAbs, we cloned, sequenced, and expressed the variable regions of this group of antibodies as functional F(ab) fragments. The nucleotide and deduced amino acid sequences of the expressed light chain (Vκ) germline gene assignments confidently belonged to germline family Vκ1A, gene bb1.1 and GenBank accession number EF672207 while heavy chain variable region V(H) genes belonged to V(H) 1/V(H) J558, gene V130.3 and GenBank accession number EF672221. A well-established expression system based on the pARA7 vector was examined for its ability to produce catalytically active antibodies. Recombinant F(ab) (rF(ab) ) fragments were purified and their hydrolyzing activity was analyzed against supercoiled pUC19 plasmid DNA (scDNA). The study of rF(ab) provides important information about the potential catalytic activities of antibodies whose structure allows us to understand their basis of catalysis. Molecular surface analysis and docking studies were performed on the molecular interactions between the antibodies and poly(dA9), poly(dG9), poly(dT9), and poly(dC9) oligomers. Surface analysis identified the important sequence motifs at the binding sites, and different effects exerted by arginine and tyrosine residues at different positions in the light and heavy chains. This study demonstrates the potential usefulness of the protein DNA surrogate in the investigation of the origin of anti-DNA antibodies. These studies may define important features of DNA catAbs.     

pH dependence and protein selectivity of poly(ethyleneimine)/poly(acrylic acid) multilayers studied by in situ ATR-FTIR spectroscopy

The selective interaction between polyelectrolyte multilayers (PEM) consecutively adsorbed from poly(ethyleneimine) (PEI) and poly(acrylic acid) (PAC) and a binary mixture containing concanavalin A (COA) and lysozyme (LYZ) based on electrostatic interaction is reported. The composition and structure of the PEM and the uptake of proteins were analyzed by in situ attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy, and the morphology and thickness were characterized by atomic force microscopy (AFM) and ellipsometry. The PEM dissociation degree and charge state and the protein adsorption were shown to be highly dependent on the outermost layer type and the pH in solution. High protein uptake was obtained under electrostatically attractive conditions. This was used to bind selectively one protein from a binary mixture of LYZ/COA. In detail it could be demonstrated that six-layered PEM-6 at pH = 7.3 showed a preferential sorption of positively charged LYZ, while at PEM-5 and pH = 7.3 negatively charged COA could be selectively bound. No protein sorption from the binary mixture was observed at pH = 4.0 for both PEM, when COA, LYZ, and the outermost PEI layer of PEM-5 were positively charged or the outermost PAC layer of PEM-6 was neutral. Furthermore, from factor analysis of the spectral data the higher selectivity was found for PEM-5 compared to PEM-6. Increasing the ionic strength revealed a drastic decrease in the selectivity of both PEM. Evidence was found that the proteins were predominantly bound at the surface and to a minor extent in the bulk phase of PEM. These results suggest possible working regimes and application fields of PEI/PAC multilayer assemblies related to the preparative separation of binary and multicomponent protein mixtures (biofluids, food) as well as to the design of selective protein-resistant surfaces.     

Reduction of wall adsorption in capillary zone electrophoresis of a basic single-chain antibody fragment by a cationic polymeric buffer additive

Reduction of adsorptive protein-wall interactions by poly(diallyldimethyl ammonium chloride), a permanently cationic polymer, at a concentration of 0.5% (w/v) is demonstrated for a basic single-chain antibody fragment (scFv, pI about 9.5) even in the range of physiological pH of around 7. The polymer additive forms a positively charged layer at the silica surface which reverses electroosmosis and leads to electrostatic repulsion of the positively charged basic protein.     

