Effective binding of Tb3+ and La3+ cations on the donor side of Mn-depleted photosystem II.

The interaction of Tb3+ and La3+ cations with different photosystem II (PSII) membranes (intact PSII, Ca-depleted PSII (PSII[-Ca]) and Mn-depleted PSII (PSII[-Mn]) membranes) was studied. Although both lanthanide cations (Ln3+) interact only with Ca2+-binding site of oxygen-evolving complex (OEC) in PSII and PSII(-Ca) membranes, we found that in PSII(-Mn) membranes both Ln3+ ions tightly bind to another site localized on the oxidizing side of PSII. Binding of Ln3+ cations to this site is not protected by Ca2+ and is accompanied by very effective inhibition of Mn2+ oxidation at the high-affinity (HA) Mn-binding site ([Mn2+ + H2O2] couple was used as a donor of electrons). The values of the constant for inhibition of electron transport Ki are equal to 2.10 ± 0.03 µM for Tb3+ and 8.3 ± 0.4 µM for La3+, whereas OEC inhibition constant in the native PSII membranes is 323 ± 7 µM for Tb3+. The value of Ki for Tb3+ corresponds to Ki for Mn2+ cations in the reaction of diphenylcarbazide oxidation via HA site (1.5 µM) presented in the literature. Our results suggest that Ln3+ cations bind to the HA Mn-binding site in PSII(-Mn) membranes like Mn2+ or Fe2+ cations. Taking into account the fact that Mn2+ and Fe2+ cations bind to the HA site as trivalent cations after light-induced oxidation and the fact that Mn cation bound to the HA site (Mn4) is also in trivalent state, we can suggest that valency may be important for the interaction of Ln3+ with the HA site.

A NiRhS fuel cell catalyst - lessons from hydrogenase.

We present a novel fuel cell heterogeneous catalyst based on rhodium, nickel and sulfur with power densities 5-28% that of platinum. The NiRhS heterogeneous catalyst was developed via a homogeneous model complex of the [NiFe]hydrogenases (H2ases) and can act as both the cathode and anode of a fuel cell.

Understanding the chemistry of the artificial electron acceptors PES, PMS, DCPIP and Wurster's Blue in methanol dehydrogenase assays.

Methanol dehydrogenases (MDH) have recently taken the spotlight with the discovery that a large portion of these enzymes in nature utilize lanthanides in their active sites. The kinetic parameters of these enzymes are determined with a spectrophotometric assay first described by Anthony and Zatman 55 years ago. This artificial assay uses alkylated phenazines, such as phenazine ethosulfate (PES) or phenazine methosulfate (PMS), as primary electron acceptors (EAs) and the electron transfer is further coupled to a dye. However, many groups have reported problems concerning the bleaching of the assay mixture in the absence of MDH and the reproducibility of those assays. Hence, the comparison of kinetic data among MDH enzymes of different species is often cumbersome. Using mass spectrometry, UV-Vis and electron paramagnetic resonance (EPR) spectroscopy, we show that the side reactions of the assay mixture are mainly due to the degradation of assay components. Light-induced demethylation (yielding formaldehyde and phenazine in the case of PMS) or oxidation of PES or PMS as well as a reaction with assay components (ammonia, cyanide) can occur. We suggest here a protocol to avoid these side reactions. Further, we describe a modified synthesis protocol for obtaining the alternative electron acceptor, Wurster's blue (WB), which serves both as EA and dye. The investigation of two lanthanide-dependent methanol dehydrogenases from Methylorubrum extorquens AM1 and Methylacidiphilum fumariolicum SolV with WB, along with handling recommendations, is presented. Lanthanide-dependent methanol dehydrogenases. Understanding the chemistry of artificial electron acceptors and redox dyes can yield more reproducible results.

Potential complementation effects of two disease-associated mutations in tetrameric glutaryl-CoA dehydrogenase is due to inter subunit stability-activity counterbalance.

