The multistep oxidation of cholesterol to pregnenolone by human cytochrome P450 11A1 is highly processive.

Cytochrome P450 (P450, CYP) 11A1 is the classical cholesterol side chain cleavage enzyme (P450scc) that removes six carbons of the side chain, the first and rate-limiting step in the synthesis of all mammalian steroids. The reaction is a 3-step, 6-electron oxidation that proceeds via formation of 22R-hydroxy (OH) and 20R,22R-(OH)2 cholesterol, yielding pregnenolone. We expressed human P450 11A1 in bacteria, purified the enzyme in the absence of nonionic detergents, and assayed pregnenolone formation by HPLC-mass spectrometry of the dansyl hydrazone. The reaction was inhibited by the nonionic detergent Tween 20, and several lipids did not enhance enzymatic activity. The 22R-OH and 20R,22R-(OH)2 cholesterol intermediates were bound to P450 11A1 relatively tightly, as judged by steady-state optical titrations and koff rates. The electron donor adrenodoxin had little effect on binding; the substrate cholesterol showed a ∼5-fold stimulatory effect on the binding of adrenodoxin to P450 11A1. Presteady-state single-turnover kinetic analysis was consistent with a highly processive reaction with rates of intermediate oxidation steps far exceeding dissociation rates for products and substrates. The presteady-state kinetic analysis revealed a second di-OH cholesterol product, separable by HPLC, in addition to 20R,22R-(OH)2 cholesterol, which we characterized as a rotamer that was also converted to pregnenolone at a similar rate. The first oxidation step (at C-22) is the slowest, limiting the overall rate of cleavage. d3-Cholesterol showed no kinetic deuterium isotope effect on C-22, indicating that C-H bond cleavage is not rate-limiting in the first hydroxylation step.

Probing the role of protein conformational changes in the mechanism of prenylated-FMN-dependent phenazine-1-carboxylic acid decarboxylase.

Phenazine-1-carboxylic acid decarboxylase (PhdA) is a prenylated-FMN-dependent (prFMN) enzyme belonging to the UbiD family of decarboxylases. Many UbiD-like enzymes catalyze (de)carboxylation reactions on aromatic rings and conjugated double bonds and are potentially valuable industrial catalysts. We have investigated the mechanism of PhdA using a slow turnover substrate, 2,3-dimethylquinoxaline-5-carboxylic acid (DQCA). Detailed analysis of the pH dependence and solvent deuterium isotope effects associated with the reaction uncovered unusual kinetic behavior. At low substrate concentrations, a substantial inverse solvent isotope effect (SIE) is observed on Vmax/KM of ∼ 0.5 when reaction rates of DQCA in H2O and D2O are compared. Under the same conditions, a normal SIE of 4.15 is measured by internal competition for proton transfer to the product. These apparently contradictory results indicate that the SIE values report on different steps in the mechanism. A proton inventory analysis of the reaction under Vmax/KM and Vmax conditions points to a "medium effect" as the source of the inverse SIE. Molecular dynamics simulations of the effect of D2O on PhdA structure support that D2O reduces the conformational lability of the enzyme and results in a more compact structure, akin to the active, "closed" conformer observed in crystal structures of some UbiD-like enzymes. Consistent with the simulations, PhdA was found to be more stable in D2O and to bind DQCA more tightly, leading to the observed rate enhancement under Vmax/KM conditions.

Rapid reaction studies on the chemistry of flavin oxidation in urocanate reductase.

Urocanate reductase (UrdA) is a bacterial flavin-dependent enzyme that reduces urocanate to imidazole propionate, enabling bacteria to use urocanate as an alternative respiratory electron acceptor. Elevated serum levels of imidazole propionate are associated with the development of type 2 diabetes, and, since UrdA is only present in humans in gut bacteria, this enzyme has emerged as a significant factor linking the health of the gut microbiome and insulin resistance. Here, we investigated the chemistry of flavin oxidation by urocanate in the isolated FAD domain of UrdA (UrdA') using anaerobic stopped-flow experiments. This analysis unveiled the presence of a charge-transfer complex between reduced FAD and urocanate that forms within the dead time of the stopped-flow instrument (∼1 ms), with flavin oxidation subsequently occurring with a rate constant of ∼60 s-1. The pH dependence of the reaction and analysis of an Arg411Ala mutant of UrdA' are consistent with Arg411 playing a crucial role in catalysis by serving as the active site acid that protonates urocanate during hydride transfer from reduced FAD. Mutational analysis of urocanate-binding residues suggests that the twisted conformation of urocanate imposed by the active site of UrdA' facilitates urocanate reduction. Overall, this study provides valuable insight into the mechanism of urocanate reduction by UrdA.

