Gx Values Research Articles

Synergistic analysis of wake effect of two cylinders on energy harvesting characteristics of piezoelectric flag

In this study, the effect of asymmetric wake flow regime of two side-by-side cylindrical bluff bodies on power output is experimentally examined by using a piezoelectric flag. Different synchronization modes of the flag with wake flow are observed. It is demonstrated that the streamwise gap between the flag and cylinders (Gx), and the center-to-center gap between cylinders have a significant impact on the flag's dynamical behavior that results in a fluctuation in the power output of the piezoelectric flag. The levels of output power are analyzed by varying the Gx and the cross-stream or lateral gap (N/d) between the two cylinders. N/d values from 1.0 to 2.0 for different values of Gx (2.0 ≤ Gx ≤ 4.0) are experimentally tested. The comparison of the flapping response at each point is made to ascertain the impact of the harvester's dynamic behavior on the output energy. The power generated at each point is recorded for all cases and a comparative analysis is made to find the optimal configuration. Limited research is conducted in the past to enhance the energy output by using the bluff body with the improved wake dynamics. Hence, two cylinders are employed in a uniform flow and crosswise gap between cylinders is varied to change the characteristics of the wake region. The cylinder arrangement with N/d = 1.0, shows continuous oscillations and higher output power persisting for 2.0 ≤ Gx ≤ 4.0. The monotonic rise in power output is observed till Gx = 4.0. The stated configurations with N/d = 1.0 gives a significant advantage over a single-cylinder-based energy harvester as a kinetic source of fluid energy harvester from the flowing fluid. The output power became almost doubled with an increase of 95% approximately using side-by-side arrangement.

Heat transfer enhancement in a channel flow using two wall-mounted flexible flags with a confined cylinder

A heat transfer system to improve the thermal performance of a channel flow with two symmetrically wall-mounted flexible flags is proposed by arranging a confined cylinder in an upstream region of the flags. The effects of the horizontal gap distance between the cylinder center and the wall-mounted position of the downstream flags (Gx) and the bending rigidity of the flags (γ) on the vortex dynamics are examined based on numerical simulations of the fluid-structure-thermal interaction using a penalty-immersed boundary method. Four distinct flapping modes of the two downstream flags are identified in the regime map of (Gx, γ): an out-of-phase flapping mode with symmetric motion of the flags (OS mode) and an out-of-phase flapping mode with non-symmetric motion of the flags (ON mode), and an in-phase flapping mode (I mode) and combined flapping mode in which the I mode and ON mode are presented alternately with time (ION mode). Heat transfer systems consisting of two flags under the OS and ON modes achieve improved thermal efficiency compared to that for a clean channel system (CS) due to vortex-to-vortex interactions promoting fluid mixing between the core cold fluid and near-wall hot fluid. However, the thermal efficiency for the systems in the I and ION modes decreases due to the small increase in the heat transfer rate for the I mode and a large deficit of the mechanical energy loss for the ION mode, respectively. When the values of Gx, γ and the Reynolds number are optimal, the present system in the OS mode results in an increase of the heat transfer rate by approximately 177% with an accompanying increase of the thermal efficiency up to 24% compared to that for the CS (despite an increase of the mechanical energy loss of 1021%).

Spacecraft launch and re-entry: Effects of simulated +Gx acceleration on cardiorespiratory parameters

