High-energy Intermediates Research Articles

Legionella pneumophila couples fatty acid flux to microbial differentiation and virulence

During its life cycle, Legionella pneumophila alternates between at least two phenotypes: a resilient, infectious form equipped for transmission and a replicative cell type that grows in amoebae and macrophages. Considering its versatility, we postulated that multiple cues regulate L. pneumophila differentiation. Beginning with a Biolog Phenotype MicroArray screen, we demonstrate that excess short-chain fatty acids (SCFAs) trigger replicative cells to cease growth and activate their panel of transmissive traits. To co-ordinate their response to SCFAs, L. pneumophila utilizes the LetA/LetS two-component system, but not phosphotransacetylase or acetyl kinase, two enzymes that generate high-energy phosphate intermediates. Instead, the stringent response enzyme SpoT appears to monitor fatty acid biosynthesis to govern transmission trait expression, as an altered distribution of acylated acyl carrier proteins correlated with the SpoT-dependent differentiation of cells treated with either excess SCFAs or the fatty acid biosynthesis inhibitors cerulenin and 5-(tetradecyloxy)-2-furoic acid. We postulate that, by exploiting the stringent response pathway to couple cellular differentiation to its metabolic state, L. pneumophila swiftly acclimates to stresses encountered in its host or the environment, thereby enhancing its overall fitness.

Metal Binding Kinetics of Bi-Histidine Sites Used in ψ Analysis: Evidence of High-Energy Protein Folding Intermediates

The zinc-specific fluorophore, Zinpyr-1, is used in competition assays to determine the kinetic and thermodynamic parameters of Zn2+ binding to engineered bi-histidine sites located in ubiquitin and the B domain of protein A (BdpA). These binding sites are used in psi analysis studies to investigate structure formation in the folding transition state identified by the change in folding rate upon addition of metal ions. For ubiquitin, the on-rate binding constant and binding affinity for a site located along an alpha-helix are measured to be approximately 10(7) M-1 s-1 and 3 microM, respectively. For a site located across two beta-strands, the metal binding affinity was too weak to measure in the dye competition assays (Kd > 55 microM). The equilibrium-determined values for the Zn2+-induced stabilization of ubiquitin and BdpA match the values derived from changes in the global folding and unfolding rates. Therefore, metal ion binding is in fast equilibrium during the transit over the free energy barrier. Accordingly, the folding rate must be slower than the product of the fractional population of a high-energy intermediate with the metal site formed and the metal binding on-rate constant. The known folding rate of 20 s-1 at 1.5 M guanidinium chloride in 400 microM Zn2+ provides an upper bound for the stability of such intermediates (DeltaG(U-I) < 4 kcal/mol). These results support a view of the apparent two-state protein folding reaction surface as a fast pre-equilibrium between the denatured state and a series of high-energy species. The net folding rate is a product of the equilibrium constant of the highest-energy species and a transmission rate. For ubiquitin, we estimate the transmission rate to be approximately 10(4) s-1. Implications for the role of unfolded chain diffusion on folding rates and barrier heights are discussed.

Concerted and Stepwise Proton‐Coupled Electron Transfers in Aquo/Hydroxo Complex Couples in Water: Oxidative Electrochemistry of [OsII(bpy)2(py)(OH2)]2+

Successive oxidation of transition metal(II) aqua complexes (M(II)OH(2) to M(III)OH) is a domain in which proton-coupled electron transfer reactions are extremely common. The mechanism of these PCET reactions-concerted or stepwise-is an important issue in the understanding and design of natural or artificial systems catalyzing the formation of dioxygen by four-electron oxidation of water. Concerted proton-coupled electron transfer from an aqua metal(II) to a hydroxo metal(III) complex requires the close proximity of a proton-accepting group with a pK value between those of the aqua complexes. Otherwise, stepwise electron-proton or proton-electron pathways involving high-energy intermediates are followed. Concerted proton-electron pathways involving water as proton-acceptor or proton-donor group are inefficient. Cyclic voltammetry of the title complex in buffered aqueous solution and re-examination of previous results for the same complex attached to an electrode surface are used to establish these conclusions, which provide a starting point on the route to higher degrees of oxidation, such as those involved in the catalysis of water oxidation.