Human 2'-Deoxynucleoside 5'-Phosphate N-Hydrolase 1: The Catalytic Roles of Tyr24 and Asp80.

The human enzyme 2'-deoxynucleoside 5'-phosphate N-hydrolase 1 (HsDNPH1) catalyses the hydrolysis of 5-hydroxymethyl-2'-deoxyuridine 5'-phosphate to generate 5-hydroxymethyluracil and 2-deoxyribose-5-phosphate via a covalent 5-phospho-2-deoxyribosylated enzyme intermediate. HsDNPH1 is a promising target for inhibitor development towards anticancer drugs. Here, site-directed mutagenesis of conserved active-site residues, followed by HPLC analysis of the reaction and steady-state kinetics are employed to reveal the importance of each of these residues in catalysis, and the reaction pH-dependence is perturbed by each mutation. Solvent deuterium isotope effects indicate no rate-limiting proton transfers. Crystal structures of D80N-HsDNPH1 in unliganded and substrate-bound states, and of unliganded D80A- and Y24F-HsDNPH1 offer atomic level insights into substrate binding and catalysis. The results reveal a network of hydrogen bonds involving the substrate and the E104-Y24-D80 catalytic triad and are consistent with a proposed mechanism whereby D80 is important for substrate positioning, for helping modulate E104 nucleophilicity, and as the general acid in the first half-reaction. Y24 positions E104 for catalysis and prevents a catalytically disruptive close contact between E104 and D80.

Isotope Effects and the Atmosphere.

Chemical physics plays a large role in determining the isotopic compositions of gases in Earth's atmosphere, which in turn provide fundamental insights into the sources, sinks, and transformations of atmospheric gases and particulates and their influence on climate. This review focuses on the kinetic and photolysis isotope effects relevant to understanding the isotope compositions of atmospheric ozone, carbon dioxide, methane, nitrous oxide, and other gases and their historical context. The discussion includes non-mass-dependent isotope compositions of oxygen-containing species and a brief overview of the recent growth of clumped isotope measurements at natural isotopic abundances, that is, of molecules containing more than one rare isotope. The intention is to introduce chemistry researchers to the field of using isotope compositions as tracers of atmospheric chemistry and climate both today and back in time through ice and rock records and to highlight the outstanding research questions to which experimental and theoretical physical chemists can contribute.

A nuclear configuration interaction approach to study nuclear spin effects: an application to ortho- and para-3 He2 @C60.

We introduce a non-orthogonal configuration interaction approach to investigate nuclear quantum effects on energies and densities of confined fermionic nuclei. The Hamiltonian employed draws parallels between confined systems and many-electron atoms, where effective non-Coulombic potentials represent the interactions of the trapped particles. One advantage of this method is its generality, as it offers the potential to study the nuclear quantum effects of various confined species affected by effective isotropic or anisotropic potentials. As a first application, we analyze the quantum states of two 3 He atoms encapsulated in C60 . At the Hartree-Fock level, we observe the breaking of spin and spatial symmetries. To ensure wavefunctions with the correct symmetries, we mix the broken-symmetry Hartree-Fock states within the non-orthogonal configuration interaction expansion. Our proposed approach predicts singly and triply degenerate ground states for the singlet (para-3 He2 @C60 ) and triplet (ortho-3 He2 @C60 ) nuclear spin configurations, respectively. The ortho-3 He2 @C60 ground state is 5.69 cm-1 higher in energy than the para-3 He2 @C60 ground state. The nuclear densities obtained for these states exhibit the icosahedral symmetry of the C60 embedding potential. Importantly, our calculated energies for the lowest 85 states are in close agreement with perturbation theory results based on a harmonic oscillator plus rigid rotor model of 3 He2 @C60 .