Membrane potential of primitive red cells from chick embryo is a proton potential

The membrane potential of primitive red cells from 4- and 6-day old chick embryos has been determined using the fluorescent dye Dis-C3-(5). At day 4 the membrane potential Em was -44 mV for pH 7.4 and 20 degrees C and -36 mV at day 6. Both values are far removed from the equilibrium potential for chloride, which is about -14 mV at day 6. Changes in the external potassium, sodium or chloride concentration were without effect on the membrane potential, except at very high potassium concentrations, where a small but significant depolarization was observed at day 6. The measurements gave the same results in the absence or presence of the anion exchange blocking agent DIDS. Three pieces of evidence indicate that the membrane potential of primitive red cells is primarily caused by an electrogenic H+ conductance: 1) The measured membrane potential of -36 mV at day 6 is close to the previously determined proton equilibrium potential (Baumann and Haller, 1983) EH + of -36 mV. 2) Addition of the electrosilent Cl-/OH- exchanger tributyltin causes a significant depolarization of about 20 mV at day 4 and about 14 mV at day 6. 3) Measurement of hydrogen ion fluxes demonstrate a potential dependent proton conductance, which increases with depolarization. These results indicate that large qualitative differences exist with regard to the mechanisms involved in the generation of membrane potential and hydrogen distribution between red cell and plasma of embryonic and adult chicken.     

The sites of superoxide anion generation in higher plant mitochondria.

An impermeable charged paramagnetic amphiphile 4-(dodecyl dimethyl ammonium)-1-oxyl-2,2,6,6-tetramethyl piperidine bromide can be used as a probe of membrane surface potentials. Upon energization of photosystem II or photosystems I + II in illuminated spinach chloroplast thylakoids, a decrease occurs in the potential of the outer surface of these membranes of up to 14 mV.     

Influence of ammonium chloride feeding time and light intensity on the cultivation of Spirulina (Arthrospira) platensis

This study dealt with the influence of both the feeding time and light intensity on the fed-batch culture of the cyanobacterium Spirulina (Arthrospira) platensis using ammonium chloride as a nitrogen source. For this purpose, a 2(2) plus star central composite experimental design combined with response surface methodology was employed, and the maximum cell concentration (X(m)), the cell productivity (P(X)), and the yield of biomass on nitrogen (Y(X/N)) were selected as the response variables. The optimum values of X(m) (1,833 mg L(-1)) and Y(X/N) (5.9 g g(-1)) estimated by the model at light intensity of 13 klux and feeding time of 17.2 days were very close to those obtained experimentally under these conditions (X(m) = 1,771 +/- 41 mg L(-1); Y(X/N) = 5.7 +/- 0.17 g g(-1)). The cell productivity was a decreasing function of the ammonium chloride feeding time and a quadratic function of the light intensity. The protein and lipid contents of dry biomass collected at the end of cultivations were shown to decrease with increasing light intensity.     

Phosphonium-containing polyelectrolytes for nonviral gene delivery

Nonviral gene therapy focuses intensely on nitrogen-containing macromolecules and lipids to condense and deliver DNA as a therapeutic for genetic human diseases. For the first time, DNA binding and gene transfection experiments compared phosphonium-containing macromolecules with their respective ammonium analogs. Conventional free radical polymerization of quaternized 4-vinylbenzyl chloride monomers afforded phosphonium- and ammonium-containing homopolymers for gene transfection experiments of HeLa cells. Aqueous size exclusion chromatography confirmed similar absolute molecular weights for all polyelectrolytes. DNA gel shift assays and luciferase expression assays revealed phosphonium-containing polymers bound DNA at lower charge ratios and displayed improved luciferase expression relative to the ammonium analogs. The triethyl-based vectors for both cations failed to transfect HeLa cells, whereas tributyl-based vectors successfully transfected HeLa cells similar to Superfect demonstrating the influence of the alkyl substituent lengths on the efficacy of the gene delivery vehicle. Cellular uptake of Cy5-labeled DNA highlighted successful cellular uptake of triethyl-based polyplexes, showing that intracellular mechanisms presumably prevented luciferase expression. Endocytic inhibition studies using genistein, methyl β-cyclodextrin, or amantadine demonstrated the caveolae-mediated pathway as the preferred cellular uptake mechanism for the delivery vehicles examined. Our studies demonstrated that changing the polymeric cation from ammonium to phosphonium enables an unexplored array of synthetic vectors for enhanced DNA binding and transfection that may transform the field of nonviral gene delivery.     