Design and Synthesis of 23-Amino- 24,25,26,27-tetrakisnorlanosterolacetate and Derivatives as New Antimycotic Agents

In search of new antimycotic agents acting as ergosterol biosynthesis inhibitors we investigated widely new and optimized reaction pathways for preparing different types of triazasteroids [1, 2]. The various mono-, bi-, tri- and tetracyclic triazasteroidal compounds are designed as stable aza-analogues of carbo-cationic high energy intermediates (HEIA) of fungal enzymes [1, 2].

Mitochondria, Bioenergetics, and the Epigenome in Eukaryotic and Human Evolution

Studies on the origin of species have focused largely on anatomy, yet animal populations are generally limited by energy. Animals can adapt to available energy resources at three levels: (1) evolution of different anatomical forms between groups of animals through nuclear DNA (nDNA) mutations, permitting exploitation of alternative energy reservoirs and resulting in new species with novel niches, (2) evolution of different physiologies within intraspecific populations through mutations in mitochondrial DNA (mtDNA) and nDNA bioenergetic genes, permitting adjustment to energetic variation within a species' niche, and (3) epigenomic regulation of dispersed bioenergetic genes within an individual via mitochondrially generated high-energy intermediates, permitting individual adjustment to environmental fluctuations. Because medicine focuses on changes within our species, clinically relevant variation is more likely to involve changes in bioenergetics than anatomy. This may explain why mitochondrial diseases and epigenomic diseases frequently have similar phenotypes and why epigenomic diseases are being found to involve mitochondrial dysfunction. Therefore, common complex diseases may be the result of changes in any of a large number of mtDNA and nDNA bioenergetic genes or to altered epigenomic regulation of these bioenergetic genes. All of these changes result in similar bioenergetic failure and consequently related phenotypes.

Synthetic, Mechanistic, and Computational Investigations of Nitrile-Alkyne Cross-Metathesis

The terminal nitride complexes NW(OC(CF 3) 2Me) 3(DME) ( 1-DME), [Li(DME) 2][NW(OC(CF 3) 2Me) 4] ( 2), and [NW(OCMe 2CF 3) 3] 3 ( 3) were prepared in good yield by salt elimination from [NWCl 3] 4. X-ray structures revealed that 1-DME and 2 are monomeric in the solid state. All three complexes catalyze the cross-metathesis of 3-hexyne with assorted nitriles to form propionitrile and the corresponding alkyne. Propylidyne and substituted benzylidyne complexes RCW(OC(CF 3) 2Me) 3 were isolated in good yield upon reaction of 1-DME with 3-hexyne or 1-aryl-1-butyne. The corresponding reactions failed for 3. Instead, EtCW(OC(CF 3)Me 2) 3 ( 6) was prepared via the reaction of W 2(OC(CF 3)Me 2) 6 with 3-hexyne at 95 degrees C. Benzylidyne complexes of the form ArCW(OC(CF 3)Me 2) 3 (Ar = aryl) then were prepared by treatment of 6 with the appropriate symmetrical alkyne ArCCAr. Three coupled cycles for the interconversion of 1-DME with the corresponding propylidyne and benzylidyne complexes via [2 + 2] cycloaddition-cycloreversion were examined for reversibility. Stoichiometric reactions revealed that both nitrile-alkyne cross-metathesis (NACM) cycles as well as the alkyne cross-metathesis (ACM) cycle operated reversibly in this system. With catalyst 3, depending on the aryl group used, at least one step in one of the NACM cycles was irreversible. In general, catalyst 1-DME afforded more rapid reaction than did 3 under comparable conditions. However, 3 displayed a slightly improved tolerance of polar functional groups than did 1-DME. For both 1-DME and 3, ACM is more rapid than NACM under typical conditions. Alkyne polymerization (AP) is a competing reaction with both 1-DME and 3. It can be suppressed but not entirely eliminated via manipulation of the catalyst concentration. As AP selectively removes 3-hexyne from the system, tandem NACM-ACM-AP can be used to prepare symmetrically substituted alkynes with good selectivity, including an arylene-ethynylene macrocycle. Alternatively, unsymmetrical alkynes of the form EtCCR (R variable) can be prepared with good selectivity via the reaction of RCN with excess 3-hexyne under conditions that suppress AP. DFT calculations support a [2 + 2] cycloaddition-cycloreversion mechanism analogous to that of alkyne metathesis. The barrier to azametalacyclobutadiene ring formation/breakup is greater than that for the corresponding metalacyclobutadiene. Two distinct high-energy azametalacyclobutadiene intermediates were found. These adopted a distorted square pyramidal geometry with significant bond localization.