The Synergy between Nuclear Magnetic Resonance and Density Functional Theory Calculations.

This paper deals with the synergy between Nuclear Magnetic Resonance (NMR) spectroscopic investigations and DFT calculations, mainly of NMR parameters. Both the liquid and the solid states are discussed here. This text is a mix of published results supplemented with new findings. This paper deals with examples in which useful results could not have been obtained without combining NMR measurements and DFT calculations. Examples of such cases are tautomeric systems in which NMR data are calculated for the tautomers; hydrogen-bonded systems in which better XH bond lengths can be determined; cage compounds for which assignment cannot be made based on NMR data alone; revison of already published structures; ionic compounds for which reference data are not available; assignment of solid-state spectra and crystal forms; and the creation of libraries for biological molecules. In addition to these literature cases, a revision of a cage structure and substituent effects on pyrroles is also discussed.

Application of the Bland-Altman and Receiver Operating Characteristic (ROC) Approaches to Study Isotope Effects in Gas Chromatography-Mass Spectrometry Analysis of Human Plasma, Serum and Urine Samples.

The Bland-Altman approach is one of the most widely used mathematical approaches for method comparison and analytical agreement. This work describes, for the first time, the application of Bland-Altman to study 14N/15N and 1H/2H (D) chromatographic isotope effects of endogenous analytes of the L-arginine/nitric oxide pathway in human plasma, serum and urine samples in GC-MS. The investigated analytes included arginine, asymmetric dimethylarginine, dimethylamine, nitrite, nitrate and creatinine. There was a close correlation between the percentage difference of the retention times of the isotopologs of the Bland-Altman approach and the area under the curve (AUC) values of the receiver operating characteristic (ROC) approach (r = 0.8619, p = 0.0047). The results of the study suggest that the chromatographic isotope effects in GC-MS result from differences in the interaction strengths of H/D isotopes in the derivatives with the hydrophobic stationary phase of the GC column. D atoms attenuate the interaction of the skeleton of the molecules with the lipophilic GC stationary phase. Differences in isotope effects in plasma or serum and urine in GC-MS are suggested to be due to a kind of matrix effect, and this remains to be investigated in forthcoming studies using Bland-Altman and ROC approaches.

Reactivity of Superbasic Carbanions Generated via Reductive Radical-Polar Crossover in the Context of Photoredox Catalysis.

Photocatalytic reactions involving a reductive radical-polar crossover (RRPCO) generate intermediates with carbanionic reactivity. Many of these proposed intermediates resemble highly reactive organometallic compounds. However, conditions of their formation are generally not tolerated by their isolated organometallic versions and often a different reactivity is observed. Our investigations on their nature and reactivity under commonly used photocatalytic conditions demonstrate that these intermediates are indeed best described as free, superbasic carbanions capable of deprotonating common polar solvents usually assumed to be inert such as acetonitrile, dimethylformamide, and dimethylsulfoxide. Their basicity not only towards solvents but also towards electrophiles, such as aldehydes, ketones, and esters, is comparable to the reactivity of isolated carbanions in the gas-phase. Previously unsuccessful transformations thought to result from a lack of reactivity are explained by their high reactivity towards the solvent and weakly acidic protons of reaction partners. An intuitive explanation for the mode of action of photocatalytically generated carbanions is provided, which enables methods to verify reaction mechanisms proposed to involve an RRPCO step and to identify the reasons for the limitations of current methods.

Synthesis of Partially Fluorinated Alkyl Triflates by Electrochemical Fluorination (Simons Process).