Glutaric Aciduria Type I (GA-I), is an autosomal recessive neurometabolic disease caused by mutations in the GCDH gene that encodes for glutaryl-CoA dehydrogenase (GCDH), a flavoprotein involved in the metabolism of tryptophan, lysine and hydroxylysine. Although over 200 disease mutations have been reported a clear correlation between genotype and phenotype has been difficult to establish. To contribute to a better molecular understanding of GA-I we undertook a detailed molecular study on two GCDH disease-related variants, GCDH-p.Arg227Pro and GCDH-p.Val400Met. Heterozygous patients harbouring these two mutations have increased residual enzymatic activity in relation to homozygous patients with only one of the mutations, suggesting a complementation effect between the two. Combining biochemical, biophysical and structural methods we here establish the effects of these mutations on protein folding, stability and catalytic activity. We show that both variants retain the overall protein fold, but with compromised enzymatic activities. Detailed enzyme kinetic studies reveal that GCDH-p.Arg227Pro has impaired function due to deficient substrate affinity as evidenced by its higher Km, and that the lower activity in GCDH-p.Val400Met results from weaker interactions with its physiological redox partner (electron transfer flavoprotein). Moreover, the GCDH-p.Val400Met variant has a significantly lower thermal stability (ΔTm ≈ 9 °C), and impaired binding of the FAD cofactor in relation to wild-type protein. On these grounds, we provide a rational for the possible interallelic complementation observed in heterozygous patients based on the fact that in GCDH, the low active p.Arg227Pro variant contributes to stabilize the tetramer while the structurally unstable p.Val400Met variant compensates for enzyme activity.

Characterization of pyranose oxidase variants for bioelectrocatalytic applications.

Pyranose oxidase (POx) catalyzes the oxidation of d-glucose to 2-ketoglucose with concurrent reduction of oxygen to H2O2. POx from Trametes ochracea (ToPOx) is known to react with alternative electron acceptors including 1,4-benzoquinone (1,4-BQ), 2,6-dichlorophenol indophenol (DCPIP), and the ferrocenium ion. In this study, enzyme variants with improved electron acceptor turnover and reduced oxygen turnover were characterized as potential anode biocatalysts. Pre-steady-state kinetics of the oxidative half-reaction of ToPOx variants T166R, Q448H, L545C, and L547R with these alternative electron acceptors were evaluated using stopped-flow spectrophotometry. Higher kinetic constants were observed as compared to the wild-type ToPOx for some of the variants. Subsequently, the variants were immobilized on glassy carbon electrodes. Cyclic voltammetry measurements were performed to measure the electrochemical responses of these variants with glucose as substrate in the presence of 1,4-BQ, DCPIP, or ferrocene methanol as redox mediators. High catalytic efficiencies (Imaxapp/KMapp) compared to the wild-type POx proved the potential of these variants for future bioelectrocatalytic applications, in biosensors or biofuel cells. Among the variants, L545C showed the most desirable properties as determined kinetically and electrochemically.

In silico and crystallographic studies identify key structural features of biliverdin IXß reductase inhibitors having nanomolar potency.

Heme cytotoxicity is minimized by a two-step catabolic reaction that generates biliverdin (BV) and bilirubin (BR) tetrapyrroles. The second step is regulated by two non-redundant biliverdin reductases (IXα (BLVRA) and IXß (BLVRB)), which retain isomeric specificity and NAD(P)H-dependent redox coupling linked to BR's antioxidant function. Defective BLVRB enzymatic activity with antioxidant mishandling has been implicated in metabolic consequences of hematopoietic lineage fate and enhanced platelet counts in humans. We now outline an integrated platform of in silico and crystallographic studies for the identification of an initial class of compounds inhibiting BLVRB with potencies in the nanomolar range. We found that the most potent BLVRB inhibitors contain a tricyclic hydrocarbon core structure similar to the isoalloxazine ring of flavin mononucleotide and that both xanthene- and acridine-based compounds inhibit BLVRB's flavin and dichlorophenolindophenol (DCPIP) reductase functions. Crystallographic studies of ternary complexes with BLVRB-NADP+-xanthene-based compounds confirmed inhibitor binding adjacent to the cofactor nicotinamide and interactions with the Ser-111 side chain. This residue previously has been identified as critical for maintaining the enzymatic active site and cellular reductase functions in hematopoietic cells. Both acridine- and xanthene-based compounds caused selective and concentration-dependent loss of redox coupling in BLVRB-overexpressing promyelocytic HL-60 cells. These results provide promising chemical scaffolds for the development of enhanced BLVRB inhibitors and identify chemical probes to better dissect the role of biliverdins, alternative substrates, and BLVRB function in physiologically relevant cellular contexts.