Improved Performance and Stability of Inverted Organic Solar Cells with Sol–Gel Processed,Amorphous Mixed Metal Oxide Electron Extraction Layers Comprising Alkaline Earth Metals

Stability of organic photovoltaic devices (OPVs) is a limiting factor for their commercialization and still remains a major challenge whilst power conversion efficiencies are now approaching minimum requirements. The inverted organic solar cell (iOSC) architecture shows promising potential for improving significantly the cell's working lifetime. However, when solution processed ZnO is used as electron extraction layer, an undesirable light‐soaking step is commonly required before the device reaches a non‐permanent maximum performance. This work investigates the use of Sr and Ba doped ZnO films, ZnSrO and ZnBaO, formed by sol‐gel deposition using molecular alkoxide precursor solutions, as electron extraction layers in a model iOSCs with poly [3‐hexylthiophene] (P3HT): [6, 6]‐phenyl C60 butyl acid methyl ester (PCBM) as the active layer. We show that using these ternary oxides the light‐soaking step can be circumvented by preventing a dipole forming between the oxide and the active organic layer as supported by electroabsorption spectroscopy measurements of the device built‐in field. It is suggested that Sr or Ba doping results in suppression/reduction of the oxygen adsorption at mobile oxygen vacancy sites on the metal oxide surface. Like in thin film transistor (TFT) applications, where materials like InGaZnO are rapidly becoming an important technology, the use of amorphous, mixed metal oxides allows improving the performance and stability of interfacial charge extraction layers for organic solar cells.     

Gene delivery using biodegradable polyelectrolyte microcapsules prepared through the layer-by-layer technique

Biodegradable and non‐biodegradable microcapsules were prepared via the layer‐by‐layer (LbL) technique consisting of the polyelectrolyte pairs of dextran sulphate/poly‐L ‐arginine and poly(styrene sulfonate)/poly(allylamine hydrochloride), respectively, in an attempt to encapsulate plasmid DNA (pDNA) for efficient transfection into NIH 3T3 cells. Results indicated the retention of bioactivity in the encased pDNA, as well as a correlation between the level of in vitro gene expression and biodegradability properties of polyelectrolyte. Furthermore, the incorporation of iron oxide nanoparticles within the polyelectrolyte layers significantly improved the in vitro transfection efficiency of the microcapsules. As a novel pDNA delivery system, the reported biodegradable microcapsules provide useful insight into plasmid‐based vaccination and where there is a prerequisite to deliver genes into cells capable of phagocytosis. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1088–1094, 2012     

Effect of Polyelectrolytes on Catalytic Activity of Alcohol Dehydrogenase

Fluorescent and optical spectroscopy were used to study the interaction of alcohol dehydrogenase (ADH) with negatively charged polystyrene sulfonate (PSS) and dextran sulfate (DS), as well as positively charged poly(diallyldimethylammonium) (PDADMA). As found, DS and PDADMA did not affect the structural and catalytic enzyme properties. In contrast, PSS slightly decreased the protein self-fluorescence over 1 h of incubation, which is associated with partial destruction of its quaternary (globular) structure. Investigation of the ADH activity with and without PSS showed its dependency on the incubation time and the PSS presence. Sodium chloride (2.0 and 0.2 M) or ammonium sulfate (0.1 M) added to the reaction mixture did not completely protect the enzyme quaternary structure from the PSS action. However ammonium sulfate or 0.2 M sodium chloride stabilized the enzyme and partially inhibited the negative PSS effect.     

