Single Reaction Pathway Research Articles

Dynamics of co-substrate pools can constrain and regulate metabolic fluxes.

Cycling of co-substrates, whereby a metabolite is converted among alternate forms via different reactions, is ubiquitous in metabolism. Several cycled co-substrates are well known as energy and electron carriers (e.g. ATP and NAD(P)H), but there are also other metabolites that act as cycled co-substrates in different parts of central metabolism. Here, we develop a mathematical framework to analyse the effect of co-substrate cycling on metabolic flux. In the cases of a single reaction and linear pathways, we find that co-substrate cycling imposes an additional flux limit on a reaction, distinct to the limit imposed by the kinetics of the primary enzyme catalysing that reaction. Using analytical methods, we show that this additional limit is a function of the total pool size and turnover rate of the cycled co-substrate. Expanding from this insight and using simulations, we show that regulation of these two parameters can allow regulation of flux dynamics in branched and coupled pathways. To support these theoretical insights, we analysed existing flux measurements and enzyme levels from the central carbon metabolism and identified several reactions that could be limited by the dynamics of co-substrate cycling. We discuss how the limitations imposed by co-substrate cycling provide experimentally testable hypotheses on specific metabolic phenotypes. We conclude that measuring and controlling co-substrate dynamics is crucial for understanding and engineering metabolic fluxes in cells.

A Kinetic Model of Acrylamide Formation inside of the Heat Boundary Layer

The Maillard reaction is important for the formation of color and aroma in heat-processed foods, but it may also be associated with the formation of toxic compounds, such as acrylamide (AA) and hydroxymethylfurfural (HMF). In an acetic acid food system, the major characteristics of the heating process are heat transfer and mass transfer. During high-temperature heating, an acetic acid system contains a heat boundary layer (HBL). For the food system, AA and HMF formations are complex on the inside of the HBL. To assess the effects of chitosan addition on the inhibition of AA formation, a kinetic model in the food system was developed that combined the Langmuir adsorption mechanism, the coverage function, the relative efficiency of chitosan dispersion, and the step function to predict the entire heating time. The inhibition of AA formation in asparagine-glucose-chitosan solution is dependent on chitosan addition and chitosan dispersion on the inside of the HBL. For extending heating duration, AA forms with high loading of glucose and asparagine were significantly higher than those with low loading. The effect of the asparagine on the AA content is related to one reaction pathway in the AA formation. However, the effect of sugar reduction on the AA content is due to two reaction pathways. For heating circumstances, a process prediction with different glucose and asparagine loadings was developed. • A heat process prediction with different Glu and Asp loadings was developed. • Model formed from Langmuir adsorption, coverage function and chitosan dispersion. • AA forms in Asp-Glu-chitosan system dependent on chitosan added and dispersed in HBL. • Influence of asp is due to a single reaction pathway in the formation of AA. • Influence of reducing sugar is on two reaction pathways in the formation of AA.

(Invited) Towards First-Principles Prediction of Early SEI Formation

Despite decades of work, there is still considerable uncertainty regarding the major components of the solid-electrolyte interface (SEI) and its dynamic formation mechanism as a function of electrolyte and anode composition. Here we present a new data-driven first-principles framework using a combination of high-throughput calculations, reaction networks, machine learning and microkinetic modeling. Our automated methodology is based on a systematic generation of relevant species using a general fragmentation/recombination procedure which provides the basis for a vast thermodynamic reaction landscape, calculated with density functional theory. We explore this landscape using stochastic methods and shortest pathfinding algorithms, which yield the most likely reaction pathways which are then refined with transition state calculations and kinetic information. The results of the framework show promise in being able to automatically recover previous insights on single reaction pathways, as well as successfully predicting the early dynamics and competitive nature of the SEI formation. As examples, we present formation mechanisms of LEMC as compared to LEDC and recover the Peled-like separation of the SEI into inorganic and organic domains resulting from rich reactive competition. By conducting accelerated simulations at elevated temperature, we track SEI evolution, confirming the postulated reduction of lithium ethylene monocarbonate to dilithium ethylene monocarbonate and hydrogen gas. These findings furnish fundamental insights into the dynamics of SEI formation and demonstrate a path forward toward a predictive understanding of electrochemical passivation.