Yeast sterol C24-methyltransferase: Role of highly conserved tyrosine-81 in catalytic competence studied by site-directed mutagenesis and thermodynamic analysis

The role of Try-81 in the reaction catalyzed by Saccharomyces cerevisiae sterol 24-C-methyltransferase (Erg6p) was investigated kinetically and for product differences against a panel of position-81 mutants in which Tyr was substituted with Trp, Phe, Ile, Leu, Val and Ala. The residue chosen for mutation is one that was reported previously to accept fecosterol and yield a set 24-ethyl (idene) sterol products typical of plants, showing the amino acid residue is located close to the transient C25 carbocation intermediate in the active site. One group of mutants (aromatic) tested with the natural substrate zymosterol accelerated the C-methylation reaction ( k cat/ K m) whereas the other group of mutants (aliphatics) decreased catalytic competence as the amino acid side chain was downsized. Mutating to aromatic and assaying with the substrate analog designed as a suicide substrate 26,27-dehydrozymosterol favored C26-monol formation, whereas mutating to the aliphatic of smaller size favored C26-diol formation (a measure of enzyme alkylation). In no case was zymosterol converted to an intermediate that formed a C25-diol. Thermodynamic analysis (determination of E a, Δ G ‡, Δ H ‡ and TΔ S ‡) for the C-methylation reaction performed by these enzymes assayed with the substrate and its analog or zymosterol paired with the “charged’ high energy intermediate (HEI) analogs 24( R,S)25,epiminolanosterol and 25-azalanosterol or “neutral” membrane insert ergosterol showed that mutation to aromatics can reduce inhibitor potency (measured as K m/ K i), yet catalysis can improve in Trp81 by the introduction of a gain in free energy associated with stabilization of the transition state of a rate-controlling step directed toward turnover. Alternatively, mutation to the smaller aliphatic amino acid side chains led to a destabilization in the active site structure which was accompanied by increases in the partition ratios associated with abortive complex formation. The results are explained by consideration of the functional differences attributed to Tyr81 substitution to aromatics and aliphatics of different size involved with cation-π or hydrogen bonding interactions and in the activation barriers required of differing side chain conformations to orient the reactants in the direction of turnover versus enzyme inactivation.

GUARDING AGAINST TOO MUCH LIGHT

BY STUDYING and mimicking photosynthesis, researchers hope to unlock and harness the secrets of how plants protect themselves from too much sunlight. The work might have application in the development of energy-efficient solar cells. At Arizona State University, a team of researchers has designed a self-regulating molecule that boosts or slows down a charge transfer process in response to changes in light intensity. Meanwhile, a team at Lawrence Berkeley National Laboratory (LBNL) and the University of California, Berkeley, is closer to figuring out the details of the natural protective mechanism. In photosynthesis, an ultraefficient charge transfer reaction converts virtually all incoming light energy into electrochemical energy. But such efficiency can be dangerous to the plant when there’s too much light. “If the light gets too bright, then the reaction center works too fast and produces a lot of high-energy intermediates that don’t have anywhere to go because the system is jammed up down the ...

Evidence for Concerted Pathways in Ion-Pairing Coupled Electron Transfers

Ion-pairing with electro-inactive metal ions may change drastically the thermodynamic and kinetic reactivity of electron transfer in chemical and biochemical processes. Besides the classical stepwise pathways (electron-transfer first, followed by ion-pairing or vice versa), ion-pairing may also occur concertedly with electron transfer. The latter pathway avoids high-energy intermediates but a key issue is that of the kinetic price to pay to benefit from this thermodynamic advantage. A model is proposed leading to activation/driving force relationships characterizing such concerted associative electron transfers for intermolecular and intramolecular homogeneous reactions and for electrochemical reactions. Contrary to previous assertions, the driving force of the reaction (defined as the opposite of the reaction standard free energy), as well as the intrinsic barrier, does not depend on the concentration of the ion-pairing agent, which simply plays the role of one of the reactants. Besides solvent and intramolecular reorganization, the energy of the bond being formed is the main component of the intrinsic barrier. Application of these considerations to reactions reported in recent literature illustrates how concerted ion-pairing electron-transfer reactions can be diagnosed and how competition between stepwise and concerted pathways can be analyzed. It provided the first experimental evidence of the viability of concerted ion-pairing electron-transfer reactions.

