Jumpstarting the cytochrome P450 catalytic cycle with a hydrated electron.

Cytochrome P450cam (CYP101Fe) regioselectively hydroxylates camphor. Possible hydroxylating intermediates in the catalytic cycle of this well-characterized enzyme have been proposed on the basis of experiments carried out at very low temperatures and shunt reactions, but their presence has not yet been validated at temperatures above 0 °C during a normal catalytic cycle. Here, we demonstrate that it is possible to mimic the natural catalytic cycle of CYP101Fe by using pulse radiolysis to rapidly supply the second electron of the catalytic cycle to camphor-bound CYP101[FeO] Judging by the appearance of an absorbance maximum at 440 nm, we conclude that CYP101[FeOOH] (compound 0) accumulates within 5 μs and decays rapidly to CYP101Fe, with a of 9.

Activation of Signaling Cascades by Weak Extremely Low Frequency Electromagnetic Fields.

Background/aims: Results from recent studies suggest that extremely low frequency magnetic fields (ELF-MF) interfere with intracellular signaling pathways related to proliferative control. The mitogen-activated protein kinases (MAPKs), central signaling components that regulate essentially all stimulated cellular processes, include the extracellular signal-regulated kinases 1/2 (ERK1/2) that are extremely sensitive to extracellular cues. Anti-phospho-ERK antibodies serve as a readout for ERK1/2 activation and are able to detect minute changes in ERK stimulation.

Signaling by sulfur-containing molecules. Quantitative aspects.

There is currently interest in sulfur-containing molecules that may or may not play a role in signaling. We have collected relevant thermodynamic data, namely standard Gibbs energies of formation and electrode potentials at pH 7, and used these to construct a Frost diagram. Thermodynamic data not available in the literature could be estimated with reasonable confidence.

Extremely low-frequency magnetic fields and risk of childhood leukemia: A risk assessment by the ARIMMORA consortium.

Exposure to extremely low-frequency magnetic fields (ELF-MF) was evaluated in an International Agency for Research on Cancer (IARC) Monographs as "possibly carcinogenic to humans" in 2001, based on increased childhood leukemia risk observed in epidemiological studies. We conducted a hazard assessment using available scientific evidence published before March 2015, with inclusion of new research findings from the Advanced Research on Interaction Mechanisms of electroMagnetic exposures with Organisms for Risk Assessment (ARIMMORA) project. The IARC Monograph evaluation scheme was applied to hazard identification.

Redox properties and activity of iron-citrate complexes: evidence for redox cycling.

Iron in iron overload disease is present as non-transferrin-bound iron, consisting of iron, citrate, and albumin. We investigated the redox properties of iron citrate by electrochemistry, by the kinetics of its reaction with ascorbate, by ESR, and by analyzing the products of reactions of ascorbate with iron citrate complexes in the presence of H2O2 with 4-hydroxybenzoic acid as a reporter molecule for hydroxylation. We report -0.

Concurrent cooperativity and substrate inhibition in the epoxidation of carbamazepine by cytochrome P450 3A4 active site mutants inspired by molecular dynamics simulations.

Cytochrome P450 3A4 (CYP3A4) is the major human P450 responsible for the metabolism of carbamazepine (CBZ). To explore the mechanisms of interactions of CYP3A4 with this anticonvulsive drug, we carried out multiple molecular dynamics (MD) simulations, starting with the complex of CYP3A4 manually docked with CBZ. On the basis of these simulations, we engineered CYP3A4 mutants I369F, I369L, A370V, and A370L, in which the productive binding orientation was expected to be stabilized, thus leading to increased turnover of CBZ to the 10,11-epoxide product.

Peroxynitrous acid: controversy and consensus surrounding an enigmatic oxidant.

The isomerisation of ONOOH to NO(3)(-) and H(+), some oxidations and all hydroxylations and nitrations of aromatic compounds are first-order in ONOOH and zero-order in the compounds that are modified. These reactions are widely believed to proceed via homolysis of ONOOH into HO˙ and NO(2)˙ to an extent of ca. 30%.

Nitration activates tyrosine toward reaction with the hydrated electron.

3-Nitrotyrosine has been reported as an important biomarker of oxidative stress that may play a role in a variety of diseases. In this work, transient UV-visible absorption spectra and kinetics observed during the reaction of the hydrated electron, e(aq)(-), with 3-nitrotyrosine and derivatives thereof were investigated. The absorption spectra show characteristics of aromatic nitro anion radicals.

Otto Warburg's contributions to current concepts of cancer metabolism.

Otto Warburg pioneered quantitative investigations of cancer cell metabolism, as well as photosynthesis and respiration. Warburg and co-workers showed in the 1920s that, under aerobic conditions, tumour tissues metabolize approximately tenfold more glucose to lactate in a given time than normal tissues, a phenomenon known as the Warburg effect. However, this increase in aerobic glycolysis in cancer cells is often erroneously thought to occur instead of mitochondrial respiration and has been misinterpreted as evidence for damage to respiration instead of damage to the regulation of glycolysis.

Damage to fuel cell membranes. Reaction of HO* with an oligomer of poly(sodium styrene sulfonate) and subsequent reaction with O(2).