Product specific thermal degradation kinetics of bisphenol F epoxy in inert and oxidative atmospheres using evolved gas analysis–mass spectrometry

Knowledge of the degradation kinetics for polymer materials is important for understanding thermal stability. Here, evolved gas analysis–mass spectrometry and pyrolysis gas-chromatography-mass spectrometry were evaluated for the potential to deliver additional insight into thermal degradation kinetics of diglycidal ether of bisphenol F (DGEBF) epoxy thermoset under inert and oxidative atmospheres. Degradation products of selected precursor ions were evaluated for their uniqueness to the specific precursor using extracted ion thermographs. Unique mass peaks, solely attributed to a single reaction pathway of a specific product, were determined from extracted ion thermographs and used to determine both activation energy (Ea) and pre-exponential factors for the specific primary reaction pathways. These primary reaction pathways for DGEBF epoxy degradation were then evaluated in the context of transition state theory (TST) and related transition state enthalpies (ΔH‡) and entropies (ΔS‡) of activation to further elucidate the degradation process. It was determined under pyrolysis conditions, as suggested by the Ea, the formation of bisphenol F monomer was the rate-limiting step toward the formation of xanthene and phenol. In contrast, under thermo-oxidative conditions, reactions involving oxygen containing species were identified as the rate-limiting step for all observed products based on the large negative ΔS‡ calculated from TST. This work demonstrates a powerful combination of technique and theory that can provide new insight into the degradation of polymer materials.

Experiments and Direct Dynamics Simulations That Probe η2-Arene/Aryl Hydride Equilibria of Tungsten Benzene Complexes.

Key steps in the functionalization of an unactivated arene often involve its dihaptocoordination by a transition metal followed by insertion into the C-H bond. However, rarely are the η2-arene and aryl hydride species in measurable equilibrium. In this study, the benzene/phenyl hydride equilibrium is explored for the {WTp(NO)(PBu3)} (Bu = n-butyl; Tp = trispyrazoylborate) system as a function of temperature, solvent, ancillary ligand, and arene substituent. Both face-flip and ring-walk isomerizations are identified through spin-saturation exchange measurements, which both appear to operate through scission of a C-H bond. The effect of either an electron-donating or electron-withdrawing substituent is to increase the stability of both arene and aryl hydride isomers. Crystal structures, electrochemical measurements, and extensive NMR data further support these findings. Static density functional theory calculations of the benzene-to-phenyl hydride landscape suggest a single linear sequence for this transformation involving a sigma complex and oxidative cleavage transition state. Static DFT calculations also identified an η2-coordinated benzene complex in which the arene is held more loosely than in the ground state, primarily through dispersion forces. Although a single reaction pathway was identified by static calculations, quasiclassical direct dynamics simulations identified a network of several reaction pathways connecting the η2-benzene and phenyl hydride isomers, due to the relatively flat energy landscape.

A systems-biology approach to molecular machines: Exploration of alternative transporter mechanisms

Motivated by growing evidence for pathway heterogeneity and alternative functions of molecular machines, we demonstrate a computational approach for investigating two questions: (1) Are there multiple mechanisms (state-space pathways) by which a machine can perform a given function, such as cotransport across a membrane? (2) How can additional functionality, such as proofreading/error-correction, be built into machine function using standard biochemical processes? Answers to these questions will aid both the understanding of molecular-scale cell biology and the design of synthetic machines. Focusing on transport in this initial study, we sample a variety of mechanisms by employing Metropolis Markov chain Monte Carlo. Trial moves adjust transition rates among an automatically generated set of conformational and binding states while maintaining fidelity to thermodynamic principles and a user-supplied fitness/functionality goal. Each accepted move generates a new model. The simulations yield both single and mixed reaction pathways for cotransport in a simple environment with a single substrate along with a driving ion. In a “competitive” environment including an additional decoy substrate, several qualitatively distinct reaction pathways are found which are capable of extremely high discrimination coupled to a leak of the driving ion, akin to proofreading. The array of functional models would be difficult to find by intuition alone in the complex state-spaces of interest.

