Kinetics of the transhydrogenase reaction catalyzed by mitochondrial NADH:ubiquinone oxidoreductase (complex I)

The kinetics of the NADH3'-acetylpyridine adenine dinucleotide (APAD⁺) transhydrogenase reaction (DD-reaction) catalyzed by different preparations of mitochondrial NADH-dehydrogenase (submitochondrial particles (SMP), purified Complex I, and three-subunit fragment of Complex I (FP)) have been studied. Complex I (in SMP or in purified preparation) catalyzes two NADHAPAD⁺ reactions with different rates and nucleotide affinities. Reaction 1 has high affinity to APAD⁺ (K _m = 7 M, for SMP) and low rate (V _m = 0.2 mol/min per mg protein, for SMP) and occurs with formation of a ternary complex. Reaction 2 has much higher rate and considerably lower affinity for oxidized nucleotide (V _m = 1.7 mol/min per mg protein and K _m = 160 M, for SMP). FP catalyzes only reaction 1. ADP-ribose inhibits reaction 1 with mixed type inhibition (competitive with non-competitive) with respect to NADH and APAD⁺. Rhein competes with both substrates. The results suggest that at least two nucleotide-binding sites exist in Complex I.     

Crystallographic and pre-steady-state kinetics studies on binding of NADH to wild-type and isoniazid-resistant enoyl-ACP(CoA) reductase enzymes from Mycobacterium tuberculosis

An understanding of isoniazid (INH) drug resistance mechanism in Mycobacterium tuberculosis should provide significant insight for the development of newer anti-tubercular agents able to control INH-resistant tuberculosis (TB). The inhA-encoded 2-trans enoyl-acyl carrier protein reductase enzyme (InhA) has been shown through biochemical and genetic studies to be the primary target for INH. In agreement with these results, mutations in the inhA structural gene have been found in INH-resistant clinical isolates of M.tuberculosis, the causative agent of TB. In addition, the InhA mutants were shown to have higher dissociation constant values for NADH and lower values for the apparent first-order rate constant for INH inactivation as compared to wild-type InhA. Here, in trying to identify structural changes between wild-type and INH-resistant InhA enzymes, we have solved the crystal structures of wild-type and of S94A, I47T and I21V InhA proteins in complex with NADH to resolutions of, respectively, 2.3A, 2.2A, 2.0 A, and 1.9A. The more prominent structural differences are located in, and appear to indirectly affect, the dinucleotide binding loop structure. Moreover, studies on pre-steady-state kinetics of NADH binding have been carried out. The results showed that the limiting rate constant values for NADH dissociation from the InhA-NADH binary complexes (k(off)) were eleven, five, and tenfold higher for, respectively, I21V, I47T, and S94A INH-resistant mutants of InhA as compared to INH-sensitive wild-type InhA. Accordingly, these results are proposed to be able to account for the reduction in affinity for NADH for the INH-resistant InhA enzymes.     

Folding mechanism of the (H3-H4)2 histone tetramer of the core nucleosome

To further understand oligomeric protein assembly, the folding and unfolding kinetics of the H3-H4 histone tetramer have been examined. The tetramer is the central protein component of the core nucleosome, which is the basic unit of DNA compaction into chromatin in the eukaryotic nucleus. This report provides the first kinetic folding studies of a protein containing the histone fold dimerization motif, a motif observed in several protein-DNA complexes. Previous equilibrium unfolding studies have demonstrated that, under physiological conditions, there is a dynamic equilibrium between the H3-H4 dimer and tetramer species. This equilibrium is shifted predominantly toward the tetramer in the presence of the organic osmolyte trimethylamine-N-oxide (TMAO). Stopped-flow methods, monitoring intrinsic tyrosine fluorescence and far-UV circular dichroism, have been used to measure folding and unfolding kinetics as a function of guanidinium hydrochloride (GdnHCl) and monomer concentrations, in 0 and 1 M TMAO. The assignment of the kinetic phases was aided by the study of an obligate H3-H4 dimer, using the H3 mutant, C110E, which destabilizes the H3-H3' hydrophobic four-helix bundle tetramer interface. The proposed kinetic folding mechanism of the H3-H4 system is a sequential process. Unfolded H3 and H4 monomers associate in a burst phase reaction to form a dimeric intermediate that undergoes a further, first-order folding process to form the native dimer in the rate-limiting step of the folding pathway. H3-H4 dimers then rapidly associate with a rate constant of > or =10(7) M(-1)sec(-1) to establish a dynamic equilibrium between the fully assembled tetramer and folded H3-H4 dimers.     

