Evidence That the P\u3csub\u3ei\u3c/sub\u3e Release Event Is the Rate-Limiting Step in the Nitrogenase Catalytic Cycle

Nitrogenase reduction of dinitrogen (N2) to ammonia (NH3) involves a sequence of events that occur upon the transient association of the reduced Fe protein containing two ATP molecules with the MoFe protein that includes electron transfer, ATP hydrolysis, Pi release, and dissociation of the oxidized, ADP-containing Fe protein from the reduced MoFe protein. Numerous kinetic studies using the nonphysiological electron donor dithionite have suggested that the rate-limiting step in this reaction cycle is the dissociation of the Fe protein from the MoFe protein. Here, we have established the rate constants for each of the key steps in the catalytic cycle using the physiological reductant flavodoxin protein in its hydroquinone state. The findings indicate that with this reductant, the rate-limiting step in the reaction cycle is not protein–protein dissociation or reduction of the oxidized Fe protein, but rather events associated with the Pi release step. Further, it is demonstrated that (i) Fe protein transfers only one electron to MoFe protein in each Fe protein cycle coupled with hydrolysis of two ATP molecules, (ii) the oxidized Fe protein is not reduced when bound to MoFe protein, and (iii) the Fe protein interacts with flavodoxin using the same binding interface that is used with the MoFe protein. These findings allow a revision of the rate-limiting step in the nitrogenase Fe protein cycle

Engineering tyrosine-based electron flow pathways in proteins: The case of aplysia myoglobin

Tyrosine residues can act as redox cofactors that provide an electron transfer ("hole-hopping") route that enhances the rate of ferryl heme iron reduction by externally added reductants, for example, ascorbate. Aplysia fasciata myoglobin, having no naturally occurring tyrosines but 15 phenylalanines that can be selectively mutated to tyrosine residues, provides an ideal protein with which to study such through-protein electron transfer pathways and ways to manipulate them. Two surface exposed phenylalanines that are close to the heme have been mutated to tyrosines (F42Y, F98Y). In both of these, the rate of ferryl heme reduction increased by up to 3 orders of magnitude. This result cannot be explained in terms of distance or redox potential change between donor and acceptor but indicates that tyrosines, by virtue of their ability to form radicals, act as redox cofactors in a new pathway. The mechanism is discussed in terms of the Marcus theory and the specific protonation/deprotonation states of the oxoferryl iron and tyrosine. Tyrosine radicals have been observed and quantified by EPR spectroscopy in both mutants, consistent with the proposed mechanism. The location of each radical is unambiguous and allows us to validate theoretical methods that assign radical location on the basis of EPR hyperfine structure. Mutation to tyrosine decreases the lipid peroxidase activity of this myoglobin in the presence of low concentrations of reductant, and the possibility of decreasing the intrinsic toxicity of hemoglobin by introduction of these pathways is discussed. © 2012 American Chemical Society

Ascorbic acid: A useful reductant to avoid oxidation of catecholamines in electrophysiological experiments in vitro?

The actions of the reductant ascorbic acid on rat neocortical neurons in vitro was investigated by means of intracellular recordings. At a concentration (500 μM), which reduced the magnitude of dopamine degradation in oxygen-saturated saline solutions by about 50%, ascorbic acid reversibly depressed synaptic potentials and enhanced direct excitability of cortical neurons. The latter effect was not reversible within the observation period. Ascorbic acid did not alter membrane potential and input resistance of the neurons. On the basis of our results we conclude that ascorbic acid is not a useful reductant to avoid oxidation of catecholamines in oxygen-saturated solutions used in electrophysiological experiments in vitro