Optoplasmonic characterisation of reversible disulfide interactions at single thiol sites in the attomolar regime

Probing individual chemical reactions is key to mapping reaction pathways. Trace analysis of sub-kDa reactants and products is obfuscated by labels, however, as reaction kinetics are inevitably perturbed. The thiol-disulfide exchange reaction is of specific interest as it has many applications in nanotechnology and in nature. Redox cycling of single thiols and disulfides has been unresolvable due to a number of technological limitations, such as an inability to discriminate the leaving group. Here, we demonstrate detection of single-molecule thiol-disulfide exchange using a label-free optoplasmonic sensor. We quantify repeated reactions between sub-kDa thiolated species in real time and at concentrations down to 100’s of attomolar. A unique sensing modality is featured in our measurements, enabling the observation of single disulfide reaction kinetics and pathways on a plasmonic nanoparticle surface. Our technique paves the way towards characterising molecules in terms of their charge, oxidation state, and chirality via optoplasmonics.

Reaction Rates in Nitromethane under High Pressure from Density Functional Tight Binding Molecular Dynamics Simulations.

We use density functional tight binding (DFTB) molecular dynamics (MD) simulations to determine the reaction rates of nitromethane CH3NO2 (NM) under high pressure (P = 14-28 GPa), and temperature (T = 1450-1850 K). DFTB-MD simulations performed with the same initial conditions (P0, T0) reveal a stochastic behavior, both in terms of reaction times and chemical paths. By running series of MD simulations, we are able to obtain average reaction times with quantified errors and devise a simple two-step model for NM explosion: ignition/explosion. While our model bypasses the chemical complexity due to the numerous reaction paths and intermediates observed during reactions, the chemistry is accounted for via the accurate parameterization of the DFTB model, and our results suggest a single main reaction pathway for the pressure range considered here, dominated in the earlier stages by the formation of the aci-ion, CH2NOO-. By fitting our data to a Frank-Kamenetskii model, we extract prefactors and pressure-independent activation energies and volumes for the ignition and explosion stages. A two-step model is then built and compared to experimental observations. Single and two-step Arrhenius models are also provided for comparison with literature data. This work presents an efficient way of investigating the reactivity of high explosives by performing electronic structure-based MD simulations and provides reaction rates for simplified models that can be implemented into hydrocodes.

Developing cellulosic waste products as platform chemicals: protecting group chemistry of α-glucoisosaccharinic acid

Alpha and beta-glucoisosaccharinic acids ((2S,4S)-2,4,5-trihydroxy-2-(hydroxymethyl)pentanoic acid and (2R,4S)-2,4,5-trihydroxy-2-(hydroxymethyl)pentanoic acid) which are produced when cellulosic materials are treated with aqueous alkali are potentially valuable platform chemicals. Their highly functionalised carbon skeleton, with fixed chirality at C-2 and C-4, makes them ideal starting materials for use in synthesis. In order to assess the potential of these saccharinic acids as platform chemicals we have explored the protecting group chemistry of the lactone form of alpha-glucoisosaccharinic acid (α-GISAL). We report here the use of single and multiple step reaction pathways leading to the regioselective protection of the three different hydroxyl groups of α-GISAL. We report strategies for protecting the three different hydroxyl groups individually or in pairs. We also report the synthesis of a range of tri-O-protected α-GISAL derivatives where a number of the products contain orthogonal protecting groups.

