scholarly journals Spectral characterization and chemical modification of FMN-containing ascorbyl free-radical reductase from Pleurotus ostreatus

1999 ◽  
Vol 341 (3) ◽  
pp. 755-763 ◽  
Author(s):  
Seong-Woon YU ◽  
Yeon-Ran KIM ◽  
Sa-Ouk KANG
Keyword(s):  
Charge Transfer ◽  
Free Radical ◽  
Molecular Mass ◽  
Pleurotus Ostreatus ◽  
Catalytic Mechanism ◽  
Thiol Group ◽  
Prosthetic Group ◽  
Ascorbyl Free Radical ◽  
Lysine Residues ◽  
Thiolate Anion

Ascorbyl free-radical reductase was purified 1143-fold with an overall yield of 9.9% from the cytosolic fraction of Pleurotus ostreatus. The native enzyme had a molecular mass of 127 kDa and SDS/PAGE revealed that the enzyme consists of two subunits, each with a molecular mass of 62 kDa. The enzyme utilized only NADH as an electron donor. The enzyme was highly specific for ascorbyl free radical as an electron acceptor and capable of catalysing the reduction of ferricyanide and 2,6-dichloroindophenol as artificial electron acceptors. The apparent Km values of the enzyme towards NADH and ascorbyl free radical were 35±0.22 and 2.1±0.03 μM, respectively. The catalytic mechanism of this enzyme is of Ping Pong type. The enzyme contained FMN as a prosthetic group and showed the characteristic absorption spectrum ascribed to the charge-transfer interaction of thiolate anion with FMN. The enzyme contained eight cysteine residues per monomer and was inactivated more rapidly by mercurials than by thiol-alkylating reagents. Kinetic analysis of the inactivation process revealed that the enzyme had 1 mol of thiol group/mol of subunit in the active site with a pKa of 6.9. The modification of the thiol group of the enzyme caused the loss of charge-transfer absorbance centred at 540 nm and blocked the electron-transfer process from NADH to FMN. The modification of lysine, arginine and histidine residues led to the loss of its activity. Unlike the active enzyme, the fluorescence quenching of NADH was not observed in the lysine-modified enzyme, which implies that lysine residues can participate in the interaction with NADH.

Mechanistic Insights into Fe Catalyzed α-C-H Oxidations of Tertiary Amines

2019 ◽  
Author(s):  
Christopher J. Legacy ◽  
Frederick T. Greenaway ◽  
Marion Emmert
Keyword(s):  
Free Radical ◽  
Catalytic Mechanism ◽  
Catalyst System ◽  
Oxidation Products ◽  
Tertiary Amines ◽  
Catalyst Activation ◽  
Rate Determining Step ◽  
Fe Catalyst ◽  
Ligand Coordination ◽  
Rate Kinetics

We report detailed mechanistic investigations of an iron-based catalyst system, which allows the α-C-H oxidation of a wide variety of amines, including acyclic tertiary aliphatic amines, to afford dealkylated or amide products. In contrast to other catalysts that affect α-C-H oxidations of tertiary amines, the system under investigation employs exclusively peroxy esters as oxidants. More common oxidants (e.g. tBuOOH) previously reported to affect amine oxidations via free radical pathways do not provide amine α-C-H oxidation products in combination with the herein described catalyst system. Motivated by this difference in reactivity to more common free radical systems, the investigations described herein employ initial rate kinetics, kinetic profiling, Eyring studies, kinetic isotope effect studies, Hammett studies, ligand coordination studies, and EPR studies to shed light on the Fe catalyst system. The obtained data suggest that the catalytic mechanism proceeds through C-H abstraction at a coordinated substrate molecule. This rate-determining step occurs either at an Fe(IV) oxo pathway or a 2-electron pathway at a Fe(II) intermediate with bound oxidant. We further show via kinetic profiling and EPR studies that catalyst activation follows a radical pathway, which is initiated by hydrolysis of PhCO3 tBu to tBuOOH in the reaction mixture. Overall, the obtained mechanistic data support a non-classical, Fe catalyzed pathway that requires substrate binding, thus inducing selectivity for α-C-H functionalization.<br>

scholarly journals Anion polarity-induced dual oxidation states in a dual-layered purely organic paramagnetic charge-transfer salt, (TTF)3(PO-CON(CH3)C2H4SO3)2, where PO = 2,2,5,5-tetramethyl-3-pyrrolin-1-oxyl free radical