Reactivation of NADH Dehydrogenase (Complex I) Inhibited by 1-Methyl-4-(4'-Alkylphenyl)pyridinium Analogues: A Clue to the Nature of the Inhibition Site

Abstract: Expression of the neurotoxicity of 1-methyl-4-phenyl-1.2,3,6-tetrahydropyridine, following oxidation to l-methyl-4-phenylpyridinium ion (MPP⁺), is believed to involve inhibition of mitochondrial electron transport from NADH dehydrogenase (complex l) to ubquinone. MPP⁺ and its analogues have been shown to Mock electron transport at or near the same site as two powerful inhibitors of mitochondrial respiration, rotenone and piericidin A. All three types of inhibitors combine at two sites on NADH dehydrogenase, a hydrophilic and hydrophobic one, and occupancy of both sites is required for complete inhibition. Tetraphenylboron anion (TPB⁻) in catalytic amounts is known to increase the effectiveness of positively charged MPP⁺ analogues in blodclng mitochondrial respiration. A part of this effect involves facitation of the entry of MPP⁺ oongeners into the hydrophobic site by ion pairing, as has been demonstrated in studies with submitochondrial particles (electron transport particles). This communication documents the fact that TPB⁻, when present in molar excess over the MPP⁺ analogues, reverses the inhibition. This seems to involve again strong ion pairing. removal of the inhibitory analogue from one to the two binding sites, and concentration of the inhibitor in the membrane, so that only the hydrophobic binding site remains occupied, resulting in lowering of the inhibiti to 30–40%.     

Synthesis and characterisation of the carboxylate linked osmium clusters [{Os3H(CO)10}2(CO2CH2CO2)], [{Os3H(CO)10}2(CO2C2H4CO2)], [{Os3H(CO)10}2(C4O4)] and [{Os3H(CO)10}2(C4O4){Co2(CO)6}]

Reactions between the activated cluster [Os₃(CO)₁₀(NCMe)₂] and malonic acid, succinic acid and dicarboxylic acetylene, respectively, lead to the formation of the linked cluster complexes [{Os₃H(CO)₁₀}₂(CO₂CH₂CO₂)] (1), [{Os₃H(CO)₁₀}₂(CO₂C₂H₄CO₂)] (2), and [{Os₃H(CO)₁₀}₂(C₄O₄)] (3) in good yield. Cluster 3 was subsequently treated with [Co₂(CO)₈] and this results in the addition of a “Co₂(CO)₆” group giving [{Os₃H(CO)₁₀}₂(C₂O₄){Co₂(CO)₆}] (4). The X-ray crystal structures are reported for 2–4. In each structure the two triangular triosmium units are linked by the carboxylate groups and within each complex the carboxylate groups are chelating and bridge two osmium atoms.     

Investigations of the {ReO} core: A '2+2' complex from bidentate and potentially trident ligands: [ReO(η-HOC(6)H(4)-2-CH(2)NC(6)H(4)S)(η-SC(5)H(4)N)(PPh(3))

The reaction of [ReOCl(3)(PPh(3))(2)] with N-(2-hydroxybenzyl)-2-mercaptoaniline (H(3)hbma) (2) and 2-mercaptopyridine in hot CHCl yields [ReO(η(2)-HOC(6)H(4)-2-CH(2)NC(6)H(4)S)(η(2)-SC(5)H(4)N)(PPh(3))] (3). The structure of 3 consists of distorted octahedral Re(V) monomers. The coordination geometry at the rhenium is defined by a terminal oxo-group, the nitrogen and sulfur donors of the chelating mercaptopyridine, the nitrogen and sulfur donors of a bidentate (Hhbma)(2-) ligand, and the phosphorus of the PPh(3) group. The -C(6)H(4)OH arm of (Hhbma)(2-) is pendant, and the coordinated nitrogen of this ligand is present as a deprotonated amido nitrogen.     