Introduction: In the spaceflight, during launch and re-entry, the crew is exposed to acceleration ranging from +4Gx to +8Gx in nominal conditions. This study was conducted to assess the changes in cardiorespiratory parameters, namely, heart rate (HR), electrocardiogram (ECG), respiratory rate (RR), and SpO2 on exposure to simulated +Gx acceleration. Material and Methods: Fifteen randomly selected healthy male volunteers participated in the study. They were exposed to a simulated acceleration profile consisting of two peaks in the high-performance human centrifuge; first peak of +4Gx for 30 s and second peak of+8Gx for 30 s. The cardiorespiratory parameters were monitored and recorded during the acceleration exposure. The data were compiled and analyzed using one-way repeated measures ANOVA. Results: Significant increase in HR was observed on exposure to +4Gx (110.4 ± 16.7 bpm; P < 0.001) in comparison to the baseline value (80.5 ± 7.5 bpm). However, the changes in the HR at +8Gx were not significant in comparison to baseline as well as +4Gx values. On the other hand, RR indicated a significant increase on exposure to +8Gx (25.2 ± 5.8 breaths/min) in comparison to the baseline (15.1 ± 1.6 breaths/min; P = 0.001) and +4Gx (19.0 ± 6.1 breaths/min; P = 0.009) values. SpO2 showed a significant reduction at +8Gx (94.2 ± 3.8%) in comparison to baseline (98.9 ± 0.3%; P = 0.004) and +4Gx (96.9 ± 1.5%; P = 0.003). ECG did not show any evidence of arrhythmia during the exposure to +Gx acceleration. Conclusion: The insignificant changes in the HR at peak of +8Gx indicate less pronounced effects on the smaller hydrostatic gradient in +Gx acceleration unlike +Gz acceleration. However, the findings of the study point towards a significant increase in respiratory rate and reduction in SpO2 at +8Gx.

Monte Carlo simulation of chemistry following radiolysis with TOPAS-nBio

Simulation of water radiolysis and the subsequent chemistry provides important information on the effect of ionizing radiation on biological material. The Geant4 Monte Carlo toolkit has added chemical processes via the Geant4-DNA project. The TOPAS tool simplifies the modeling of complex radiotherapy applications with Geant4 without requiring advanced computational skills, extending the pool of users. Thus, a new extension to TOPAS, TOPAS-nBio, is under development to facilitate the configuration of track-structure simulations as well as water radiolysis simulations with Geant4-DNA for radiobiological studies. In this work, radiolysis simulations were implemented in TOPAS-nBio. Users may now easily add chemical species and their reactions, and set parameters including branching ratios, dissociation schemes, diffusion coefficients, and reaction rates. In addition, parameters for the chemical stage were re-evaluated and updated from those used by default in Geant4-DNA to improve the accuracy of chemical yields. Simulation results of time-dependent and LET-dependent primary yields Gx (chemical species per 100 eV deposited) produced at neutral pH and 25 °C by short track-segments of charged particles were compared to published measurements. The LET range was 0.05–230 keV µm−1. The calculated Gx values for electrons satisfied the material balance equation within 0.3%, similar for protons albeit with long calculation time. A smaller geometry was used to speed up proton and alpha simulations, with an acceptable difference in the balance equation of 1.3%. Available experimental data of time-dependent G-values for agreed with simulated results within 7% ± 8% over the entire time range; for over the full time range within 3% ± 4%; for H2O2 from 49% ± 7% at earliest stages and 3% ± 12% at saturation. For the LET-dependent Gx, the mean ratios to the experimental data were 1.11 ± 0.98, 1.21 ± 1.11, 1.05 ± 0.52, 1.23 ± 0.59 and 1.49 ± 0.63 (1 standard deviation) for , , H2, H2O2 and , respectively. In conclusion, radiolysis and subsequent chemistry with Geant4-DNA has been successfully incorporated in TOPAS-nBio. Results are in reasonable agreement with published measured and simulated data.

Hydrogen bond network between amino acid radical intermediates on the proton-coupled electron transfer pathway of E. coli α2 ribonucleotide reductase.

Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides in all organisms. In all Class Ia RNRs, initiation of nucleotide diphosphate (NDP) reduction requires a reversible oxidation over 35 Å by a tyrosyl radical (Y122•, Escherichia coli) in subunit β of a cysteine (C439) in the active site of subunit α. This radical transfer (RT) occurs by a specific pathway involving redox active tyrosines (Y122 ⇆ Y356 in β to Y731 ⇆ Y730 ⇆ C439 in α); each oxidation necessitates loss of a proton coupled to loss of an electron (PCET). To study these steps, 3-aminotyrosine was site-specifically incorporated in place of Y356-β, Y731- and Y730-α, and each protein was incubated with the appropriate second subunit β(α), CDP and effector ATP to trap an amino tyrosyl radical (NH2Y•) in the active α2β2 complex. High-frequency (263 GHz) pulse electron paramagnetic resonance (EPR) of the NH2Y•s reported the gx values with unprecedented resolution and revealed strong electrostatic effects caused by the protein environment. 2H electron–nuclear double resonance (ENDOR) spectroscopy accompanied by quantum chemical calculations provided spectroscopic evidence for hydrogen bond interactions at the radical sites, i.e., two exchangeable H bonds to NH2Y730•, one to NH2Y731• and none to NH2Y356•. Similar experiments with double mutants α-NH2Y730/C439A and α-NH2Y731/Y730F allowed assignment of the H bonding partner(s) to a pathway residue(s) providing direct evidence for colinear PCET within α. The implications of these observations for the PCET process within α and at the interface are discussed.

Single photon emission in the red spectral range from a GaAs-based self-assembled quantum dot

Low temperature micro-photoluminecence and second-order single photon correlation experiments were performed on individual self-assembled In0.47Al0.34Ga0.19As/Al0.3Ga0.7As/GaAs quantum dots emitting in the range of 680–780 nm. Emission lines originating from exciton, biexciton, and charge exciton confined in the same dot could be identified. The derived exciton fine structure splitting is ∼125 μeV, whereas the biexciton and charge exciton binding energies are ∼4 and ∼9 meV, respectively. The photon correlation statistics measured for the exciton emission exhibited a clear antibunching with the value of gX−X2(0)=0.30±0.05, confirming unambiguously that such quantum dots act as true single photon quantum emitters.

A microcontroller-based interface circuit for lossy capacitive sensors

This paper introduces and analyses a low-cost microcontroller-based interface circuit for lossy capacitive sensors, i.e. sensors whose parasitic conductance (Gx) is not negligible. Such a circuit relies on a previous circuit also proposed by the authors, in which the sensor is directly connected to a microcontroller without using either a signal conditioner or an analogue-to-digital converter in the signal path. The novel circuit uses the same hardware, but it performs an additional measurement and executes a new calibration technique. As a result, the sensitivity of the circuit to Gx decreases significantly (a factor higher than ten), but not completely due to the input capacitances of the port pins of the microcontroller. Experimental results show a relative error in the capacitance measurement below 1% for Gx < 200 nS, which is quite remarkable considering the simplicity of the circuit proposed. The measurement of a commercial capacitive humidity sensor subjected to condensation (in order to have a significant value of Gx) shows the effectiveness of the circuit.

An Oxyferrous Heme/Protein-based Radical Intermediate Is Catalytically Competent in the Catalase Reaction of Mycobacterium tuberculosis Catalase-Peroxidase (KatG)

A mechanism accounting for the robust catalase activity in catalase-peroxidases (KatG) presents a new challenge in heme protein enzymology. In Mycobacterium tuberculosis, KatG is the sole catalase and is also responsible for peroxidative activation of isoniazid, an anti-tuberculosis pro-drug. Here, optical stopped-flow spectrophotometry, rapid freeze-quench EPR spectroscopy both at the X-band and at the D-band, and mutagenesis are used to identify catalase reaction intermediates in M. tuberculosis KatG. In the presence of millimolar H2O2 at neutral pH, oxyferrous heme is formed within milliseconds from ferric (resting) KatG, whereas at pH 8.5, low spin ferric heme is formed. Using rapid freeze-quench EPR at X-band under both of these conditions, a narrow doublet radical signal with an 11 G principal hyperfine splitting was detected within the first milliseconds of turnover. The radical and the unique heme intermediates persist in wild-type KatG only during the time course of turnover of excess H2O2 (1000-fold or more). Mutation of Met255, Tyr229, or Trp107, which have covalently linked side chains in a unique distal side adduct (MYW) in wild-type KatG, abolishes this radical and the catalase activity. The D-band EPR spectrum of the radical exhibits a rhombic g tensor with dual gx values (2.00550 and 2.00606) and unique gy (2.00344) and gz values (2.00186) similar to but not typical of native tyrosyl radicals. Density functional theory calculations based on a model of an MYW adduct radical built from x-ray coordinates predict experimentally observed hyperfine interactions and a shift in g values away from the native tyrosyl radical. A catalytic role for an MYW adduct radical in the catalase mechanism of KatG is proposed.