CrystEngComm ◽  
2011 ◽  
Vol 13 (17) ◽  
pp. 5281 ◽  
Author(s):  
Hiroki Akutsu ◽  
Atsushi Kawamura ◽  
Jun-ichi Yamada ◽  
Shin'ichi Nakatsuji ◽  
Scott S. Turner
Keyword(s):  
Charge Transfer ◽  
Free Radical ◽  
Oxidation States

scholarly journals Antioxidant/Oxidant Status and Cardiac Function in Bradykinin B1- and B2-Receptor Null Mice

2013 ◽  
pp. 511-517 ◽  
Author(s):  
S. DELEMASURE ◽  
N. BLAES ◽  
C. RICHARD ◽  
R. COUTURE ◽  
M. BADER ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Free Radical ◽  
Cardiovascular Diseases ◽  
Vitamin C ◽  
Cardiac Function ◽  
Cardiac Phenotype ◽  
Plasma Antioxidant Capacity ◽  
Ascorbyl Free Radical ◽  
Plasma Antioxidant ◽  
Oxidant Status

Kinin-vasoactive peptides activate two G-protein-coupled receptors (R), B1R (inducible) and B2R (constitutive). Their complex role in cardiovascular diseases could be related to differential actions on oxidative stress. This study investigated impacts of B1R or B2R gene deletion in mice on the cardiac function and plasma antioxidant and oxidant status. Echocardiography-Doppler was performed in B1R (B1R-/-) and B2R (B2R-/-) deficient and wild type (WT) adult male mice. No functional alteration was observed in B2R-/- hearts. B1R-/- mice had significantly lowered fractional shortening and increased isovolumetric contraction time. The diastolic E and A waves velocity ratio was similar in all mice groups. Thus B1R-/- mice provide a model of moderate systolic dysfunction, whereas B2R-/- mice displayed a normal cardiac phenotype. Plasma antioxidant capacity (ORAC) was significantly decreased in both B1R-/- and B2R-/- mice whereas the vitamin C levels were decreased in B2R-/- mice only. Plasma ascorbyl free radical was significantly higher in B1R-/- compared to WT and B2R-/- mice. Therefore, the oxidative stress index, ascorbyl free radical to vitamin C ratio, was increased in both B1R-/- and B2R-/- mice. Hence, B1R and B2R deficiency are associated with increased oxidative stress, but there is a differential imbalance between free radical production and antioxidant defense. The interrelationship between the differential B1R and B2R roles in oxidative stress and cardiovascular diseases remain to be investigated.

scholarly journals Identification and Analysis of Genes Involved in Anaerobic Toluene Metabolism by Strain T1: Putative Role of a Glycine Free Radical

1998 ◽  
Vol 64 (5) ◽  
pp. 1650-1656 ◽  
Author(s):  
Peter W. Coschigano ◽  
Thomas S. Wehrman ◽  
L. Y. Young
Keyword(s):  
Free Radical ◽  
Amino Acid ◽  
Molecular Mass ◽  
Cysteine Residue ◽  
Open Reading Frames ◽  
Site Directed Mutagenesis ◽  
Pyruvate Formate Lyase ◽  
Calculated Molecular Mass ◽  
Acid Protein ◽  
Amino Acid Protein

ABSTRACT The denitrifying strain T1 is able to grow with toluene serving as its sole carbon source. Two mutants which have defects in this toluene utilization pathway have been characterized. A clone has been isolated, and subclones which contain tutD and tutE, two genes in the T1 toluene metabolic pathway, have been generated. ThetutD gene codes for an 864-amino-acid protein with a calculated molecular mass of 97,600 Da. The tutE gene codes for a 375-amino-acid protein with a calculated molecular mass of 41,300 Da. Two additional small open reading frames have been identified, but their role is not known. The TutE protein has homology to pyruvate formate-lyase activating enzymes. The TutD protein has homology to pyruvate formate-lyase enzymes, including a conserved cysteine residue at the active site and a conserved glycine residue that is activated to a free radical in this enzyme. Site-directed mutagenesis of these two conserved amino acids shows that they are also essential for the function of TutD.

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