Cationic Liposome-Mediated Gene Delivery Via Systemic Administration

Abstract

Cationic liposomes have been studied as a potential carrier for delivering genes to cells for the purpose of gene therapy. This report summarizes our efforts to characterize the in vivo expression of transgene delivered by cationic liposomes via intravenous administrtion. Using a CMV driven gene expression system containing cDNA of luciferase or green fluorescence protein gene as a reporter and two commonly used cationic lipids, 2, 3-dioleoyloxypropyl-1-trimethyl ammonium chloride (DOTMA) and 2, 3-dioleoyloxyl-1-trimethylammonium propanyl chloride (DOTAP), we demonstrate that a significant level of gene expression can be obtained in different organs including the lung, heart, spleen, liver and kidneys following intravenous administration in the mouse. Our finding show that the transfection efficiency of cationic liposomes is determined by the structure of the cationic lipids, the lipid composition of liposomes and cationic lipid to DNA ratio. Furthermore, gene expression was short in duration, peaked between 4-24 hours post injection, and dropped to less than 1% of the peak level within a 4 day period. Experiments with repeated injections revealed that cells initially transfected by the first transfection were not fully responsive to the subsequent second transfection for approximately 14 days.     

Cell interactions with polyelectrolyte multilayer films

The short-term interactions of chondrosarcoma cells with polyelectrolyte multilayer films built up by the alternate adsorption of poly(L-lysine) (PLL) and poly(L-glutamic acid) (PGA) was studied in the presence and in the absence of serum. The films and their interaction with serum proteins were first characterized by means of optical waveguide lightmode spectroscopy, quartz crystal microbalance, and zeta potential measurements. In a serum-containing medium, the detachment forces measured by the micropipet technique were about eight times smaller on PGA-ending than on PLL-ending films. For these latter ones, the adhesion force decreased when the film thickness increased. In a serum-free medium, the differences between the negative- and positive-ending films were enhanced: adhesion forces on PLL-ending films were 40-100% higher, whereas no cellular adherence was found on PGA-terminating films. PGA-ending films were found to prevent the adsorption of serum proteins, whereas important protein adsorption was always observed on PLL-ending films. These results show how cell interactions with polyelectrolyte films can be tuned by the type of the outermost layer, the presence of proteins, and the number of layers in the film.     

Significant increase in Young׳s modulus of ATDC5 cells during chondrogenic differentiation induced by PAMPS/PDMAAm double-network gel: Comparison with induction by insulin

A double-network (DN) gel, which was composed of poly(2-acrylamido-2-methylpropanesulfonic acid) and poly(N,N′-dimethyl acrylamide) (PAMPS/PDMAAm), has the potential to induce chondrogenesis both in vitro and in vivo. The present study investigated the biomechanical and biological responses of chondrogenic progenitor ATDC5 cells cultured on the DN gel. ATDC5 cells were cultured on a polystyrene surface without insulin (Culture 1) and with insulin (Culture 2), and on the DN gel without insulin (Culture 3). The cultured cells were evaluated using micropipette aspiration for cell Young?s modulus and qPCR for gene expression of chondrogenic and actin organization markers on days 3, 7 and 14. On day 3, the cells in Culture 3 formed nodules, in which the cells exhibited an actin cortical layer inside them, and gene expression of type-II collagen, aggrecan, and SOX9 was significantly higher in Culture 3 than Cultures 1 and 2 (p<0.05). Young?s modulus in Culture 3 was significantly higher than that in Culture 1 throughout the testing period (p<0.05) and that in Culture 2 on day 14 (p<0.01). There was continuous expression of actin organization markers in Culture 3. This study highlights that the cells on the DN gel increased the modulus and mRNA expression of chondrogenic markers at an earlier time point with a greater magnitude compared to those on the polystyrene surface with insulin. This study also demonstrates a possible strong interrelation among alteration of cell mechanical properties, changes in actin organization and the induction of chondrogenic differentiation.     