Understanding and modeling the hydrogen-abstraction from dimethyl ether by the methyl radical with torsional anharmonicity

Hybrid density functionals M06-2X and BMK in combination with the MG3S basis set, along with double-hybrid density functional B2PLYP with the TZVP basis set, were employed to better characterize the hydrogen atom transferring from dimethyl ether (DME) to the methyl radical. The distinct orientations of hydrogen atoms (in-plane and out-of-plane ones) in the DME molecule were taken into account. Density functional energetics was validated by comparison with the high-level CBS-QB3, G4, and G4MP2 calculations. Kinetic calculations were performed by means of the conventional transition-state theory (TST), canonical variational TST (CVT), and improved CVT (ICVT) over a wide temperature range 200–2600K to clarify the available experimental measurements, and tunneling effect and anharmonic torsion were also included. Two transition structures are located with hydrogen-abstraction occurring at the in-plane and out-of-plane hydrogen positions, which are in fact related via the relative motion of the CH3⋯H⋯CH2 and OCH3 moieties about the CO bond. Based on a single reaction pathway, the M06-2X/MG3SICVT rate constants with multidimensional small-curvature tunneling correction and proper treatment of anharmonic torsions compare well with most of the available experimental data. Variational effects on the computed rate constants are found to be negligibly small. Activation energies for the CH3+DME reaction increase substantially with temperature, and exhibit a nonlinear dependence on the temperature. Therefore, rate constants are fitted to the four-parameter expression k(T)=8.59×10-16(T/300)5.11exp[-19.65(T+140.90)/R(T2+140.902)] cm3molecule−1s−1 over the broad temperature range.

Understanding reaction processes for n-heptane over 10Mo2C/SZ catalyst

Supported carbided molybdena catalysts was prepared by treating MoO3/sulfated zirconia in methane/hydrogen (1:4 volumetric ratio) at 923K. Characterisation shows the oxide to be different from the carbide phase. Carburisation did not induce phase change of the support with the tetragonal phase remaining dominant but did results in some changes to the textural properties. Lewis acid site density was constant in transforming from oxide to carbide forms while Brønsted acidity site densities was diminished by ca 10% to give a Brønsted/Lewis density ratio 2.46 in the carbide form. The ratio of hydrogenation sites to (Brønsted) acid sites on the carbidic form of the catalyst was 0.12. Increasing temperature and decreasing WHSV augmented heptane conversion but leads to multiple cracking. Analysis of the product distribution as a function of conversion or as a function of temperature implied that the reaction did not simply proceed via a single consecutive reaction pathway Conversion increased the research octane number (RON) due to of the increased fraction of pentane isomers.

Branched Motifs Enable Long-Range Interactions in Signaling Networks through Retrograde Propagation

Branched structures arise in the intra-cellular signaling network when a molecule is involved in multiple enzyme-substrate reaction cascades. Such branched motifs are involved in key biological processes, e.g., immune response activated by T-cell and B-cell receptors. In this paper, we demonstrate long-range communication through retrograde propagation between branches of signaling pathways whose molecules do not directly interact. Our numerical simulations and experiments on a system comprising branches with JNK and p38MAPK as terminal molecules respectively that share a common MAP3K enzyme MEKK3/4 show that perturbing an enzyme in one branch can result in a series of changes in the activity levels of molecules “upstream” to the enzyme that eventually reaches the branch-point and affects other branches. In the absence of any evidence for explicit feedback regulation between the functionally distinct JNK and p38MAPK pathways, the experimentally observed modulation of phosphorylation amplitudes in the two pathways when a terminal kinase is inhibited implies the existence of long-range coordination through retrograde information propagation previously demonstrated in single linear reaction pathways. An important aspect of retrograde propagation in branched pathways that is distinct from previous work on retroactivity focusing exclusively on single chains is that varying the type of perturbation, e.g., between pharmaceutical agent mediated inhibition of phosphorylation or suppression of protein expression, can result in opposing responses in the other branches. This can have potential significance in designing drugs targeting key molecules which regulate multiple pathways implicated in systems-level diseases such as cancer and diabetes.