Iron-Sulfur Cluster N5 Is Coordinated by an HXXXCXXCXXXXXC Motif in the NuoG Subunit of Escherichia coli NADH:Quinone Oxidoreductase (Complex I)

NADH:quinone oxidoreductase (complex I) plays a central role in cellular energy metabolism, and its dysfunction is found in numerous human mitochondrial diseases. Although the understanding of its structure and function has been limited, the x-ray crystal structure of the hydrophilic part of Thermus thermophilus complex I recently became available. It revealed the localization of all redox centers, including 9 iron-sulfur clusters and their coordinating ligands, and confirmed the predictions mostly made by Ohnishi et al. (Ohnishi, T., and Nakamaru-Ogiso, E. (2008) Biochim. Biophys. Acta 1777, 703-710) based on various EPR studies. Recently, Yakovlev et al. (Yakovlev, G., Reda, T., and Hirst, J. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 12720-12725) claimed that the EPR signals from clusters N4, N5, and N6b were misassigned. Here we identified and characterized cluster N5 in the Escherichia coli complex I whose EPR signals had never been detected by any group. Using homologous recombination, we constructed mutant strains of H101A, H101C, H101A/C114A, and cluster N5 knock-out. Although mutant NuoEFG subcomplexes were dissociated from complex I, we successfully recovered these mutant NuoCDEFG subcomplexes by expressing the His-tagged NuoCD subunit, which had a high affinity to NuoG. The W221A mutant was used as a control subcomplex carrying wild-type clusters. By lowering temperatures to around 3 K, we finally succeeded in detecting cluster N5 signals in the control for the first time. However, no cluster N5 signals were found in any of the N5 mutants, whereas EPR signals from all other clusters were detected. These data confirmed that, contrary to the misassignment claim, cluster N5 has a unique coordination with His(Cys)(3) ligands in NuoG.

Characterization of the Tyrosine-Z Radical and Its Environment in the Spin-Coupled S2TyrZ• State of Photosystem II from Thermosynechococcus elongatus

The Mn4Ca cluster of photosystem II (PSII) goes through five sequential oxidation states (S0-S4) in the water oxidation process that also involves a tyrosine radical intermediate (TyrZ*). An S2TyrZ* state in which the Mn4Ca cluster and TyrZ* are magnetically coupled to each other and which is characterized by a distinct "split-signal" EPR spectrum can be generated in acetate-treated PSII. This state was examined by high-field EPR (HFEPR) in PSII from Thermosynechococcus elongatus isolated from a D2-Tyr160Phe mutant to avoid spectral contributions from TyrD*. In contrast to the same state in plants, both antiferromagnetic and ferromagnetic spin-spin couplings were observed. The intrinsic g values of TyrZ* in the coupled state were directly measured from the microwave frequency dependence of the HFEPR spectrum. The TyrZ* gx value in the antiferromagnetic centers was 2.0083, indicating that the coupled radical was in a less electropositive environment than in Mn-depleted PSII. Two gx values were found in the ferromagnetically coupled centers, 2.0069 and 2.0079. To put these values in perspective, the second redox-active tyrosine, TyrD*, was examined in various electrostatic environments. The TyrD* gx value changed from 2.0076 in the wild type to 2.0095 when the hydrogen bond from histidine 189 to TyrD* was removed using the D2-His189Leu mutant, indicating a change to a significantly less electropositive environment. BLY3P/6-31+G** density functional calculations on the hydrogen-bonded p-ethylphenoxy radical-imidazole supermolecular model complex showed that the entire range of Tyr* gx values, from 2.0065 to 2.0095, could be explained by the combined effects of hydrogen bonding and the dielectric constant of the local protein environment.

