Electrochemical Carbonylation of Organoiron Methyl Complex: A Study of Reaction Intermediates

The one-electron reduction of CpFe(CO)2CH3 has been investigated by voltammetry and Fourier transform IR spectroelectrochemistry. The reduction initiates the insertion of CO ligand in the FeCH3 bond. The dissociation of a CO group proceeds in a parallel reaction. Reaction intermediates, the acyl derivative and released CO, form the radical anion of a complex CpFe(CO)2(COCH3) which is able to reduce the parent compound. The reversible redox potential − 1.8 V of CpFe(CO)2(COCH3) allows the regeneration of its radical anion which drives a catalytic cycle. The lifetime of intermediates is shortened by side reactions, one of which is the migration of the acyl group from the central atom to the cyclopentadienyl ring. This explains the apparent discrepancy between products observed in preparative scale electrolysis and the absence of catalytic effects in routine voltammetric experiments

Endothelial Nitric Oxide Synthase Oxygenase on Lipid Nanodiscs: A Nano-Assembly Reflecting Native-Like Function of eNOS

© 2017 Elsevier Inc. Endothelial nitric oxide synthase (eNOS) is a membrane-anchored enzyme. To highlight the potential role and effect of membrane phospholipids on the structure and activity of eNOS, we have incorporated the recombinant oxygenase subunit of eNOS into lipid nanodiscs. Two different size distribution modes were detected by multi-angle dynamic light scattering both for empty nanodiscs, and nanodiscs-bound eNOSoxy. The calculated hydrodynamic diameter for mode 1 species was 9.0 nm for empty nanodiscs and 9.8 nm for nanodisc bound eNOSoxy. Spectroscopic Griess assay was used to measure the enzymatic activity. Remarkably, the specific activity of nanodisc-bound eNOSoxy is ∼65% lower than the activity of free enzyme. The data shows that the nano-membrane environment affects the catalytic properties of eNOS heme domain

Charge-Pairing Interactions Control The Conformational Setpoint and Motions of The FMN Domain in Neuronal Nitric Oxide Synthase

The NOS (nitric oxide synthase; EC 1.14.13.39) enzymes contain a C-terminal flavoprotein domain [NOSred (reductase domain of NOS)] that binds FAD and FMN, and an N-terminal oxygenase domain that binds haem. Evidence suggests that the FMN-binding domain undergoes large conformational motions to shuttle electrons between the NADPH/FAD-binding domain [FNR (ferredoxin NADP-reductase)] and the oxygenase domain. Previously we have shown that three residues on the FMN domain (Glu(762), Glu(816) and Glu(819)) that make charge-pairing interactions with the FNR help to slow electron flux through nNOSred (neuronal NOSred). In the present study, we show that charge neutralization or reversal at each of these residues alters the setpoint [K-eq(A)], of the NOSred conformational equilibrium to favour the open (FMN-deshielded) conformational state. Moreover, computer simulations of the kinetic traces of cytochrome c reduction by the mutants suggest that they have higher conformational transition rates (1.5-4-fold) and rates of interflavin electron transfer (1.5-2-fold) relative to wild-type nNOSred. We conclude that the three charge-pairing residues on the FMN domain govern electron flux through nNOSred by stabilizing its closed (FMN-shielded) conformational state and by retarding the rate of conformational switching between its open and closed conformations

Elevation of serum soluble E-selectin and VCAM-1 in severe asthma.

OBJECTIVE: To investigate the significance of circulating adhesion molecules associated with leucocyte-endothelial cell interactions in asthma, serum levels of soluble E (sE)-selectin, soluble P (sP)-selectin, soluble L (sL)-selectin, and soluble vascular cell adhesion molecule-1 (sVCAM-1) were measured in mild, moderate and severe asthma. METHOD: Serum levels of sE-selectin, sP-selectin, sL-selectin, and sVCAM-1 were measured in 32 women with asthma and 30 healthy donors using an enzyme-linked immunosorbent assay method. Twenty patients were suffering from severe asthma, and 12 from mild/moderate asthma. RESULTS: Serum sE-selectin and sVCAM-1 levels from patients with asthma were significantly higher than those observed in healthy donors (p < 0.01). The levels of sP-selectin were the same as those of controls. The level of sE-selectin exhibited an important increase in the severe asthmatic patients compared with mild/moderate asthma (p < 0.01). The sVCAM-1 level was increased in severe asthma when compared with healthy controls. There was no correlation between the levels of soluble selectins and the age of the patients. A significant correlation was found between sE-selectin and sVCAM-1 levels. CONCLUSION: These data indicate that circulating soluble forms of the selectins may have different kinetics during the clinical course of asthma, suggesting that they may reflect different inflammatory pathways in severe asthma. Both sVCAM-1 and sE-selectin may be useful immunological markers for monitoring disease activity in asthma