A scalable straightforward synthesis of monofluoro- and difluoromethyl triflate CF3 SO2 OCH2 F (MH2F ) and CF3 SO2 OCHF2 (MHF2 ) through electrochemical fluorination (ECF, Simons process) of methyl triflate MH3 in anhydrous hydrogen fluoride at nickel anodes is presented. The ECF method is also feasible for the preparation of the deuterated analogues CF3 SO2 OCD2 F (MD2F ) and CF3 SO2 OCDF2 (MD2F ). Surprisingly, no H/D exchange occurs during ECF of CF3 SO2 OCD3 (MD3 ); this provides further evidence for a NiF3 /NiF4 -mediated ECF mechanism. The ECF of selected partially fluorinated ethyl triflates is described, and electrochemical fluorination of CF3 SO2 OCH2 CF3 (EH2F3 ) leads to the until now unknown chiral CF3 SO2 OCHFCF3 (EHFF3 ). The analogous fluoromethyl and fluoroethyl nonaflates are also accessible by ECF. This study contains detailed spectroscopic, structural, and thermal data on (fluoro)methyl and fluoro(ethyl) triflates.

Impact of Catalyst Deuteration on the Reactivity of Chiral Phase-Transfer Organocatalysts.

Site-specifically deuterated organocatalysts were prepared and found to show improved reactivity over the non-deuterated analogs. Two privileged C2 -symmetric chiral binaphthyl-modified tetraalkylammonium salts were selected for this study. The stability of these phase-transfer catalysts was generally improved by site-specific deuteration, though the degree of improvement was structure dependent. In particular, a large secondary kinetic isotope effect was observed for the tetradeuterated phase-transfer catalyst. The performance of these deuterated catalysts in the asymmetric catalytic alkylation of amino acid derivatives was better than that of non-deuterated analogs at low catalyst loadings. The results suggest that catalyst deuteration is a promising strategy for enhancing the stability and performance of organocatalysts.

CH3 CN@open-C60 : An Effective Inner-Space Modification and Isotope Effect Inside a Nano-Sized Flask.

Despite several small molecules being encapsulated inside cage-opened fullerene derivatives, such species have not considerably affected the structures and properties of the outer carbon cages. Herein, we achieved an effective inner-space modification for an open-cage C60 derivative by insertion of a neutral CH3 CN molecule into the cavity. The CH3 CN@open-C60 thus obtained showed an enhanced polarity, thus affording an easy separation from a mixture containing the empty cage by column chromatography on silica gel, without the preparative HPLC that was needed for previous cases. The less negative reduction potentials with respect to those of empty cage reflect the decreased energy level of the LUMO, which is supported by the DFT calculations. NMR spectroscopy, single-crystal X-ray analysis, and theoretical calculations revealed that both the presence of the encapsulated CH3 CN and cage deformation caused by the CH3 CN play an essential role in the change of the electronic properties. Furthermore, the favored binding affinity of deuterated acetonitrile CD3 CN with internal C60 surface is discussed.

The Reaction of Muonium with Hydrogen Peroxide in Aqueous Solution.

Rate constants for the reactions of muonium (Mu) (the ultralight isotope of the hydrogen atom) with H2 O2 in H2 O and D2 O2 in D2 O have been determined at various temperatures and pH (pD) values. The data are consistent with the three reactions: Mu + H 2 O 2 → k 1 M products ${{\rm{Mu}} + {\rm{H}}_2 {\rm{O}}_2 \mathop \rightarrow \limits ^{{k_{1{\rm{M}}} }}_{} {\rm{products}}}$ , Mu + HO 2 - → k 2 M products ${{\rm{Mu}} + {\rm{HO}}_2^ - \mathop \rightarrow \limits ^{{k_{2{\rm{M}}} }}_{} {\rm{products}}}$ , Mu + O - → k 3 M products ${{\rm{Mu}} + {\rm{O}}^ - \mathop \rightarrow \limits ^{{k_{3{\rm{M}}} }}_{} {\rm{products}}}$ and the equivalent for the deuterated entities. A significant positive H/D isotope effect was found for the undissociated peroxide, while for the anions the effect was negligible or slightly in the opposite direction. In addition, for concentrated solutions of peroxide a study of the muon spin polarization as a function of applied transverse magnetic field yielded results consistent with the rate constants determined from the direct decay measurements, and indicated that the reaction products are diamagnetic, most likely MuH and MuOH, i. e., no muoniated radical products are formed. These results are potentially relevant for management of the radiolysis products in nuclear industry.