Biphasic kinetics in the reaction between amino acids or glutathione and the chromium acetate cluster, [Cr3O(OAc)6]

Kinetics for the breakdown of the trinuclear chromium acetate cluster with a series of monoprotic and diprotic amino acid ligands and with glutathione in aqueous media have been investigated spectrophotometrically at pH 3.5–5.5 and in a temperature range of 45–60 °C. Under pseudo-first-order conditions, reactions with these ligands exhibited biphasic kinetic behavior that can be accounted for by a consecutive two-step reaction, A → B → C, where A is assumed to be a forced ion pair, B an intermediate and C is the product; experimental data fit to a biexponential equation for the transformation. Rates for k_short, k_long, and k_obs were determined by manual extrapolation of absorbance data or curve-fitting routines; associated activation parameters for each step of the reaction were calculated using the Eyring equation. Rates for the first and second steps of the reaction are on the order of 10⁻⁴ and 10⁻⁵ s⁻¹, respectively. The large negative values of ΔS^‡ and smaller ΔH^‡ in the first step indicate an associative step, while high positive values of ΔS^‡ in the second step indicate dissociation. To account for the results mechanistically, the results are interpreted to be a first step of ligand exchange with a pseudo-axial aqua ligand, followed by a dissociative step involving acetate or oxo ligand displacement. The dissociative step is the rate determining step, with k_obs ≈ k_long.The results demonstrate reaction pathways that are available to the Cr(III) metal centers that may be physiologically relevant in the ligand-rich environment of biological systems. Under general conditions Cr(III) clusters may be expected to be broken down, unless some unique biological environment stabilizes the cluster. The present study has application to the processes related to Cr(III) transport and excretion, to potential mechanisms of Cr(III) action in a biological setting, and to the pharmacokinetics of Cr(III) supplements for animal and human consumption.     

Expression of genes encoding the luciferase from Photobacterium leiognathi in Escherichia coli Rosetta (DE3) and its application in NADH detection

Cloning of genes encoding the luciferase from Photobacterium leiognathi YL in Escherichia coli Rosetta (DE3) was performed successfully and the expressed forms of lux AB were purified to homogeneity. Experimental measurements revealed that luciferase from Photobacterium leiognathi YL has good thermal stability and a high residual activity at extreme pH values, which are extremely important for its various ecological, industrial and medical applications. Furthermore, we made a first attempt for quantitative detection of NADH by recombinant E. coli Rosetta (DE3) coupled enzyme system. A good linear relationship between luminescence intensity and NADH with low (1–12 nmol/L) and high (10–500 nmol/L) concentration was observed, whose standard curve was y = 772.97× + 4041.1, R² = 0.9884 and y = 1710× + 4.99 × 10⁵, R² = 0.9727, respectively. Our results demonstrate a high sensitivity of recombinant E. coli coupled enzyme system to NADH on the basis of high soluble expression of recombinant luciferase and continuous and stable expression of some NAD(P)H‐dependent flavin mononucleotide (FMN) reductases.     

Synthesis of isochroman-4-ones and 2H-pyran-3(6H)-ones by gold-catalyzed oxidative cycloalkoxylation of alkynes

Gold catalysis is a convenient tool to oxidatively functionalize alkyne into a range of valuable compounds. In this article, we report a new access to isochroman-4-one and 2H-pyran-3(6H)-one derivatives that involves a gold-catalyzed oxidative cycloalkoxylation of an alkyne in the presence of a pyridine N-oxide. The reaction proceeds under mild conditions, is relatively efficient and exhibits a high functional group compatibility.     

Phenylalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) and catechins (flavan-3-ols) accumulation in tea

Phenylalanine ammonia-lyase and cinnamate 4-hydroxylase are important enzymes in allocating significant amounts of carbon from phenylalanine into the biosynthesis of several important secondary metabolites. Tea is an important crop of commerce known for its beverage and medicinally important flavonoid compounds, mainly catechins. As metabolic flux for the operation of the flavonoid pathway is maintained through the activities of PAL and C4H, thus, catechins biosynthesis in tea is critically dependent on the products of these enzymes. We examined the expression of PAL and C4H. Sequence encoding CsPAL was isolated from tea by polymerase chain reaction using sequence information available at the NCBI GenBank. Sequence encoding C4H was isolated from tea by using differential display of mRNA and rapid amplification of cDNA ends technology. CsC4H (AY641731) comprised of 1,352 bp full-length cDNA with open reading frame of 1,173 bp encoding 390 amino acids. Catechin contents decreased in response to drought stress (DS), abscisic acid (ABA), and gibberellic acid (GA₃) treatments but increased in response to wounding. The expression of CsPAL and CsC4H showed the same behavior under the above treatments and was also in accordance with the catechin contents. A positive correlation between catechin contents and gene expression suggested a critical role of the enzymes in catechins biosynthesis and a crosstalk between phenylpropanoid and flavonoid pathways.     

Characterization of the mechanism of the NADH-dependent polysulfide reductase (Npsr) from Shewanella loihica PV-4: Formation of a productive NADH-enzyme complex and its role in the general mechanism of NADH and FAD-dependent enzymes

The NADH-dependent polysulfide reductase (Npsr) from Shewanella loihica PV-4 is a member of the single cysteine-containing subset of the family of disulfide reductases represented by glutathione reductase. We have determined the kinetics of the reductive half-reaction of the enzyme with NADH using stopped-flow spectroscopy and kinetic isotope effects, and these results indicate that the reductive and oxidative half-reactions are both partially rate-limiting for enzyme turnover. During reaction with NADH, the reduced nucleotide appears to bind rapidly in an unproductive conformation, followed by the formation of a productive E·NADH complex and subsequent electron transfer to FAD. F161 of Npsr fills the space in which the nicotinamide ring of NADH would be expected to bind. We have shown that while this residue is not absolutely required for catalysis, it does assist in the forward commitment to catalysis through its role in the reductive half reaction, where it appears to enhance hydride transfer in the productive E·NADH complex. While the fluorescence and absorbance spectra of the stable redox forms of the wild-type and F161A mutant enzymes are similar, intermediates formed during reduction and turnover have different characteristics and appear to indicate that the enzyme–NADH complex formed just prior to hydride transfer on the F161A enzyme has weaker FAD–NADH interactions than the wild-type enzyme, consistent with a “looser” enzyme–NADH complex. The 2.7 Å crystal structure of the F161A mutant was determined, and shows that the nicotinamide ring of NADH would have the expected freedom of motion in the more open NADH binding cavity.     

Interaction of 1-Methyl-4-Phenylpyridinium Ion (MPP+) and Its Analogs with the Rotenone/Piericidin Binding Site of NADH Dehydrogenase

Nigrostriatal cell death in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease results from the inhibition of mitochondrial respiration by 1-methyl-4-phenylpyridinium (MPP+). MPP+ blocks electron flow from NADH dehydrogenase to coenzyme Q at or near the same site as do rotenone and piericidin and protects against binding of and loss of activity due to these inhibitors. The 4'-analogs of MPP+ showed increasing affinity for the site with increasing length of alkyl chain, with the lowest Ki, for 4'-heptyl-MPP+, being 6 microM. The 4'-analogs compete with rotenone for the binding site in a concentration-dependent manner. They protect the activity of the enzyme from inhibition by piericidin in parallel to preventing its binding, indicating that the analogs and piericidin bind at the same inhibitory site(s). The optimum protection, however, was afforded by 4'-propyl-MPP+. The lesser protection by the more lipophilic MPP+ analogs with longer alkyl chains may involve a different orientation in the hydrophobic cleft, allowing rotenone and piericidin to still bind even when the pyridinium cation is in a position to interrupt electron flow from NADH to coenzyme Q.     