Potential Pitfalls of Using Epichlorohydrin as Starting Material for Oxazolidinone Antibacterials

The importance of oxazolidinone functional group has been well documented in synthetic and medicinal chemistry. In the latter area, recent interests are focused on N-arylsubstituted oxazolidin-2-one structures, as exemplified by linezolid and eperezolid, which represent a new class of antibacterials with activity against drug-resistant Grampositive pathogens, including MRSA. Biologically active enantiomers of the oxazolidinone antibacterials are those with the (S)-configuration. Synthesis of the (S)-enantiomers have been achieved in enantiopure form starting from various glycidol derivatives such as (R)-glycidyl butyrate, (S)and (R)-epichlorohydrin, N-Boc-glycidylamine, and more recently, (R)-glycidyl tosylate. Other enantiopure starting materials include 3-chloro-1,2-propanediol, mannitol, protected glyceraldehyde, and aziridine-2carboxamide. Several routes employing asymmetric reactions have also been reported. Of those enantiopure starting materials, epichlorohydrin and glycidyl tosylate are particularly useful as they are doubly activated (at both C-1 and C-3) toward nucleophilic substitutions. The tandem, three-step sequence of the epoxide ring-opening, carbamate formation and oxazolidin-2one ring-closure (sometimes referred to as “1,3-cycloaddition”) represents an atom-economic key step in the synthesis of oxazolidinone antibacterials (Scheme 1). Yu et al. first employed this process with epichlorohydrin as the starting material; we then followed it up with glycidyl tosylate, and showed that this crystalline and storage-stable compound was a viable starting material for oxazolidinone antibacterials. Our work with glycidyl tosylate reminded us how the issues of regioand stereochemistry were entwined together for the doubly activated glycidol derivatives. It also prompted us to wonder how these issues had played out in Yu’s work with epichlorohydrin. As the details were not discussed in their original communication, we decided to reexamine the tandem, three-step sequence with epichlorohydrin as the starting material. The 1,3-cycloaddition reaction of (R)-epichlorohydrin and phenyl isocyanate was performed with a catalytic amount of LiBr. In the absence of detailed experimental procedure in the original work, also to make a meaningful comparison with our findings with glycidyl tosylate, we maintained the reaction conditions as close as possible to the ones employed in the glycidyl tosylate process [in THF (0.067 M) at reflux with phenyl isocyanate (3 eq) and LiBr (0.1 eq)]. Epichlorohydrin underwent the tandem, three-step sequence more slowly with LiBr than glycidyl tosylate did with LiI. The reaction took 8 h to complete (vs ~30 min for glycidyl tosylate/LiI). The desired chloro oxazolidinone product was isolated in 93% yield, seemingly validating the results in the original report. A careful examination, however, revealed the presence of a by-product, suggesting concurrent sidereaction pathway(s) and potential complications in the process. In order to gain a fuller picture of the process, we postulated possible reaction pathways for the 1,3-cycloaddition reaction of epichlorohydrin/PhNCO/LiBr and set out to assess the significance of each pathway quantitatively (Scheme 2). Model reactions revealed that the products could undergo secondary displacements, not one-way, but in both ways, hence Paths IV and V in addition to Path III. This raised an extra issue for the epichlorohydrin process. In the glycidyl tosylate process, a single reaction pathway was responsible for the formation of the desired product, whose stereochemical integrity was therefore maintained through the entire course of the reaction. In the epichlorohydrin process, on the other hand, we now needed to consider multiple pathways for the formations of the desired as well as undesired products (Paths I, IV and V for 2; Paths II and III for 3), each resulting in a distinct stereochemical outcome. Significance of these pathways may be assessed in a time study in which the enantiomeric purities as well as the chemical yields of both products would be determined at various reaction times.

One RNA plays three roles to provide catalytic activity to a group I intron lacking an endogenous internal guide sequence.