[Competition between redox mediator and oxygen in the microbial fuel cell].

The maximal rates and effective constants of 2,6-dichlorphenolindophenol and oxygen reduction by bacterim Gluconobacter oxydans in bacterial fuel cells under different conditions were evaluated. In an open-circuit mode, the rate of 2,6-dichlorphenolindophenol reduction coupled with ethanol oxidation under oxygen and nirogen atmospheres were 1.0 and 1.1 µM s­1 g­1, respectively. In closed-circuit mode, these values were 0.4 and 0.44 µM s­1 g­1, respectively. The initial rate of mediator reduction with the use of membrane fractions of bacteria in oxygen and nitrogen atmospheres in open-circuit mode were 6.3 and 6.9 µM s­1 g­1, whereas these values in closed-circuit mode comprised 2.2 and 2.4 µM s­1 g­1, respectively. The oxygen reduction rates in the presence and absence of 2,6-dichlorphenolindophenol were 0.31 and 0.32 µM s­1 g­1, respectively. The data obtained in this work demonstrated independent electron transfer from bacterial redox centers to the mediator and the absence of competition between the redox mediator and oxygen. The results can make it possible to reduce costs of microbial fuel cells based on activity of acetic acid bacteria G. oxydans.

Oxygen-Evolving Porous Glass Plates Containing the Photosynthetic Photosystem II Pigment-Protein Complex.

The development of artificial photosynthesis has focused on the efficient coupling of reaction at photoanode and cathode, wherein the production of hydrogen (or energy carriers) is coupled to the electrons derived from water-splitting reactions. The natural photosystem II (PSII) complex splits water efficiently using light energy. The PSII complex is a large pigment-protein complex (20 nm in diameter) containing a manganese cluster. A new photoanodic device was constructed incorporating stable PSII purified from a cyanobacterium Thermosynechococcus vulcanus through immobilization within 20 or 50 nm nanopores contained in porous glass plates (PGPs). PSII in the nanopores retained its native structure and high photoinduced water splitting activity. The photocatalytic rate (turnover frequency) of PSII in PGP was enhanced 11-fold compared to that in solution, yielding a rate of 50-300 mol e(-)/(mol PSII·s) with 2,6-dichloroindophenol (DCIP) as an electron acceptor. The PGP system realized high local concentrations of PSII and DCIP to enhance the collisional reactions in nanotubes with low disturbance of light penetration. The system allows direct visualization/determination of the reaction inside the nanotubes, which contributes to optimize the local reaction condition. The PSII/PGP device will substantively contribute to the construction of artificial photosynthesis using water as the ultimate electron source.

Identification of KPNB1 as a Cellular Target of Aminothiazole Derivatives with Anticancer Activity.