High‐Performance Perovskite Solar Cells with Enhanced Environmental Stability Based on a (p‐FC6H4C2H4NH3)2[PbI4] Capping Layer

Supported by the density functional theory (DFT) calculations, for the first time, a fluorinated aromatic cation, 2‐(4‐fluorophenyl)ethyl ammonium iodide (FPEAI), is introduced to grow in situ a low dimensional perovskite layer atop 3D perovskite film with excess PbI₂. The resulted (p‐FC₆H₄C₂H₄NH₃)₂[PbI₄] perovskite functions as a protective capping layer to protect the 3D perovskite from moisture. In the meantime, the thin layer facilitates charge transfer at the interfaces, thereby reducing the nonradiative recombination pathways. Laser scanning confocal microscopy unveils visually the distribution of the 2D perovskite layer on top of the 3D perovskite. When employing the 3D–2D perovskite as the absorbing layer in the photovoltaic cells, a high power conversion efficiency of 20.54% is realized. Superior device performance and moisture stability are observed with the modified perovskite over the whole stability test period.     

Synthesis of novel 2-phenylsulfonylhydrazono-3-(2',3',4',6'-tetra-O-acetyl-beta-d-glucopyranosyl)thiazolidine-4-ones from thiosemicarbazide precursors

To develop novel biologically active organic compounds possessing a sugar moiety, a series of 2-phenylsulfonylhydrazono-3-(2',3',4',6'-tetra-O-acetyl-beta-d-glucopyranosyl)thiazolidine-4-one were synthesized via reaction of the thiosemicarbazide with ethyl bromoacetate. Their chemical structures were characterized by (1)H and (13)C NMR spectroscopy, elemental analysis and MS. The bioassay results indicated that some of these compound exhibit moderate fungicidal and herbicidal activities. Furthermore, the effect of various solvents at reflux temperature on the reactions of ethyl bromoacetate with the related thiosemicarbazides was investigated.     

The monoamine oxidase inhibition properties of C6-mono- and N3/C6-disubstituted derivatives of 4(3H)-quinazolinone

Parkinson’s disease is characterised by the death of the nigrostriatal neurons and depletion of striatal dopamine. The standard symptomatic therapy consists of dopamine replacement with l-dopa, the metabolic precursor of dopamine, which represents the most effective treatment. Since monoamine oxidase (MAO) B is a key dopamine metabolising enzyme in the brain, MAO-B inhibitors are often used as adjuvants to l-dopa. In addition to the symptomatic benefits offered by MAO-B inhibitors, these drugs may also possess neuroprotective properties and possibly delay the progression of Parkinson’s disease. Based on the therapeutic use of MAO-B inhibitors, the present study evaluates a series of mono- and disubstituted derivatives of 4(3H)-quinazolinone as potential inhibitors of recombinant human MAO-A and MAO-B. Twelve C6-monosubstituted and nine N3/C6-disubstituted 4(3H)-quinazolinone derivatives were synthesised, which led to the discovery of novel quinazolinone derivatives with micromolar and submicromolar activities as inhibitors of MAO-B. The most potent mono- and disubstituted derivatives exhibited IC₅₀ values of 6.35 μM (7f) and 0.685 μM (8b), respectively. This study identifies suitable substitution patterns for the design of 4(3H)-quinazolinone derivatives as MAO-B inhibitors.     

Differences in drought sensitivities and photosynthetic limitations between co-occurring C3 and C4 (NADP-ME) Panicoid grasses

Background and Aims

The success of C₄ plants lies in their ability to attain greater efficiencies of light, water and nitrogen use under high temperature, providing an advantage in arid, hot environments. However, C₄ grasses are not necessarily less sensitive to drought than C₃ grasses and are proposed to respond with greater metabolic limitations, while the C₃ response is predominantly stomatal. The aims of this study were to compare the drought and recovery responses of co-occurring C₃ and C₄ NADP-ME grasses from the subfamily Panicoideae and to determine stomatal and metabolic contributions to the observed response.

Methods

Six species of locally co-occurring grasses, C₃ species Alloteropsis semialata subsp. eckloniana, Panicum aequinerve and Panicum ecklonii, and C₄ (NADP-ME) species Heteropogon contortus, Themeda triandra and Tristachya leucothrix, were established in pots then subjected to a controlled drought followed by re-watering. Water potentials, leaf gas exchange and the response of photosynthetic rate to internal CO₂ concentrations were determined on selected occasions during the drought and re-watering treatments and compared between species and photosynthetic types.