Catalytic RNA molecules possess simultaneously a genotype and a phenotype. However, a single RNA genotype has the potential to adopt two or perhaps more distinct phenotypes as a result of differential folding and/or catalytic activity. Such multifunctionality would be particularly significant if the phenotypes were functionally inter-related in a common biochemical pathway. Here, this phenomenon is demonstrated by the ability of the Azoarcus group I ribozyme to function when its canonical internal guide sequence (GUG) has been removed from the 5′ end of the molecule, and added back exogenously in trans. The presence of GUG triplets in non-covalent fragments of the ribozyme allow trans-splicing to occur in both a reverse splicing assay and a covalent self-assembly assay in which the internal guide sequence (IGS)-less ribozyme can put itself together from two of its component pieces. Analysis of these reactions indicates that a single RNA fragment can perform up to three distinct roles in a reaction: behaving as a portion of a catalyst, behaving as a substrate, and providing an exogenous IGS. This property of RNA to be multifunctional in a single reaction pathway bolsters the probability that a system of self-replicating molecules could have existed in an RNA world during the origins of life on the Earth.

Oxygen−Oxygen Bond Formation by the Ru-Hbpp Water Oxidation Catalyst Occurs Solely via an Intramolecular Reaction Pathway

A thorough kinetics investigation of the Ru-Hbpp water oxidation catalyst has been carried out at temperatures in the range 10-40 degrees C. Four oxidative electron-transfer processes that take the catalyst from its initial II,II oxidation state up to the formal IV,IV oxidation state were kinetically characterized and the corresponding activation parameters determined. Once the IV,IV oxidation state is reached, two additional slower kinetic processes take place, corresponding to the formation of an intermediate that finally evolves oxygen and regenerates the initial Ru-Hbpp catalyst. These two kinetic processes were also fully characterized with respect to the evaluation of their rate constants and activation parameters. Furthermore, (18)O labeling experiments were performed with different degrees of labeled catalyst and solvent, and the (16)O(2)/(16)O(18)O/(18)O(2) isotopic distribution of the generated molecular oxygen was calculated. These results clearly point to the existence of a single intramolecular reaction pathway for the formation of the oxygen-oxygen bond in the case of the Ru-Hbpp catalyst.

Radiation–oxidation mechanisms: Volatile organic degradation products from polypropylene having selective C-13 labeling studied by GC/MS

PP samples, in which the three unique carbon atom positions along the chain were selectively labeled with C-13, have been subjected to γ-irradiation in the presence of oxygen, and the resulting organic volatile products analyzed by GC/MS. The isotopic labeling patterns in 33 organic degradation compounds have been assigned by comparison of the four mass spectra for each compound (from unlabeled PP, and from the three labeled PP materials). The volatile products have been “mapped” onto their positions of origin from the PP macromolecule, and insights have been gained into the chemistry through which these compounds must have formed. Most products show high specificity of isotopic labeling, indicating a single dominant reaction pathway. Oxidation chemistry occurred heavily at the C(2) tertiary carbon, with chemistry also at C(1) methylene. Methyl ketones are in abundance, along with alcohols, some aliphatic hydrocarbons, and other compound types. The C(3) methyl carbon remained attached to its original C(2) position in all catenated degradation products, and underwent no chemistry. However, products containing “non-catenated” carbons (i.e., not bonded to any other carbon atom) consisted entirely of a mix of C(3) and C(1). By examination of the labeling patterns, many products could be assigned to two successive chain scission events in close proximity, while others are clearly seen to arise from cleavage, followed by radical–radical recombination reactions. Interestingly, the former products (two chain scissions) are all found to have an odd number of carbon atoms along their chain, while the latter (scission followed by radical–radical reaction) all have an even number of carbons. An explanation of this odd/even phenomenon is provided in terms of the symmetry of the PP macromolecule.