We found that aminothiazole derivative (E)-N-(5-benzylthiazol-2-yl)-3-(furan-2-yl)acrylamide (1) has strong anticancer activity, and undertook proteomics approaches to identify the target protein of compound 1, importin ß1 (KPNB1). A competitive binding assay using fluorescein-labeled 1 showed that 1 has strong binding affinity for KPNB1 (Kd : ∼20 nm). Furthermore, through western blotting assays for KPNB1, KPNA2, EGFR, ErbB2, and STAT3, we confirmed that 1 has inhibitory effects on the importin pathway. KPBN1 appears to be overexpressed in several cancer cells, and siRNA-induced inhibition of KPNB1 shows significant inhibition of cancer cell proliferation, while leaving non-cancerous cells unaffected. Therefore, compound 1 is a promising new lead for the development of KPNB1-targeted anticancer agents. Fluorescein-labeled 1 could be a useful quantitative probe for the development of novel KPNB1 inhibitors.

Photocatalytic parameters and kinetic study for degradation of dichlorophenol-indophenol (DCPIP) dye using highly active mesoporous TiO2 nanoparticles.

Highly active mesoporous TiO2 of about 6nm crystal size and 280.7m(2)/g specific surface areas has been successfully synthesized via controlled hydrolysis of titanium butoxide at acidic medium. It was characterized by means of XRD (X-ray diffraction), SEM (scanning electron microscopy), TEM (transmission electron microscopy), FT-IR (Fourier transform infrared spectroscopy), TGA (thermogravimetric analysis), DSC (differential scanning calorimetry) and BET (Brunauer-Emmett-Teller) surface area. The degradation of dichlorophenol-indophenol (DCPIP) under ultraviolet (UV) light was studied to evaluate the photocatalytic activity of samples. The effects of different parameters and kinetics were investigated. Accordingly, a complete degradation of DCPIP dye was achieved by applying the optimal operational conditions of 1g/L of catalyst, 10mg/L of DCPIP, pH of 3 and the temperature at 25±3°C after 3min under UV irradiation. Meanwhile, the Langmuir-Hinshelwood kinetic model described the variations in pure photocatalytic branch in consistent with a first order power law model. The results proved that the prepared TiO2 nanoparticle has a photocatalytic activity significantly better than Degussa P-25.

Acceleration of reaction in charged microdroplets.

Using high-resolution mass spectrometry, we have studied the synthesis of isoquinoline in a charged electrospray droplet and the complexation between cytochrome c and maltose in a fused droplet to investigate the feasibility of droplets to drive reactions (both covalent and noncovalent interactions) at a faster rate than that observed in conventional bulk solution. In both the cases we found marked acceleration of reaction, by a factor of a million or more in the former and a factor of a thousand or more in the latter. We believe that carrying out reactions in microdroplets (about 1-15 µm in diameter corresponding to 0·5 pl - 2 nl) is a general method for increasing reaction rates. The mechanism is not presently established but droplet evaporation and droplet confinement of reagents appear to be two important factors among others. In the case of fused water droplets, evaporation has been shown to be almost negligible during the flight time from where droplet fusion occurs and the droplets enter the heated capillary inlet of the mass spectrometer. This suggests that (1) evaporation is not responsible for the acceleration process in aqueous droplet fusion and (2) the droplet-air interface may play a significant role in accelerating the reaction. We argue that this 'microdroplet chemistry' could be a remarkable alternative to accelerate slow and difficult reactions, and in conjunction with mass spectrometry, it may provide a new arena to study chemical and biochemical reactions in a confined environment.

Evaluation of 2,6-dichlorophenolindophenol acetate as a substrate for acetylcholinesterase activity assay.

Ellman's method is a standard protocol for the determination of cholinesterases activity. Though the method is ready for laboratory purposes, it has some drawbacks as well. In the current article, 2,6-dichloroindophenol acetate is performed as a chromogenic substrate suitable for acetylcholinesterase (AChE) activity examination. Michaelis constant and maximal velocity for 2,6-dichloroindophenol acetate were determined (38.0 µM and 244 pkat) and compared to the values for acetythiocholine (K(m) 0.18 mM; V(max) 5.1 nkat). Docking for 2,6-dichloroindophenol acetate and human AChE was done as well. In conclusion, 2,6-dichloroindophenol acetate seems to be suitable chromogenic substrate for AChE and spectrophotometry and based on this it can be easily performed whenever AChE activity should be tested.