Determination of the Structural Environment of the Tyrosyl Radical in Prostaglandin H2 Synthase-1: A High Frequency ENDOR/EPR Study

The catalytically active tyrosyl radical which gives rise to the "wide doublet" (WD1) signal in ovine Prostaglandin H2 Synthase-1 has been studied using high frequency (HF) pulsed ENDOR and EPR. A hydrogen-bonded deuteron was directly detected in HFENDOR (130 GHz) spectra of 1H2O/2H2O-exchanged samples. The HFENDOR spectral simulations required a distribution in hydrogen bond distances to achieve proper fits. This range of distances was consistent with that used to model the distribution in gX values detected in pulsed HFEPR spectra. Possible hydrogen-bonding partners, as well as implications regarding the mechanism of self-inactivation for PGHS, are discussed.

Characterization of the Iron-Sulfur Cluster N7 (N1c) in the Subunit NuoG of the Proton-translocating NADH-quinone Oxidoreductase from Escherichia coli

The proton-pumping NADH-quinone oxidoreductase from Escherichia coli houses nine iron-sulfur clusters, eight of which are found in its mitochondrial counterpart, complex I. The extra putative iron-sulfur cluster binding site with a CXXCXXXCX(27)C motif in the NuoG subunit has been assigned to ligate a [2Fe-2S] (N1c). However, we have shown previously that the Thermus thermophilus N1c fragment containing this motif ligates a [4Fe-4S] (Nakamaru-Ogiso, E., Yano, T., Ohnishi, T., and Yagi, T. (2002) J. Biol. Chem. 277, 1680-1688). In the current study, we individually inactivated four sets of the iron-sulfur binding motifs in the E. coli NuoG subunit by replacing all four ligands with Ala. Each mutant subunit, designated Delta N1b, Delta N1c, Delta N4, and Delta N5, was expressed as maltose-binding protein fusion proteins. After in vitro reconstitution, all mutant subunits were characterized by EPR. Although EPR signals from cluster N1b were not detected in any preparations, we detected two [4Fe-4S] EPR signals with g values of g(x,y,z) = 1.89, 1.94, and 2.06, and g(x,y,z) = 1.91, 1.94, and 2.05 at 6-20 K in wild type, Delta N1b, and Delta N5. The former signal was assigned to cluster N4, and the latter signal was assigned to cluster N1c because of their disappearance in Delta N4 and Delta N1c. Confirming that a [4Fe-4S] cluster ligates to the N1c motif, we propose to replace its misleading [2Fe-2S] name, N1c, with "cluster N7." In addition, because these mutations differently affected the assembly of peripheral subunits by in trans complementation analysis with the nuoG knock-out strain, the implicated structural importance of the iron-sulfur binding domains is discussed.

Purification and characterization of the HndA subunit of NADP-reducing hydrogenase from Desulfovibrio fructosovorans overproduced in Escherichia coli.