Folding of the αΙΙ-spectrin SH3 domain under physiological salt conditions

The SH3 domain has often been used as a model for protein folding due to its typical two-state behaviour. However, recent experimental data at low pH as well as molecular dynamic simulations have indicated that the folding process of SH3 probably is more complicated, and may involve intermediate states. Using both kinetic and equilibrium measurements we have obtained evidence that under native-like conditions the folding of the spectrin SH3 domain does not follow a classic two-state behaviour. The curvature we observed in the Chevron plots is a strong indication of a non-linear activation energy relationship due to the presence of high-energy intermediates. In addition, circular dichroism measurements indicated that refolding after thermal denaturation did not follow the same pattern as thermal unfolding but rather implied less cooperativity and that the refolding transition increased with increasing protein concentration. Further, NMR experiments indicated that upon refolding the SH3 domain gave rise to more than one conformation. Therefore, our results suggest that the folding of the SH3 domain of αII-spectrin does not follow a classical two-state process under high-salt conditions and neutral pH. Heterogeneous folding pathways, which can include folding intermediates as well as misfolded intermediates, might give a more reasonable insight into the folding behaviour of the αII-spectrin SH3 domain.

Mechanisms of Water Oxidation from the Blue Dimer to Photosystem II

The blue dimer, cis, cis-[(bpy)2(H2O)Ru(III)ORu(III)(H2O)(bpy)2](4+), is the first designed, well-defined molecule known to function as a catalyst for water oxidation. It meets the stoichiometric requirements for water oxidation, 2H2O --> -4e(-), -4H(+) O-O, by utilizing proton-coupled electron-transfer (PCET) reactions in which both electrons and protons are transferred. This avoids charge buildup, allowing for the accumulation of multiple oxidative equivalents at the Ru-O-Ru core. PCET and pathways involving coupled electron-proton transfer (EPT) are also used to avoid high-energy intermediates. Application of density functional theory calculations to molecular and electronic structure supports the proposal of strong electronic coupling across the micro-oxo bridge. The results of this analysis provide explanations for important details of the descriptive chemistry. Stepwise e(-)/H(+) loss leads to the higher oxidation states [(bpy)2(O)Ru(V)ORu(IV)(O)(bpy)2] (3+) (Ru(V)ORu(IV)) and [(bpy)2(O)Ru(V)ORu(V)(O)(bpy)2](4+) (Ru(V)ORu(V)). Both oxidize water, Ru(V)ORu(IV) stoichiometrically and Ru(V)ORu(V) catalytically. In strongly acidic solutions (HNO3, HClO4, and HSO3CF3) with excess Ce(IV), the catalytic mechanism involves O---O coupling following oxidation to Ru(V)ORu(V), which does not build up as a detectable intermediate. Direct evidence has been found for intervention of a peroxidic intermediate. Oxidation of water by Ru(V)ORu(IV) is far slower. It plays a role late in the catalytic cycle when Ce(IV) is depleted and is one origin of anated intermediates such as [(bpy)2(HO)Ru(IV)ORu(IV)(NO3)(bpy)2](4+), which are deleterious in tying up active components in the catalytic cycle. These intermediates slowly return to [(bpy)2(H2O)Ru(IV)ORu(III)(OH2)(bpy)2](5+) with anion release followed by water oxidation. The results of a recent analysis of water oxidation in the oxygen-evolving complex (OEC) of photosystem II reveal similarities in the mechanism with the blue dimer and significant differences. The OEC resides in the thylakoid membrane in the chloroplasts of green plants, and careful attention is paid in the structure to PCET, EPT, and long-range proton transfer by sequential local proton transfers. The active site for water oxidation is a CaMn 4 cluster, which includes an appended Mn site, Mn(4), where O---O coupling is thought to occur. Photochemical electron transfer results in oxidation of tyrosine Y Z to Y Z (.), which is approximately 7 A from Mn(4). It subsequently oxidizes the OEC through the stepwise stages of the Kok cycle. O---O coupling appears to occur through an initial peroxidic intermediate formed by redox nucleophilic attack of coordinated OH(-) in Ca-OH(-) on Mn (IV)=O.