Theta-glass capillaries in electrospray ionization: rapid mixing and short droplet lifetimes.

Double-barrel wire-in-a-capillary electrospray emitters prepared from theta-glass capillaries were used to mix solutions during the electrospray process. The relative flow rate of each barrel was continuously monitored with internal standards. The complexation reaction of 18-crown-6 and K(+), introduced from opposite barrels, reaches equilibrium during the electrospray process, suggesting that complete mixing also occurs. A simplified diffusion model suggests that mixing occurs in less than a millisecond, and contributions of turbulence, estimated from times of coalescing ballistic microdroplets, suggest that complete mixing occurs within a few microseconds. This mixing time is 2 orders of magnitude less than in any mixer previously coupled to a mass spectrometer. The reduction of 2,6-dichloroindophenol by l-ascorbic acid was performed using the theta-glass emitters and monitored using mass spectrometry. On the basis of the rate constant of this reaction in bulk solution, an apparent reaction time of 274 ± 60 µs was obtained. This reaction time is an upper limit to the droplet lifetime because the surface area to volume ratio and the concentration of reagents increase as the droplet evaporates and some product formation occurs in the Taylor cone prior to droplet formation. On the basis of increases in reaction rates measured by others in droplets compared to rates in bulk solution, the true droplet lifetime is likely 1-3 orders of magnitude less than the upper limit, i.e., between 27 µs and 270 ns. The rapid mixing and short droplet lifetime achieved in these experiments should enable the monitoring of many different fast reactions using mass spectrometry.

Photoactive films of photosystem I on transparent reduced graphene oxide electrodes.

Photosystem I (PSI) is a photoactive electron-transport protein found in plants that participates in the process of photosynthesis. Because of PSI's abundance in nature and its efficiency with charge transfer and separation, there is a great interest in applying the protein in photoactive electrodes. Here, we developed a completely organic, transparent, conductive electrode using reduced graphene oxide (RGO) on which a multilayer of PSI could be deposited. The resulting photoactive electrode demonstrated current densities comparable to that of a gold electrode modified with a multilayer film of PSI and significantly higher than that of a graphene electrode modified with a monolayer film of PSI. The relatively large photocurrents produced by integrating PSI with RGO and using an opaque, organic mediator can be applied to the facile production of more economic solar energy conversion devices.

The two common polymorphic forms of human NRH-quinone oxidoreductase 2 (NQO2) have different biochemical properties.

There are two common forms of NRH-quinone oxidoreductase 2 (NQO2) in the human population resulting from SNP rs1143684. One has phenylalanine at position 47 (NQO2-F47) and the other leucine (NQO2-L47). Using recombinant proteins, we show that these variants have similar steady state kinetic parameters, although NQO2-L47 has a slightly lower specificity constant. NQO2-L47 is less stable towards proteolytic digestion and thermal denaturation than NQO2-F47. Both forms are inhibited by resveratrol, but NQO2-F47 shows negative cooperativity with this inhibitor. Thus these data demonstrate, for the first time, clear biochemical differences between the variants which help explain previous biomedical and epidemiological findings.

[Bioanode for a microbial fuel cell based on Gluconobacter oxydans inummobilized into a polymer matrix].

Acetic acid bacteria Gluconobacter oxydans subsp. industrius RKM V-1280 were immobilized into a synthetic matrix based on polyvinyl alcohol modified with N-vinylpyrrolidone and used as biocatalysts for the development ofbioanodes for microbial fuel cells. The immobilization method did not significantly affect bacterial substrate specificity. Bioanodes based on immobilized bacteria functioned stably for 7 days. The maximum voltage (fuel cell signal) was reached when 100-130 µM of an electron transport mediator, 2,6-dichlorophenolindophenol, was added into the anode compartment. The fuel cell signals reached a maximum at a glucose concentration higher than 6 mM. The power output of the laboratory model of a fuel cell based on the developed bioanode reached 7 mW/m2 with the use of fermentation industry wastes as fuel.