Based on the DNA sequence of its structural genes, clustered in the hnd operon, the NADP-reducing hydrogenase of Desulfovibrio fructosovorans is thought to be a heterotetrameric complex in which HndA and HndC constitute the NADP-reducing unit and HndD constitutes the hydrogenase unit, respectively. The weak representativity of the enzyme among cell proteins has prevented its purification. This paper discusses the purification and characterization of the HndA subunit of this unique tetrameric iron hydrogenase overproduced in Escherichia coli. The purified subunit contains 1.7 mol of non-heme iron and 1.7 mol of acid-labile sulfide/mol. EPR analysis of the reduced form of HndA indicates that it contains a single binuclear [2Fe-2S] cluster. This cluster exhibits a spectrum of rhombic symmetry with values of gx, gy, and gz equal to 1.915, 1.950, and 2. 000, respectively, and a midpoint redox potential of -395 mV. The UV-visible and EPR spectra of the [2Fe-2S] cluster indicate that HndA belongs to the [2Fe-2S] family typified by the Clostridium pasteurianum [2Fe-2S] ferredoxin. The C-terminal sequence of HndA shows 27% identity with the C-terminal sequence of the 25-kDa subunit of NADH: quinone oxidoreductase from Paracoccus denitrificans, 33% identity with the C-terminal sequence of the 24-kDa subunit from Bos taurus complex I, and 32% identity with the entire sequence of C. pasteurianum [2Fe-2S] ferredoxin. The four cysteine residues involved in HndA cluster binding have been tentatively identified on the basis of sequence identity considerations. Evidence of a HndA organization based on two independent structural domains is discussed.

Expression and Characterization of the Flavoprotein Subcomplex Composed of 50-kDa (NQO1) and 25-kDa (NQO2) Subunits of the Proton-translocating NADH-Quinone Oxidoreductase of Paracoccus denitrificans

This study reports the expression of the flavoprotein (FP) subcomplex of the proton-translocating NADH-quinone oxidoreductase (NDH-1) from Paracoccus denitrificans, which is composed of the NQO1 (50 kDa) and the NQO2 (25 kDa) subunits. The two subunits are co-expressed in Escherichia coli using a double expression plasmid system. The expressed subunits form a water-soluble heterodimer complex with 1:1 stoichiometry. The expressed complex contained one [2Fe 2S] cluster but almost no FMN or [4Fe 4S] cluster. The two latter prosthetic groups could be partially reconstituted with FMN, Na2S, and (NH4)2Fe(SO4)2 in vitro under anaerobic conditions. The reconstituted FP subcomplex showed EPR signals from two distinct species of iron-sulfur cluster. One resonance transition originates from a [2Fe-2S] cluster with g values of gx,y,z = 1.92, 1.95, and 2.00 and slow spin relaxation, which was tentatively assigned to the cluster N1a. These EPR properties are very similar to those reported for the NQO2 subunit expressed alone (Yano, T., Sled', V. D., Ohnishi, T., and Yagi, T. (1994) Biochemistry 33, 494-499). The other originates from a [4Fe 4S] cluster with g values of gx,y, z = 1.87, 1.94, and 2.04 and fast relaxing behavior, which are reminiscent of the cluster N3 in the membrane bound enzyme complex. After reconstitution with FMN, the FP subcomplex catalyzed electron transfer from NADH and from deamino-NADH to a variety of electron acceptors. The enzymatic properties of the FP subcomplex, reconstituted with FMN and iron-sulfur, correspond to those of the isolated P. denitrificans NADH-dehydrogenase complex.

A π-Donor Spectrochemical Series for X in (Me5C5)2TiX, and β-Agostic Interactions in X = Et and N(Me)Ph

The EPR and electronic spectra of d1-bent metallocene compounds of the type Cp*2TiX, where X is halide, alkoxide, amide, alkyl, or hydride and Cp* = Me5C5, have been studied. Several of these compounds are new, and those with X = N(Me)H and F were characterized by X-ray crystallography. The crystal structure of Cp*2TiN(Me)H showed that the N(Me)H ligand lies on the plane defined by Cp*(centroid)−Ti−Cp*(centroid). This is the sterically most unfavorable conformation but allows maximum Ti−N π-bonding. The anisotropic frozen solution EPR spectra were analyzed by the method used by Petersen and Dahl for the d1-metallocenes, Cp2VX2, which gives gx, gy, and gz. Although the values of gx and gz are relatively constant throughout the series, the value of gy varies with the π-donor ability of X. The π-donor series is N(Me)H ≈ NH2 ≈ OMe > OPh ≈ F > N(Me)Ph > Cl > Br > I > H. Among the known alkyls, the π-donor ability was Et > Me > n-Pr ≈ CH2CMe3 > CH2C6H5, which is rationalized, in part, by a β-agostic interaction in the case of Et. The β-agostic interaction in Cp*2TiEt and in Cp*2TiN(Me)Ph was investigated by variable-temperature EPR spectroscopy giving an enthalpy and entropy for the agostic interaction. For Cp*2TiEt the parameters for the agostic interaction are ΔH° = −1.93(3) kcal/mol and ΔS° = −6.3(2) eu, and for Cp*2TiN(Me)Ph, ΔH° = −1.5(1) kcal/mol and ΔS° = −7.9(5) eu.

