Stopped-flow Spectrophotometry Research Articles

Monitoring the reductive and oxidative half-reactions of a flavin-dependent monooxygenase using stopped-flow spectrophotometry.

Aspergillus fumigatus siderophore A (SidA) is an FAD-containing monooxygenase that catalyzes the hydroxylation of ornithine in the biosynthesis of hydroxamate siderophores that are essential for virulence (e.g. ferricrocin or N',N",N'''-triacetylfusarinine C)1. The reaction catalyzed by SidA can be divided into reductive and oxidative half-reactions (Scheme 1). In the reductive half-reaction, the oxidized FAD bound to Af SidA, is reduced by NADPH2,3. In the oxidative half-reaction, the reduced cofactor reacts with molecular oxygen to form a C4a-hydroperoxyflavin intermediate, which transfers an oxygen atom to ornithine. Here, we describe a procedure to measure the rates and detect the different spectral forms of SidA using a stopped-flow instrument installed in an anaerobic glove box. In the stopped-flow instrument, small volumes of reactants are rapidly mixed, and after the flow is stopped by the stop syringe (Figure 1), the spectral changes of the solution placed in the observation cell are recorded over time. In the first part of the experiment, we show how we can use the stopped-flow instrument in single mode, where the anaerobic reduction of the flavin in Af SidA by NADPH is directly measured. We then use double mixing settings where Af SidA is first anaerobically reduced by NADPH for a designated period of time in an aging loop, and then reacted with molecular oxygen in the observation cell (Figure 1). In order to perform this experiment, anaerobic buffers are necessary because when only the reductive half-reaction is monitored, any oxygen in the solutions will react with the reduced flavin cofactor and form a C4a-hydroperoxyflavin intermediate that will ultimately decay back into the oxidized flavin. This would not allow the user to accurately measure rates of reduction since there would be complete turnover of the enzyme. When the oxidative half-reaction is being studied the enzyme must be reduced in the absence of oxygen so that just the steps between reduction and oxidation are observed. One of the buffers used in this experiment is oxygen saturated so that we can study the oxidative half-reaction at higher concentrations of oxygen. These are often the procedures carried out when studying either the reductive or oxidative half-reactions with flavin-containing monooxygenases. The time scale of the pre-steady-state experiments performed with the stopped-flow is milliseconds to seconds, which allow the determination of intrinsic rate constants and the detection and identification of intermediates in the reaction4. The procedures described here can be applied to other flavin-dependent monooxygenases.5,6

Mechanistic studies of the reaction of bis(2,4,6-tripyridyl 1,3,5-triazine)iron(II) with triazines

Bis(2,4,6-tripyridyl 1,3,5-triazine)iron(II), \( {\text{Fe(\text{TPTZ})}}_{ 2}^{{ 2 { + }}} \) reacts with 3-(2-pyridyl)-5,6-bis(4-phenyl-sulfonicacid)-1,2,4-triazine (PDTS) and 3-(4-(4-phenylsulfonicacid)-2-pyridyl)-5,6-bis(4-phenylsulfonic-acid)-1,2,4-triazine (PPDTS) to give \( {\text{Fe(PDTS)}}_{ 3}^{ 4- } \) and \( {\text{Fe(PPDTS)}}_{ 3}^{ 7- } \) respectively. Both of these substitution reactions are fast and their kinetics were monitored by stopped-flow spectrophotometry in acetate buffers in the pH range of 3.6–5.6 at 25–45 °C. Both reactions are first order in \( {\text{Fe(TPTZ)}}_{ 2}^{{ 2 { + }}} \)and triazine, and pH has negligible effect on the rate. The kinetic data suggest that these reactions occur in an associative path and a mechanism is proposed considering both protonated and unprotonated forms of PDTS and PPDTS are very similar in reactivity. The kinetic and activation parameters have been evaluated.

Kinetics of α-Globin Binding to α-Hemoglobin Stabilizing Protein (AHSP) Indicate Preferential Stabilization of Hemichrome Folding Intermediate

Human α-hemoglobin stabilizing protein (AHSP) is a conserved mammalian erythroid protein that facilitates the production of Hemoglobin A by stabilizing free α-globin. AHSP rapidly binds to ferrous α with association (k'(AHSP)) and dissociation (k(AHSP)) rate constants of ≈10 μm(-1) s(-1) and 0.2 s(-1), respectively, at pH 7.4 at 22 °C. A small slow phase was observed when AHSP binds to excess ferrous αCO. This slow phase appears to be due to cis to trans prolyl isomerization of the Asp(29)-Pro(30) peptide bond in wild-type AHSP because it was absent when αCO was mixed with P30A and P30W AHSP, which are fixed in the trans conformation. This slow phase was also absent when met(Fe(3+))-α reacted with wild-type AHSP, suggesting that met-α is capable of rapidly binding to either Pro(30) conformer. Both wild-type and Pro(30)-substituted AHSPs drive the formation of a met-α hemichrome conformation following binding to either met- or oxy(Fe(2+))-α. The dissociation rate of the met-α·AHSP complex (k(AHSP) ≈ 0.002 s(-1)) is ∼100-fold slower than that for ferrous α·AHSP complexes, resulting in a much higher affinity of AHSP for met-α. Thus, in vivo, AHSP acts as a molecular chaperone by rapidly binding and stabilizing met-α hemichrome folding intermediates. The low rate of met-α dissociation also allows AHSP to have a quality control function by kinetically trapping ferric α and preventing its incorporation into less stable mixed valence Hemoglobin A tetramers. Reduction of AHSP-bound met-α allows more rapid release to β subunits to form stable fully, reduced hemoglobin dimers and tetramers.