Convenient microtiter plate-based, oxygen-independent activity assays for flavin-dependent oxidoreductases based on different redox dyes.

Flavin-dependent oxidoreductases are increasingly recognized as important biocatalysts for various industrial applications. In order to identify novel activities and to improve these enzymes in engineering approaches, suitable screening methods are necessary. We developed novel microtiter-plate-based assays for flavin-dependent oxidases and dehydrogenases using redox dyes as electron acceptors for these enzymes. 2,6-dichlorophenol-indophenol, methylene green, and thionine show absorption changes between their oxidized and reduced forms in the visible range, making it easy to judge visually changes in activity. A sample set of enzymes containing both flavoprotein oxidases and dehydrogenases - pyranose 2-oxidase, pyranose dehydrogenase, cellobiose dehydrogenase, D-amino acid oxidase, and L-lactate oxidase - was selected. Assays for these enzymes are based on a direct enzymatic reduction of the redox dyes and not on the coupled detection of a reaction product as in the frequently used assays based on hydrogen peroxide formation. The different flavoproteins show low Michaelis constants with these electron acceptor substrates, and therefore these dyes need to be added in only low concentrations to assure substrate saturation. In conclusion, these electron acceptors are useful in selective, reliable and cheap MTP-based screening assays for a range of flavin-dependent oxidoreductases, and offer a robust method for library screening, which could find applications in enzyme engineering programs.

Parallel measurements of reaction kinetics using ultralow-volumes.

We present a new platform for the production and manipulation of microfluidic droplets in view of measuring the evolution of a chemical reaction. Contrary to existing approaches, our device uses gradients of confinement to produce a single drop on demand and guide it to a pre-determined location. In this way, two nanoliter drops containing different reagents can be placed in contact and merged together, in order to trigger a chemical reaction. The reaction rate is extracted from an analysis of the observed reaction-diffusion front. We show that the results obtained using this platform are in excellent agreement with stopped-flow measurements, while decreasing the sample consumption 5000 fold. We also show how the device operation can be parallelized in order to react an initial sample with a range of compounds or concentrations, on a single integrated chip. This integrated chip thus further reduces sample consumption while reducing the time required for the experimental runs from hours to minutes.

Kinetic and isotopic characterization of L-proline dehydrogenase from Mycobacterium tuberculosis.

The monofunctional proline dehydrogenase (ProDH) from Mycobacterium tuberculosis performs the flavin-dependent oxidation of l-proline to Δ(1)-pyrroline-5-carboxylate in the proline catabolic pathway. The ProDH gene, prub, was cloned into the pYUB1062 vector, and the C-terminal His-tagged 37 kDa protein was expressed and purified by nickel affinity chromatography. A steady-state kinetic analysis revealed a ping-pong mechanism with an overall kcat of 33 ± 2 s(-1) and Km values of 5.7 ± 0.8 mM and 3.4 ± 0.3 µM for l-proline and 2,6-dichlorophenolindophenol (DCPIP), respectively. The pH dependence of kcat revealed that one enzyme group exhibiting a pK value of 6.8 must be deprotonated for optimal catalytic activity. Site-directed mutagenesis suggests that this group is Lys110. The primary kinetic isotope effects on V/KPro and V of 5.5 and 1.1, respectively, suggest that the transfer of hydride from l-proline to FAD is rate-limiting for the reductive half-reaction, but that FAD reoxidation is the rate-limiting step in the overall reaction. Solvent and multiple kinetic isotope effects suggest that l-proline oxidation occurs in a stepwise rather than concerted mechanism. Pre-steady-state kinetics reveal an overall kred of 88.5 ± 0.7 s(-1), and this rate is subject to a primary kinetic isotope effect of 5.2. These data confirm that the overall reaction is limited by reduced flavin reoxidation in the second half-reaction.