Structures of the Human Orotidine-5′-Monophosphate Decarboxylase Support a Covalent Mechanism and Provide a Framework for Drug Design

UMP synthase (UMPS) catalyzes the last two steps of de novo pyrimidine nucleotide synthesis and is a potential cancer drug target. The C-terminal domain of UMPS is orotidine-5'-monophosphate decarboxylase (OMPD), a cofactor-less yet extremely efficient enzyme. Studies of OMPDs from micro-organisms led to the proposal of several noncovalent decarboxylation mechanisms via high-energy intermediates. We describe nine crystal structures of human OMPD in complex with substrate, product, and nucleotide inhibitors. Unexpectedly, simple compounds can replace the natural nucleotides and induce a closed conformation of OMPD, defining a tripartite catalytic site. The structures outline the requirements drugs must meet to maximize therapeutic effects and minimize cross-species activity. Chemical mimicry by iodide identified a CO(2) product binding site. Plasticity of catalytic residues and a covalent OMPD-UMP complex prompt a reevaluation of the prevailing decarboxylation mechanism in favor of covalent intermediates. This mechanism can also explain the observed catalytic promiscuity of OMPD.

Inhibitors of Trypanosoma brucei 6-Phosphogluconate Dehydrogenase

6-Phosphogluconate dehydrogenase (6PGDH) is the third enzyme of the pentose phosphate pathway. It converts 6- phosphogluconate to ribulose 5-phosphate and concomitantly provides NADPH required for many biosynthetic and detoxification reactions. Its presence has been shown to be essential for growth of bloodstream form Trypanosoma brucei, a parasite responsible for African trypanosomiasis and it may be considered a validated drug target in this protozoan. The structure of the enzyme is known from X-ray crystallographic studies. There appears to be relatively little difference between the active sites of the trypanosomal and human enzymes, although pharmacological analysis shows that selective inhibition of an extensive nature is possible. Work has been carried out on the design of several classes of inhibitors for the T. brucei enzyme that are selective over the same enzyme from mammalian liver. One class comprises sugar derivatives, which mimic the substrate of the catalysed reaction. A second class of more potent inhibitors mimic the transition-state and high-energy intermediates of the enzymatic reaction of 6PGDH. 4-Phospho-D-erythronohydroxamate with a Ki = 10 nM is the compound with the highest affinity for the T. brucei 6PGDH reported to date, and the selectivity of 254-fold over the sheep enzyme is also the highest found. A third class of inhibitors are triphenylmethane derivatives, examples of which also show very pronounced anti-trypanosomal activity. Keywords: African trypanosomiasis, sleeping sickness, 6-phosphogluconate dehydrogenase inhibitors, hydroxamic acid derivatives, highenergy intermediate analogues, transition-state analogues, phosphonate compounds, triphenylmethane derivatives

Ligand-Directed Dynamics of Adenine Riboswitch Conformers

Riboswitches harness the structural and dynamic sophistication of RNA to coordinate specific ligand recognition with changes in gene expression. Design of molecules to manipulate riboswitch responses relies on our understanding of their RNA−ligand interactions. Here we demonstrate that for the adenine (A) riboswitch (ARNA) these interactions are highly dynamic. Given that 2-aminopurine (Ap) mimics A in its interactions with ARNA, we use the fluorescence lifetime of Ap to interrogate individual Ap-ARNA conformers (dynamic exchange times > ∼10 ns). Counter to predictions of two state and induced fit models, the ligand-bound A riboswitch is not a single, highly ordered structure: We detect at least three distinct Ap-ARNA conformers in ensemble solution. Their distribution indicates that they are not high-energy RNA folding intermediates but are instead energetically similar (ΔG < 1 kcal mol-1) conformers whose thermal stability, ligand, and Mg2+ binding affinity differ substantially. Our experimental characterization suggests that these conformers are structurally distinct locally at the ligand binding site and globally in the arrangement of the P2−P3 stems. These results correlate well with recent single molecule characterization of P2−P3 end-to-end distances and exchange rates, but contrast with recent NMR results which suggest that the highly homologous G riboswitch exhibits a static global structure both with and without ligand. These distinct dynamics may well be the root of the divergent specificity and function of the A and G riboswitches. We predict that conformational dynamics within the bound A riboswitch underlie its regulatory responses and that these dynamics are directed by ligand structure.