An understanding of the reactivity of oligomeric compounds that model fuel cell membrane materials under oxidative-stress conditions that mimic the fuel cell operating environment can identify material weaknesses and yield valuable insights into how a polymer might be modified to improve oxidative stability. The reaction of HO˙ radicals with a polymer electrolyte fuel cell membrane represents an initiation step for irreversible membrane oxidation. By means of pulse radiolysis, we measured k = (9.

Reduction of protein radicals by GSH and ascorbate: potential biological significance.

The oxidation of proteins and other macromolecules by radical species under conditions of oxidative stress can be modulated by antioxidant compounds. Decreased levels of the antioxidants glutathione and ascorbate have been documented in oxidative stress-related diseases. A radical generated on the surface of a protein can: (1) be immediately and fully repaired by direct reaction with an antioxidant; (2) react with dioxygen to form the corresponding peroxyl radical; or (3) undergo intramolecular long range electron transfer to relocate the free electron to another amino acid residue.

Electrode potentials of partially reduced oxygen species, from dioxygen to water.

We use published Gibbs energies of formation and equilibrium constants to determine electrode potentials for the partially reduced intermediates along the pathway of reduction of dioxygen to water, as well as of ozone and singlet dioxygen. The results are summarized in an oxidation state (Frost) diagram. Our review of the literature on electrode potentials leads us to revise values for the O(2)/O(2)(*-) couple to E degrees (O(2g)/O(2)(*-))=-0.

Kinetics of tyrosyl radical reduction by selenocysteine.

The rate constant for the reduction of the tyrosyl radical with selenocysteine has been measured to investigate whether selenocysteine is capable of repair of protein radicals. Tyrosyl radicals, both free in solution and in insulin, were generated by means of pulse radiolysis and laser flash photolysis in aqueous solution. The rate constant for the reaction of free N-acetyl-tyrosyl-amine radicals with selenocysteine is (8 +/- 2) x 10 (8) M (-1) s (-1), and that for tyrosyl radicals in insulin is (1.

Kinetics properties of Cu,Zn-superoxide dismutase as a function of metal content.

The kinetics of bovine Cu,Zn superoxide dismutase were studied by pulse radiolysis. To ensure the absence of catalytically active free copper, commercially obtained holo-superoxide dismutase was demetallated, and the apo-superoxide dismutase concentrations were determined by isothermal titration calorimetry prior to reconstitution with defined amounts of copper and zinc. The catalytic rate constant was determined as a function of ionic strength over the range of 4-154 mM, and of the copper and zinc content.

The preparation of apo-Cu,Zn superoxide dismutase by ion-exchange chromatography on iminodiacetic acid-sepharose.

The superoxide dismutases (EC 1.15.1.

Microsomal deacetylation of ring-hydroxylated 2-(acetylamino)fluorene isomers: effect of ring position and molecular mechanics considerations.

Metabolism of arylamides such as 2-(acetylamino)fluorene to mutagenic products is catalyzed by various liver microsomal and cytosolic enzymes. Deacylation is believed to be a deactivating pathway, and the activity of the microsomal deacetylase toward N-hydroxy-2-AAF is exceedingly greater than toward the parent 2-AAF. Another deactivating pathway is cytochrome P450-catalyzed ring hydroxylation.

The reaction of peroxynitrite with zeaxanthin.

The oxygenated carotenoids zeaxanthin and lutein, found in the macular area of the retina, may offer protection against or repair of oxidative damage associated with the degenerative diseases of aging. Since both superoxide and nitrogen monoxide, which react to form peroxynitrite, are found in the retina, we studied the reaction of peroxynitrite with zeaxanthin in liposomes. Zeaxanthin was easily incorporated into liposomes constructed from the fully saturated lipid L-alpha-dimyristoyl-phosphatidylcholine (C14:0) and from egg lecithin, and its absorbance spectrum in liposomes strongly resembles in shape and amplitude that of zeaxanthin dissolved in methanol.

Reaction of peroxynitrite with L-tryptophan.

The reaction between peroxynitrous acid (hydrogen oxoperoxonitrate) and L-tryptophan is 130 M(-1)s(-1) at 25°C. The pH dependence of the second-order rate constant shows a maximum at pH 5.1.

The kinetics of the oxidation of L-ascorbic acid by peroxynitrite.

Peroxynitrite [O = NOO-, oxoperoxonitrate(1-)bd is a strong oxidant that may be formed in vivo by the reaction of O2.- and NO(.).

Nature of the intermediate in the 3-oxo-delta 5-steroid isomerase reaction.

The role of Tyr-14 of 3-oxo-delta 5-steroid isomerase (KSI) was probed by analysis of the spectra of 3-amino-1,3,5(10)-estratrien-17 beta-ol (4) and equilenin (5) bound to the active site of KSI. The ultraviolet spectrum of 4 bound to KSI is identical to that for 4 in neutral solution. This observation indicates that Tyr-14 does not protonate the amine group of 4 at the active site.

The reaction of xanthine oxidase with aldehydic products of lipid peroxidation.

A quantitative structure-activity relationship for the reaction of xanthine oxidase with a homologous series of alpha, beta-unsaturated aldehydes, which are known to be products of lipid peroxidation, was investigated. Aldehydes in the series 2-butenal through 2-nonenal and 4-hydroxy-2-nonenal, displayed differential reactivity toward xanthine oxidase as measured by production of the superoxide radical anion. Kinetic parameters for the rate of superoxide production and substrate affinity were determined via the superoxide dismutase-sensitive reduction of cytochrome c.