Evidence for electrogenic proton extrusion by subepidermal cells of Lemna paucicostata 6746

The cell potential of Lemna paucicostata 6746 was measured between the vacuole and the external solution. The potential in the dark (-202 mV) could be depolarized with 0.1 mM dicyclohexyl carbodiimide (DCCD) or 1 mM arsenate to-81 mV. The hyperpolarization above the latter value is therefore attributed to an ATP-dependent process. The cell potential showed a significant dependence upon the pH of the external solution. The change in the potential induced by a jump in pH between two certain values, was reversible and independent of the mode of performing the pH change (stepwise or at once). The DCCD-or arsenate-depolarized potential did not respond to external pH changes. A 0.1 mM ammonium chloride solution depolarized the cell potential reversibly to-83 mV. This potential-change could be greatly reduced by simultaneous addition of 5 mM Na isobutyrate. The pH sensitivity of the cell potential is ascribed to changes in the rate of proton extrusion upon altering the proton gradient across the plasmalemma. The effects of ammonium and isobutyrate are interpreted as being the consequence of pH shifts at the inner face of the plasmalemma, caused by the permeation of the undissociated form of the weak acid or base. A critical discussion of an alternative interpretation for the ammonium effect is presented.Abbreviation DCCD N,N-dicyclohexyl carbodiimide     

Protons and calcium alter gating of the hyperpolarization-activated cation current (I(h)) in rod photoreceptors

We investigated the effects of protons and calcium ions on the voltage-dependent gating of the hyperpolarization-activated, nonselective cation channel current, I(h), in rod photoreceptors. I(h) is a cesium-sensitive current responsible for the peak-plateau sag during the rod response to bright light. The voltage dependence of I(h) activation shifted about 5 mV per pH unit, with external acidification producing positive shifts and alkalinization producing negative shifts. Increasing external [Ca(2+)] from 3 to 20 mM resulted in a large (approximately 17 mV) positive shift in I(h) activation. External [Ca(2+)] (20 mM) blocked pH-induced shifts in activation. Cytoplasmic acidification produced by 25 mM sodium acetate led to a negative shift in inactivation (-9 mV) and internal alkalinization produced with 20 mM ammonium chloride resulted in a positive shift (+6 mV). Surface charge binding and screening theory (Gouy-Chapman-Stern) accounted for the observed shifts in I(h) activation, with the best fit achieved when protons and calcium ions were assumed to bind to distinct sites on the membrane. Since light induces changes in the retinal ionic environment, these results permit us to gauge the degree to which rod light responses could be modified via alterations in I(h) activation.     

Redox and functional analysis of the Rieske ferredoxin component of the toluene 4-monooxygenase

Toluene 4-monooxygenase catalyzes the NADH- and O2-dependent hydroxylation of toluene to form p-cresol. The four-protein complex consists of a diiron hydroxylase, an oxidoreductase, a catalytic effector protein, and a Rieske-type ferredoxin (T4moC). Phylogenetic analysis suggests that T4moC is part of a clade specialized for reaction with diiron hydroxylases, possibly reflected in the conservation of W69, whose indole side chain makes close contacts with a bridging sulfide. In order to further investigate the possible origins of this specialization, T4moC, mutated variants of T4moC, and three other purified ferredoxins (the Thermus Rieske protein, the Burkholderia cepacia Rieske-type biphenyl dioxygenase ferredoxin BphF, and the Ralstonia pickettii PK01 toluene monooxygenase TbuB, the Rieske-type ferredoxin from another diiron monooxygenase complex) were studied by redox potential measurements and their ability to complement the catalytic function of the reconstituted toluene 4-monooxygenase complex. A saturation mutagenesis of T4moC W69 indicates that an aromatic residue may modulate the redox potential and is also necessary for activity and/or stability. The redox potential of T4moC was determined to be -173 mV, W69F T4moC was -139 mV, and TbuB was -150 mV. For comparison, BphF had a redox potential of -157 mV [Couture et al. (2001) Biochemistry 40, 84-92]. Of these ferredoxins, all except BphF were able to provide catalytic activity. Given the range in redox potentials observed in the active ferredoxins, shape and electrostatics are strongly implicated in the catalytic specialization. Mutagenesis of other T4moC surface residues gave further insight into possible origins of catalytic specialization. Thus R65A T4moC gave an alteration in apparent KM only, while D82A/D83A T4moC gave alterations in both apparent kcat and KM. Since the different catalytic results were obtained by mutagenesis of residues lying on different sides of the protein adjacent to the [2Fe-2S] cluster, the results suggest that two different faces of T4moC may be involved in protein-protein interactions during catalysis.