Form finds function

Assigning function to uncharacterized enzymes discovered through genome projects has provided a great challenge to the fields of informatics, enzymology and structural biology. Docking potential ligands into flexible models of protein structures and docking potential high-energy intermediates, rather than substrates, into known structures are two new computational approaches that have provided a much-needed boost to the field.

Synthesis of acyclic galactitol- and lyxitol-aminophosphonates as inhibitors of UDP-galactopyranose mutase

UDP-galactopyranose mutase (UGM) catalyzes the isomerization of UDP-galacto pyranose (UDP-Gal p) into UDP-galacto furanose (UDP-Gal f), an essential step of the mycobacterial cell wall biosynthesis. Acyclic alditol-aminophosphonates in the d-galactose and d-lyxose series were designed as mimics of high energy intermediates of the UGM catalyzed isomerization. Interestingly, the d-lyxitol-aminophosphonate displayed better inhibition properties than the d-galactitol-aminophosphonate.

An On-pathway Intermediate in the Folding of a PDZ Domain

The folding pathways of some proteins include the population of partially structured species en route to the native state. Identification and characterization of these folding intermediates are particularly difficult as they are often only transiently populated and play different mechanistic roles, being either on-pathway productive species or off-pathway kinetic traps. To define the role of folding intermediates, a quantitative analysis of the folding and unfolding rate constants over a wide range of denaturant concentration is often required. Such a task is further complicated by the reversible nature of the folding reaction, which implies the observed kinetics to be governed by a complex combination of different microscopic rate constants. Here, we tackled this problem by measuring directly the folding rate constant under highly denaturing conditions, namely by inducing the folding of a PDZ domain through a quasi-irreversible binding reaction with a specific peptide. In analogy with previous works based on hydrogen exchange experiments, we present evidence that the folding pathway of the PDZ domain involves the formation of an obligatory on-pathway intermediate. The results presented exemplify a novel type of kinetic test to detect an on-pathway folding intermediate.

Quantum Chemical Study of Atomic Structure Evolution of Cox/C60 (x ≤ 2.8) Composites

The main features of the local atomic structure of novel Cox/C60 (x<or=2.8) complex mixtures were studied using the ab initio B3LYP/6-31G* method for a set of low- and high-energy Con(C60)m (n=1, 2, m=2, 3) clusters in low and intermediate spin states. For the n = 1 isomers the spin state S=1/2 is energetically preferable, whereas the low-energy isomers of n=2 have an intermediate spin state of S=1. The eta2 (6-6 edge of C60) type of cobalt ion coordination is preferable for both the n=1 and n=2 cases. The eta2' (coordination with a 6-5 edge) and even the eta5 (C5 fragment) types can serve as low- and high-energy intermediates for the cobalt ion's migration around the C60 cage. Formation of cobalt dimers can be the final stage of evolution of Cox/C60 atomic structure approaching the equilibrium atomic geometry.

Peroxy‐Radical‐Mediated Chemiluminescence: Mechanistic Diversity and Fundamentals for Antioxidant Assay

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

Peroxy-radical-mediated chemiluminescence: mechanistic diversity and fundamentals for antioxidant assay

Herein, we give a retrospective overview and analyze recent developments in the field of chemiluminescence derived from organic oxidation processes (so called oxychemiluminescence), most prominently, mediated by peroxy radicals. As evidenced from the presented analysis, the diversities of the reaction and the excited-state-generation mechanisms are governed mainly by a diverse chemical nature of substrates being oxidized. The notable oxychemiluminescence cases, which involve peroxy radicals as key reactive species, refer to oxidation of saturated and unsaturated hydrocarbons, polymers, lipids and proteins. The general feature of the considered chemiluminescence processes pertains to a formation of high-energy cyclic intermediates (tetroxides and/or dioxetanes), whose cleavage yields electronically excited products. The considered modes of the chemiluminescence enhancement encompass the energytransfer and the electron-transfer mechanisms. Most prominent application of the discussed oxychemiluminescence phenomenon resides in a versatile chemiluminescent assay to monitor antioxidants (both their concentration and reactivity) in chemical and biological media.