Pseudomonas aeruginosa 142 uses a three-component ortho-halobenzoate 1,2-dioxygenase for metabolism of 2,4-dichloro- and 2-chlorobenzoate

Cell extracts of Pseudomonas aeruginosa 142, which was previously isolated from a polychlorinated biphenyl-degrading consortium, were shown to degrade 2,4-dichlorobenzoate, 2-chlorobenzoate, and a variety of other substituted ortho-halobenzoates by a reaction that requires oxygen, NADH, Fe(II), and flavin adenine dinucleotide. By using extracts that were chromatographically depleted of chlorocatechol and catechol 1,2-dioxygenase activities, products of the initial reaction with 2,4- or 2,5-dichlorobenzoate and 2-chlorobenzoate were identified by mass spectrometry as 4-chlorocatechol and catechol. In contrast to the well-characterized benzoate dioxygenases or the recently described 2-halobenzoate 1,2-dioxygenase from P. cepacia 2CBS (S. Fetzner, R. Müller, and F. Lingens, J. Bacteriol. 174:279-290, 1992) that possess two protein components, the P. aeruginosa enzyme was resolved by ion-exchange chromatography into three components, each of which is required for activity. To verify the distinct nature of this enzyme, we purified, characterized, and identified one component as a ferredoxin (M(r), approximately 13,000) containing a single [2Fe-2S] Rieske-type cluster (electron paramagnetic resonance spectroscopic values of gx = 1.82, gy = 1.905, and gz = 2.02 in the reduced state) that is related in sequence to ferredoxins found in the naphthalene and biphenyl three-component dioxygenase systems. By analogy to these enzymes, we propose that the P. aeruginosa ferredoxin serves as an electron carrier between an NADH-dependent ferredoxin reductase and the terminal component of the ortho-halobenzoate 1,2-dioxygenase. The broad specificity and high regiospecificity of the enzyme make it a promising candidate for use in the degradation of mixtures of chlorobenzoates.

Expression of the 25-kilodalton iron-sulfur subunit of the energy-transducing NADH-ubiquinone oxidoreductase of Paracoccus denitrificans

The energy-transducing NADH-ubiquinone (Q) oxidoreductase of Paracoccus denitrificans is composed of 14 dissimilar subunits and contains at least four iron-sulfur clusters [Yagi, T. (1993) Biochim. Biophys. Acta 1141, 1-17]. The complete DNA sequence of the gene cluster encoding the energy-transducing NADH-Q oxidoreductase of P. denitrificans has been determined. This paper reports the expression of the 25-kilodalton (kDa) (NQO2) subunit of the P. denitrificans enzyme complex in Escherichia coli and the characterization of the iron-sulfur cluster bound to the expressed subunit. The 25-kDa subunit was expressed in the cytoplasmic phase but not in the membrane fraction of E. coli cells and then purified using an affinity nickel chelation column. The purified subunit contains 1.44 mol of non-heme iron and 1.33 mol of acid-labile sulfide/mol of subunit. EPR analysis of the reduced form of this subunit indicates that the expressed subunit contains a single binuclear [2Fe-2S] cluster. This cluster exhibits a spectrum of rhombic symmetry with g values of gx,y,z = 1.913, 1.942, and 1.996, which is very similar to the spectrum of the [2Fe-2S] cluster in the resolved flavoprotein II subfraction (subunit 24 + 9 kDa) of bovine heart complex I [Ragan, C. I., Galante, Y. M., Hatefi, Y., & Ohnishi, T. (1982) Biochemistry 21, 590-594; Ohnishi, T., Ragan, C. I., & Hatefi, Y. (1985) J. Biol. Chem. 260, 2782-2788]. The assignment of the binuclear iron-sulfur cluster of the 25-kDa subunit to an EPR-visible iron-sulfur cluster in the Paracoccus NADH-Q oxidoreductase in situ is discussed.