Rumen fermentation pattern and blood metabolites of lambs from three breeds reared on pasture or feedlot

Publishe

Cholesterol Levels and Activity of Membrane Bound Proteins: Characterization by Thermal and Electrochemical Methods

The long-term goal of this investigation is to study the effects of increased cholesterol levels on the molecular activity of membrane-bound enzymes such as nitric oxide synthase, that are critical in the functioning of the cardiovascular system. In this particular investigation, we used differential scanning calorimetry (DSC) and dielectric thermal analysis (DETA) to study the effect of added cholesterol on melting/recrystallization and dielectric behavior, respectively, of phosphatidylcholine (PC) bilayered thin films. We also used electrochemical methods to investigate the effect of added cholesterol on the redox behavior of the oxygenase domain of nitric oxide synthase as a probe embedded in the PC films. The results show that added cholesterol in the PC films seems to depress the molecular dynamics as indicated by lowered current responses in the presence of cholesterol as well as a slight increase of the transition temperature in the overall two-phase regime behavior observed in PC–cholesterol films. These results are rationalized in the context of the general DSC and DETA behaviors of the PC–chol films

Distinct conformational behaviors of four mammalian dual‐flavin reductases (cytochrome P450 reductase, methionine synthase reductase, neuronal nitric oxide synthase, endothelial nitric oxide synthase) determine their unique catalytic profiles

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/109651/1/febs13073.pd

Distinct Conformational Behaviors of Four Mammalian Dual-Flavin Reductases (Cytochrome P450 Reductase, Methionine Synthase Reductase, Neuronal Nitric Oxide Synthase, Endothelial Nitric Oxide Synthase) Determine Their Unique Catalytic Profiles

Multidomain enzymes often rely on large conformational motions to function. However, the conformational setpoints, rates of domain motions and relationships between these parameters and catalytic activity are not well understood. To address this, we determined and compared the conformational setpoints and the rates of conformational switching between closed unreactive and open reactive states in four mammalian diflavin NADPH oxidoreductases that catalyze important biological electron transfer reactions: cytochrome P450 reductase, methionine synthase reductase and endothelial and neuronal nitric oxide synthase. We used stopped-flow spectroscopy, single turnover methods and a kinetic model that relates electron flux through each enzyme to its conformational setpoint and its rates of conformational switching. The results show that the four flavoproteins, when fully-reduced, have a broad range of conformational setpoints (from 12% to 72% open state) and also vary 100-fold with respect to their rates of conformational switching between unreactive closed and reactive open states (cytochrome P450 reductase \u3e neuronal nitric oxide synthase \u3e methionine synthase reductase \u3e endothelial nitric oxide synthase). Furthermore, simulations of the kinetic model could explain how each flavoprotein can support its given rate of electron flux (cytochrome c reductase activity) based on its unique conformational setpoint and switching rates. The present study is the first to quantify these conformational parameters among the diflavin enzymes and suggests how the parameters might be manipulated to speed or slow biological electron flux

Structure, bonding and morphology of hydrothermally synthesised xonotlite

The authors have systematically investigated the role of synthesis conditions upon the structure and morphology of xonotlite. Starting with a mechanochemically prepared, semicrystalline phase with Ca/Si=1, the authors have prepared a series of xonotlite samples hydrothermally, at temperatures between 200 and 250 degrees C. Analysis in each case was by X-ray photoelectron spectroscopy, environmental scanning electron microscopy and X-ray diffraction. The authors’ use of a much lower water/solid ratio has indirectly confirmed the ‘through solution’ mechanism of xonotlite formation, where silicate dissolution is a key precursor of xonotlite formation. Concerning the role of temperature, too low a temperature (~200 degrees C) fails to yield xonotlite or leads to increased number of structural defects in the silicate chains of xonotlite and too high a temperature (>250 degrees C) leads to degradation of the xonotlite structure, through leaching of interchain calcium. Synthesis duration meanwhile leads to increased silicate polymerisation due to diminishing of the defects in the silicate chains and more perfect crystal morphologies