A Review on DNA Computing Models

From a nanoscience perspective, cellular processes and their reduced in vitro imitations provide extraordinary examples for highly robust few or single molecule reaction pathways. A prime example are biochemical reactions involving DNA molecules, and the coupling of these reactions to the physical conformations of DNA. In this review, we summarise recent results on the following phenomena: We investigate the biophysical properties of DNA-looping and the equilibrium configurations of DNA-knots, whose relevance to biological processes are increasingly appreciated. We discuss how random DNA-looping may be related to the efficiency of the target search process of proteins for their specific binding site on the DNA molecule. And we dwell on the spontaneous formation of intermittent DNA nanobubbles and their importance for biological processes, such as transcription initiation. The physical properties of DNA may indeed turn out to be particularly suitable for the use of DNA in nanosensing applications. (Less)

The application of the CSB to the prediction of single step reactions and reaction pathways

Abstract Applications of the CSB (Common-Sense Builder) system for the logic-oriented and knowledge-assisted simulation of chemical reaction courses are described. We present the possibility of using the CSB for two ways of reaction simulation, i.e., as a multi-step process or as single step procedure. Results of the first simulation type are given to predict the course, and to model reaction mechanism. The second one is capable of complex chemical transformations such as multi-component and cascade reactions to generate structurally diverse products for combinatorial chemistry. In several experiments performed, we analyze the capabilities and limitations of the CSB modules and controlling tools for the examination and selective generation of solutions.

On the symmetry of the xyz vibration in the photochemistry of Pt(II) D4h complexes

A symmetry concept is proposed that links the presence of vibronic structure in UV-visible spectra in d-systems, with the identification of a single photochemical reaction pathway. It is shown that the vibronic structure on some bands of the Pt(II) D4h complexes is due to the non- degenerate, ungerade b1u (xyz) vibration that points on an intramolecular twisting process for the photochemical cis-trans isomerization. The reaction coordinate is unambiguously defined in a coordinate system where x and y bisect the metal-ligand ML2 angle.

Manganese and Iron Porphyrins Catalyze Peroxynitrite Decomposition and Simultaneously Increase Nitration and Oxidant Yield: Implications for Their Use as Peroxynitrite Scavengers in Vivo

Twelve substituted metalloporphyrins (MPs), some of which have been previously characterized with respect to superoxide dismutase and peroxynitrite decomposing activities, were evaluated for their ability to scavenge peroxynitrite in vitro at 37°C. Because the overall effectiveness of MPs as catalytic peroxynitrite scavengers is a function of (1) how fast they react with peroxynitrite, (2) how fast they cycle back to the starting compound, and (3) how well they contain or quench the reactive intermediates generated, all of these properties were evaluated and compared directly under the same conditions. Of the various MPs tested, only the iron and manganese porphyrins showed significant reactivity with peroxynitrite. The Mn(IV) intermediates resulting from oxidation by peroxynitrite were relatively stable and rereduction to the Mn(III) forms was rate-limiting to catalytic decomposition of peroxynitrite. However, in the presence of oxidizeable substrates like phenolics, rereduction of Mn(IV) forms occurred very rapidly and both the Mn- and Fe-porphyrins catalyzed nitration and oxidation by peroxynitrite. Mn- and Fe-porphyrins enhanced the yield of nitrated phenolics by peroxynitrite as much as 5-fold at pH 7.4 and up to 12-fold at pH 9.1, while total oxidative yield was more than doubled. Nitration enhancement by MPs was effectively inhibited by ascorbate, glutathione, or serum, although much higher concentrations of ascorbate were required to inhibit nitration catalyzed by either Mn or Fe tetramethylpyridyl porphyrin. Catalysis of peroxynitrite nitration by MPs appears to proceed via a radical-mediated reaction mechanism whereby the phenolic substrate rapidly reduces Mn(IV) = O or Fe[IV] = O to the +3 state to yield phenoxyl radical which then combines with the other primary product, nitrogen dioxide. Based on the rate constants and the proposed reaction mechanism, it is reasonable to suggest that Mn and Fe porphyrins could detoxify peroxynitrite in vivo by efficiently trapping the relatively unreactive peroxynitrite anion and, in effect, channeling it into a single reaction pathway which could then be more effectively scavenged by cellular reductants like ascorbate.