Characterization of the Rat Hemoglobin Thiyl Free Radical Formed upon Reaction with Phenylhydrazine

The characterization of the radical formed from rat hemoglobin (Hb) by methemoglobin-generating agents and trapped by 5,5-dimethyl-1-pyrroline N-oxide (DMPO) has shown it to be a thiyl radical. The two-electron oxidation of hemoglobin forms a ferryl species with one or more free radicals located on the globin moiety. While the radical species has not been observed by use of uv-visible spectrophotometry, the species can be detected by use of electron paramagnetic resonance spectroscopy (EPR). In previous studies, in vitro experiments have shown that the EPR signal from the rat Hb radical adduct formed by t-butyl hydroperoxide decreased following pretreatment of the oxyllb with thiol-blocking agents except for iodoacetamide. In this study the power saturation profile of the DMPO radical adduct obtained from the reaction of rat oxyHb with phenylhydrazine exhibited a pattern similar to that obtained from human oxyHb, in which the β-93 cystiene was labeled with 2,2,6,6-tetramethyl-1-piperidinyloxy-4-maleimide (4-maleimide-TEMPO). EPR spectra were taken at 77 K and computer simulations were performed. The calculated value for aNiso obtained by simulation indicates that the radical adduct is in a hydrophobic region. The value for aHiso has little structural significance, as the steric effect of the protein makes comparison with radical adducts in solution problematical. The value of gx from the rat Hb radical adduct was significantly higher than that obtained from bovine Hb, whose radical is not thiol-derived, as demonstrated by negative thiol-blocking agent experiments. A higher gxvalue is consistent with the radical adduct containing a heavy atom such as sulfur. Rat Hb was analyzed for thiol content by use of iodoacetamide, 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and uv-visible spectrophotometry. It was found that 1.15 ± 0.34 thiols/tetramer of Hb were reactive with DTNB, but not iodoacetamide.

Dry Skin of Newborn Infants: Functional Analysis of the Stratum Corneum

Our clinical observations disclosed that most newborn infants showed scaling on at least some parts of the body. To clarify the mechanism for such xerosis, we performed measurements of high-frequency conductance (Gx), which assesses the skin surface hydration and evaporative water loss from the skin. The skin of newborns showed surprisingly lower Gx values and evaporative water loss than those of adults or 1- to 6-month-old infants in a warm environment. These findings may be explained partly by low eccrine activity, which has been reported as characteristic of newborn skin. However, defective stratum corneum function, such as reduced water-holding capacity as demonstrated by the in vivo water sorption-desorption test, also seems to be responsible for the development of dry skin in newborns.

Characterization of neutrophil b‐type cytochrome in situ by electron paramagnetic resonance spectroscopy

Electron paramagnetic resonance spectroscopy at 4.2 K was successfully used to characterize neutrophil b-type cytochrome in situ. The spectra of resting neutrophils taken under aerobic conditions gave a set of characteristic signals in a high magnetic field (g = 2.85, 2.21 and 1.67) beside signals for myeloperoxidase and others. From the g values, shapes and the results of other experiments, these signals were attributed to those of cytochrome b558. The results indicate that cytochrome b558 in resting neutrophils is a hexa-coordinated ferric hemoprotein in a low-spin state. The obtained gz and gx values for the hemichrome were consistent with that of bis(imidazole)-coordinated hemoprotein.