Reduction of a-tocopherylquinone Model Compound With Various Reductant

In order to study the possibility of tranformation of a-tocopherylquinone (TQ) into a more oxidiseable compound and also to find out the recycling effect in the cells, an experiment was conducted by reducing the model compound 2-(3- hydroxy-3-methylbutyl)-3,5,6-trimethyl-1,4-benzoquinone (PQ) with various reductants. In the experiment it was shown that glutathione did not reduce PQ,nor NADH by itself, so the effective reductant in the NADH/FAD combination must have been FADH2. Thus there is a probability that in a biological system, the most probable reductant for TQ would be a flavin enzyme rather that ascorbic acid or glutathione. The non-physiological dithiothreitol was as effective as NADH/ FAD which is interesting because of its similarity to the physiologically important reduced lipoic acid. The reactivity of the various reductants used in this experiment decrease in the order of dithiothreitol ~ NADH/FAD (8/10) > sodium dithionite > NADH/FAD (2:10) > sodium ascorbate > ascorbic acid (Fig.8)

Controlled Synthesis of Silver Nanoparticles Using Double Reductants and Its Voltammetric Characteristics Study

Constructing robust silver nanoparticles (AgNPs) with good shape and dispersibility is of particular interest in analytical applications. Herein, monodispersibility AgNPs with the average size of 20 nm have been successfully prepared via one-pot method using sodium borohydride and trisodium citrate as co-reductants. The introduction of sodium borohydride greatly accelerated the rate of nucleation, which can effectively solve the problem of broad size distribution. Both shape and dispersibility of AgNPs can be effectively adjusted by simple control of refluxing time or concentrations of the sodium borohydride. We also studied the voltammetric characteristics of the AgNPs using Ag/AgCl solid-state voltammetry. An intense and stable current peak at a low potential could be obtained, which could provide a unique advantage in analytical applications. Copyright © 2016 BCREC GROUP. All rights reserved Received: 5th November 2015; Revised: 21st January 2016; Accepted: 22nd January 2016 How to Cite: Duan, Y., Xu, Z., Jiang, X. (2016). Controlled Synthesis of Silver Nanoparticles Using Double Reductants and Its Voltammetric Characteristics Study. Bulletin of Chemical Reaction Engineering & Catalysis, 11 (1): 115-119. (doi:10.9767/bcrec.11.1.433.115-119) Permalink/DOI: http://dx.doi.org/10.9767/bcrec.11.1.433.115-11

Mechanistic studies on DNA damage by minor groove binding copper–phenanthroline conjugates

Copper–phenanthroline complexes oxidatively damage and cleave nucleic acids. Copper bis-phenanthroline and copper complexes of mono- and bis-phenanthroline conjugates are used as research tools for studying nucleic acid structure and binding interactions. The mechanism of DNA oxidation and cleavage by these complexes was examined using two copper–phenanthroline conjugates of the sequence-specific binding molecule, distamycin. The complexes contained either one or two phenanthroline units that were bonded to the DNA-binding domain through a linker via the 3-position of the copper ligand. A duplex containing independently generated 2-deoxyribonolactone facilitated kinetic analysis of DNA cleavage. Oxidation rate constants were highly dependent upon the ligand environment but rate constants describing elimination of the alkali-labile 2-deoxyribonolactone intermediate were not. Rate constants describing DNA cleavage induced by each molecule were 11–54 times larger than the respective oxidation rate constants. The experiments indicate that DNA cleavage resulting from β-elimination of 2-deoxyribonolactone by copper–phenanthroline complexes is a general mechanism utilized by this family of molecules. In addition, the experiments confirm that DNA damage mediated by mono- and bis-phenanthroline copper complexes proceeds through distinct species, albeit with similar outcomes

Facile O-atom insertion into C-C and C-H bonds by a trinuclear copper complex designed to harness a singlet oxene

Two trinuclear copper [CuICuICuI(L)]1+ complexes have been prepared with the multidentate ligands (L) 3,3'-(1,4-diazepane-1,4-diyl)bis(1-((2-(dimethylamino)ethyl)(methyl)amino)propan-2-ol) (7-Me) and (3,3'-(1,4-diazepane-1,4-diyl)bis(1-((2-(diethylamino) ethyl)(ethyl) amino)propan-2-ol) (7-Et) as models for the active site of the particulate methane monooxygenase (pMMO). The ligands were designed to form the proper spatial and electronic geometry to harness a "singlet oxene," according to the mechanism previously suggested by our laboratory. Consistent with the design strategy, both [CuICuICuI(L)]1+ reacted with dioxygen to form a putative bis(µ3-oxo)CuIICuIICuIII species, capable of facile O-atom insertion across the central C-C bond of benzil and 2,3-butanedione at ambient temperature and pressure. These complexes also catalyze facile O-atom transfer to the C-H bond of CH3CN to form glycolonitrile. These results, together with our recent biochemical studies on pMMO, provide support for our hypothesis that the hydroxylation site of pMMO contains a trinuclear copper cluster that mediates C-H bond activation by a singlet oxene mechanism