Tautomerism of pyridinylbutane-1,3-diones: An NMR and DFT study.

The three possible 1-(n-pyridinyl)butane-1,3-diones (nPM) have been synthesized. Structures, tautomerism, and conformations are investigated by means of DFT calculations. 1 H and 13 C NMR spectra are assigned, and deuterium isotope effects on 13 C chemical shifts have been measured. Analysis of the isotope effects leads to the equilibrium constants of the keto-enol tautomers. Some interesting differences are seen between the three compounds and the phenyl analogs. The isotope effects can also rank the hydrogen bonds of the compounds, with the one with nitrogen in the three positions of the pyridine ring as the weakest. Structures, conformers, energies, and NMR nuclear shieldings are calculated using DFT calculations at the B3LYP/6-311++G(d,p) level.

Cations Determine the Mechanism and Selectivity of Alkaline Oxygen Reduction Reaction on Pt(111).

The proton-coupled electron transfer (PCET) mechanism of the oxygen reduction reaction (ORR) is a long-standing enigma in electrocatalysis. Despite decades of research, the factors determining the microscopic mechanism of ORR-PCET as a function of pH, electrolyte, and electrode potential remain unresolved, even on the prototypical Pt(111) surface. Herein, we integrate advanced experiments, simulations, and theory to uncover the mechanism of the cation effects on alkaline ORR on well-defined Pt(111). We unveil a dual-cation effect where cations simultaneously determine i) the active electrode surface by controlling the formation of Pt-O and Pt-OH overlayers and ii) the competition between inner- and outer-sphere PCET steps. The cation-dependent transition from Pt-O to Pt-OH determines the ORR mechanism, activity, and selectivity. These findings provide direct evidence that the electrolyte affects the ORR mechanism and performance, with important consequences for the practical design of electrochemical systems and computational catalyst screening studies. Our work highlights the importance of complementary insight from experiments and simulations to understand how different components of the electrochemical interface contribute to electrocatalytic processes.

Secondary kinetic deuterium isotope effects on unimolecular cleavage reactions: Zero-point vibrational energy and qualitative RRKM theory.

Secondary kinetic isotope effects arise as the result of transition-state zero-point vibrational energy differences. Unimolecular simple cleavage reactions of gas-phase ions in mass spectrometers allow detailed studies of isotope effects on competing reactions, particularly when examined in intramolecular competition experiments where interpretation requires very few simplifying assumptions. The zero-point energy differences reflect changes of isotope sensitive vibrational properties, and both α- and ß-secondary deuterium isotope effects are related to the sp 3 → sp 2 hybridization changes that accompany bond cleavage. Deuterium substitution three bonds or more removed from the bond broken also gives rise to isotope effects, but their origin is less easily interpreted. The magnitude and variation of the observed effects depend not only on zero-point energy differences; a number of additional factors play a role. The influence of the critical energy, the excess energy, the size of the reactant, and the presence of competing reactions can be rationalized within a simple, qualitative RRKM framework. The distinction between kinetic and thermodynamic isotope effects is not always obvious.

Carboxylate Catalyzed Isomerization of ß,γ-Unsaturated N-Acetylcysteamine Thioesters.

We demonstrate herein the capacity of simple carboxylate salts - tetrametylammonium and tetramethylguanidinium pivalate - to act as catalysts in the isomerization of ß,γ-unsaturated thioesters to α,ß-unsaturated thioesters. The carboxylate catalysts gave reaction rates comparable to those obtained with DBU, but with fewer side reactions. The reaction exhibits a normal secondary kinetic isotope effect (k1H /k1D =1.065±0.026) with a ß,γ-deuterated substrate. Computational analysis of the mechanism provides a similar value (k1H /k1D =1.05) with a mechanism where γ-reprotonation of the enolate intermediate is rate determining.

Thermally-Activated Tunneling in the Two-Water Bridge Catalyzed Tautomerization of Phosphinylidene Compounds.