Key Results

Leaves of C₄ species of grasses maintained their photosynthetic advantage until water deficits became severe, but lost their water-use advantage even under conditions of mild drought. Declining C₄ photosynthesis with water deficit was mainly a consequence of metabolic limitations to CO₂ assimilation, whereas, in the C₃ species, stomatal limitations had a prevailing role in the drought-induced decrease in photosynthesis. The drought-sensitive metabolism of the C₄ plants could explain the observed slower recovery of photosynthesis on re-watering, in comparison with C₃ plants which recovered a greater proportion of photosynthesis through increased stomatal conductance.

Conclusions

Within the Panicoid grasses, C₄ (NADP-ME) species are metabolically more sensitive to drought than C₃ species and recover more slowly from drought.     

Electrochemical investigations of platinum-methyluracil-blue and the related binuclear complex [(NH3)2Pt(MeU)2Pt(NH3)2](NO3)2

The redox behavior of the head-to-head bis(μ- (1-methyluracilato-N³,O²)-bis(cis-diammine platinum(II)) dinitrate, PtMeU, and platinum 1-methyluracil blue, PtMeUB, was studied by cyclic voltammetry (CV), rotating disk voltammetry (RDV), and controlled-potential coulometry (CPC). Redox titrimetry, electrochemistry/electron paramagnetic resonance spectroscopy (EPR), and liquid chromatography (LC) served as complementary techniques. The former reactant exhibits two-step electro-oxidation, consistent with the formation of a mixed-valence Pt(II, III) state en route to Pt(III, III). The latter also appears to oxidize to a uniform Pt(III) state. Although the oxidative-reductive electrochemistry of both reactants exhibits chemical reversibility, the heterogeneous electron-transfer kinetics are notably sluggish. The latter appears to be associated with the formation of an inhibiting film on the electrode surface. A slow conversion of PtMeU to a PtMeUB-like state was revealed by CV and LC. The complex, oligomeric nature of PtMeUB was revealed by means of gradient LC examination. Comparing oxidative and reductive electrolysis curves for PtMeUB yielded an average platinum oxidation state of 2.08. All observed behavior for PtMeUB, as well as for PtMeU, is accounted for by invoking +2 and +3 oxidation states for platinum; redox titrimetry using Ce(IV) revealed inconsequential oxidation of both of these systems beyond the III state. An estimate of molecular weight for the platinum blue was made by employing RDV in conjunction with the Einstein-Stokes equation.     

Simulation of separation of C2H6 from CH4 using zeolitic imidazolate frameworks

Separation of important chemical feedstocks, such as C₂H₆ from natural gas, can greatly benefit the petrochemical industry. In this paper, the grand canonical Monte Carlo method has been used to study the adsorption and separation of CH₄ and C₂H₆ in zeolites, isoreticular metal-organic framework-1 (IRMOF-1) and zeolitic imidazolate frameworks (ZIFs) with different topology, including soadlite, gmelinite and RHO topologies. Compared with mordenite zeolite and IRMOF-1, ZIFs and mordenite framework inverted (MFI) zeolite have better separation performance for C₂H₆/CH₄ mixtures at different mole fractions of C₂H₆. From the study of equilibrium snapshots and density distribution profiles, adsorption sites could be grouped as (1) sites with strong interactions with adsorbent and (2) sites with strong interactions with surrounding adsorbates. The gas molecules occupied the first site and then went on to occupy the second site. In CH₄/C₂H₆ mixture adsorption/separation, the adsorption of CH₄ was confined by the existence of C₂H₆. Due to energetic effect, C₂H₆ selectivity was affected by temperature at a low-pressure range, but did not change as much in a high-pressure range because of packing effect in micropore. In binary adsorption, large C₂H₆ molecules favour sites with strong adsorbent interactions. At high pressures, packing effects played an important role and it became easy for small CH₄ molecules to access the sites with strong adsorbate interactions.