Mechanisms and advancement of antifading agents for fluorescence microscopy and single-molecule spectroscopy

Modern fluorescence microscopy applications go along with increasing demands for the employed fluorescent dyes. In this work, we compared antifading formulae utilizing a recently developed reducing and oxidizing system (ROXS) with commercial antifading agents. To systematically test fluorophore performance in fluorescence imaging of biological samples, we carried out photobleaching experiments using fixed cells labeled with various commonly used organic dyes, such as Alexa 488, Alexa 594, Alexa 647, Cy3B, ATTO 550, and ATTO 647N. Quantitative evaluation of (i) photostability, (ii) brightness, and (iii) storage stability of fluorophores in samples mounted in different antifades (AFs) reveal optimal combinations of dyes and AFs. Based on these results we provide guidance on which AF should preferably be used with a specific dye. Finally, we studied the antifading mechanisms of the commercial AFs using single-molecule spectroscopy and reveal that these empirically selected AFs exhibit similar properties to ROXS AFs

Applications of nanostructured materials and biomolecules for electrocatalysis and biosensors

Electronically conducting polymers are important materials, and composites of these materials with metal nanoparticles have also been drawn significant research attention in recent years. We prepared a highly stable Agnano-Poly (3, 4-ethylenedioxythiophene) (PEDOT) nanocomposite by one-pot synthesis method. Here, 3, 4-ethylenedioxythiophene (EDOT) is used as the reductant and polystyrene sulfonate (PSS-) as a dopant for PEDOT as well as particle stabilizer for silver nanoparticles (AgNPs). Agnano–PEDOT/PSS-nanocomposite was characterized by infrared (IR) spectroscopy, transmission electron microscopy (TEM). AgNPs are distributed uniformly around PEDOT polymer with an average particle size diameter of 10–15 nm and the nanocomposite film showed catalytic activity towards 4-nitro phenol. Some types of including Ag bimetallic nanoparticles and nanostructured materials could be directly applied for the electroanalysis and biosensing applications. When you are citing the document, use the following link http://essuir.sumdu.edu.ua/handle/123456789/2045

Electrochemical processes and systems: application for tutors

The features of redox reactions and the principles of their balancing according to the medium composition are considered. The basic representations about electrochemical processes and systems are outlined. The reactions and principles of chemical sources of electric energy and electrolysis systems functioning are analyzed. A general idea is given about the chemical properties of metals, corrosion resistance in environments of various aggressiveness, and the protection principles are given. Multivariate tasks and exercises for students, and PhD student’s classroom and independent work are offered. For teachers, PhD students and students of universities of specialties "Chemical technologies and engineering", "Biotechnologies and bioengineering", "Oil and gas engineering and technologies".Розглянуто особливості окисно-відновних реакцій і принципи їх балансування залежно від складу середовища. Викладено фундаментальні уявлення про електрохімічні процеси і системи. Проаналізовано перебіг реакцій і принципи функціонування хімічних джерел електричної енергії та систем електролізу. Узагальнено уявлення щодо хімічних властивостей металів, корозійної стійкості у середовищах різної агресивності та наведено принципи організації захисту від руйнування. Запропоновано багатоваріантні завдання та вправи для аудиторної та самостійної роботи студентів і аспірантів. Розраховано на викладачів, аспірантів і студентів вищих навчальних закладів спеціальностей "Хімічні технології та інженерія”, "Біотехнології та біоінженерія", "Нафтогазова інженерія та технології"