Phosphinylidenes are an important class of organophosphorus compounds that can exhibit tautomerization between tricoordinated P(III) hydroxide (R1 R2 POH) and a pentacoordinated P(V) oxide (R1 R2 P(O)H) form. Herein we show, using the canonical variational transition state theory combined with multidimensional small-curvature tunneling approximation, the dominance of proton tunneling in the two-water-bridged tautomerizations of phosphinous acid and model phosphinylidenes comprising phosphosphinates, H-phosphonates, H-phosphinates and secondary phosphine oxides. Based on the studied system, the contribution of thermally-activated tunneling is predicted to speed up the semiclassical reaction rate by ca. threefold to as large as two orders of magnitude at 298.15 K in the gas phase. The large KIE and the concavity in the Arrhenius plots are further fingerprints of tunneling. The simulations also predicted that the rapid tunneling rate and short half-life span for the forward reaction, as opposed to the reverse reaction in fluorinated secondary phosphine oxides, would result in P(V) being elusive and only P(III) being isolable, which agrees with previous experiments where only P(III) was detected by IR and NMR spectroscopy. We also explored the role of solvent and predicted tunneling to be substantial.

The Influence of Deuterium Isotope Effects on Structural Rearrangements, Ensemble Equilibria, and Hydrogen Bonding in Protic Ionic Liquids.

We report strong isotope effects for the protic ionic liquid triethylammonium methanesulfonate [TEA][OMs] by means of deuterium solid-state NMR spectroscopy covering broad temperature ranges from 65 K to 313 K. Both isotopically labelled PILs differ in non-deuterated and fully deuterated ethyl groups of the triethyl ammonium cations. The N-D bond of both cations is used as sensitive probe for hydrogen bonding and structural ordering. The 2 H NMR line shape analysis provides the deuteron quadrupole coupling constants and the characteristics of a broad heterogeneous phase with simultaneously present static and mobile states indicating plastic crystal behavior. The temperatures where both states are equally populated differ by about 80 K for the two PILs, showing that deuteration of the ethyl groups in the trialkylammonium cations tremendously shifts the equilibrium towards the static state. In addition, it leads to a significant less cooperative transition, associated with a significantly reduced standard molar transition entropy.

Managing argon interference during measurements of 18O/16O ratios in O2 by continuous-flow isotope ratio mass spectrometry.

Monitoring changes in stable oxygen isotope ratios in molecular oxygen allows for studying many fundamental processes in bio(geo)chemistry and environmental sciences. While the measurement of [Formula: see text]O/[Formula: see text]O ratios of [Formula: see text] in gaseous samples can be carried out conveniently and from extracting moderately small aqueous samples for analyses by continuous-flow isotope ratio mass spectrometry (CF-IRMS), oxygen isotope signatures, [Formula: see text]O, could be overestimated by more than 6[Formula: see text] because of interferences from argon in air. Here, we systematically evaluated the extent of such Ar interferences on [Formula: see text]O/[Formula: see text]O ratios of [Formula: see text] for measurements by gas chromatography/IRMS and GasBench/IRMS and propose simple instrumental modifications for improved Ar and [Formula: see text] separation as well as post-measurement correction procedures for obtaining accurate [Formula: see text]O. We subsequently evaluated the consequences of Ar interferences for the quantification of O isotope fractionation in terms of isotope enrichment factors, [Formula: see text], and [Formula: see text]O kinetic isotope effects ([Formula: see text]O KIEs) in samples where [Formula: see text] is consumed and Ar:[Formula: see text] ratios increase steadily and substantially over the course of a reaction. We show that the extent of O isotope fractionation is overestimated only slightly and that this effect is typically smaller than uncertainties originating from the precision of [Formula: see text]O measurements and experimental variability. Ar interferences can become more relevant and bias [Formula: see text] values by more than [Formula: see text] in aqueous samples where fractional [Formula: see text] conversion exceeds 90%. Practically, however, such samples would typically contain less than 25 [Formula: see text]M of [Formula: see text] at ambient temperature, an amount that is close to the method detection limit of [Formula: see text]O/[Formula: see text]O ratio measurement by CF-IRMS.