Kinetic Study of the α-Tocopherol-Regeneration Reaction of Ubiquinol-10 in Methanol and Acetonitrile Solutions: Notable Effect of the Alkali and Alkaline Earth Metal Salts on the Reaction Rates

A kinetic study of regeneration reaction of α-tocopherol (α-TocH) by ubiquinol-10 has been performed in the presence of four kinds of alkali and alkaline earth metal salts (LiClO(4), NaClO(4), NaI, and Mg(ClO(4))(2)) in methanol and acetonitrile solutions, using double-mixing stopped-flow spectrophotometry. The second-order rate constants (k(r)'s) for the reaction of α-tocopheroxyl (α-Toc•) radical with ubiquinol-10 increased and decreased notably with increasing concentrations of metal salts in methanol and acetonitrile, respectively. The k(r) values increased in the order of no metal salt < NaClO(4) ~ NaI < LiClO(4) < Mg(ClO(4))(2) at the same concentration of metal salts in methanol. On the other hand, in acetonitrile, the k(r) values decreased in the order of no metal salt > NaClO(4) ~ NaI > LiClO(4) > Mg(ClO(4))(2) at the same concentration of metal salts. The metal salts having a smaller ionic radius of cation and a larger charge of cation gave a larger k(r) value in methanol, and a smaller k(r) value in acetonitrile. The effect of anion was almost negligible in both the solvents. Notable effects of metal cations on the UV-vis absorption spectrum of α-Toc• radical were observed in aprotic acetonitrile solution, suggesting complex formation between α-Toc• and metal cations. On the other hand, effects of metal cations were negligible in protic methanol, suggesting that the complex formation between α-Toc• and metal cations is hindered by the hydrogen bond between α-Toc• and methanol molecules. The difference between the reaction mechanisms in methanol and acetonitrile solutions was discussed on the basis of the results obtained. High concentrations of alkali and alkaline earth metal salts coexist with α-TocH and ubiquinol-10 in plasma, blood, and many tissues, suggesting the contribution of the metal salts to the above regeneration reaction in biological systems.

Accurate Rate Constants for Decomposition of Aqueous Nitrous Acid

Decomposition of nitrous acid in aqueous solution has been studied by stopped flow spectrophotometry to resolve discrepancies in literature values for the rate constants of the decomposition reactions. Under the conditions employed, the rate-limiting reaction step comprises the hydrolysis of NO(2). A simplified rate law based on the known elementary reaction mechanism provides an excellent fit to the experimental data. The rate constant, 1.34 × 10(-6) M(-1) s(-1), is thought to be of higher accuracy than those in the literature as it does not depend on the rate of parallel reaction pathways or on the rate of interphase mass transfer of gaseous reaction products. The activation energy for the simplified rate law was established to be 107 kJ mol(-1). Quantum chemistry calculations indicate that the majority of the large activation energy results from the endothermic nature of the equilibrium 2HNO(2) ⇆ NO + NO(2) + H(2)O. The rate constant for the reaction between nitrate ions and nitrous acid, which inhibits HNO(2) decomposition, was also determined.

Kinetics and mechanism of substitution of bis(2,4,6-tripyridyl 1,3,5-triazine)iron(II) by 2,2′,6,2″-terpyridine

The substitution of bis(2,4,6-tripyridyl 1,3,5-triazine)iron(II), \( {\text{Fe(TPTZ)}}_{ 2}^{{ 2 { + }}} \) by 2,2′,6,2″-terpyridine (terpy) occurs on a time scale of about 6 m. The kinetics of this reaction was followed by stopped-flow spectrophotometry in the pH range of 3.6–5.6 in acetate buffer. The data indicate that the reaction occurs in two consecutive steps: kinetic data for both steps were acquired simultaneously and analyzed independently. The first step is assigned to the reaction between \( {\text{Fe(TPTZ)}}_{ 2}^{{ 2 { + }}} \) and terpy to give Fe(TPTZ)(terpy)2+, followed by its reaction with another terpy molecule to give the final product, \( {\text{Fe(terpy)}}_{ 2}^{{ 2 { + }}} \). The rate of the reaction increases with increases in [terpy] and pH. The kinetic and activation parameters determined for both steps suggest that they involve both associative and dissociative paths. The ternary complex Fe(TPTZ)(terpy)2+ has been prepared, and the kinetics of its reaction with terpy suggest that this reaction is identical with the second step of the \( {\text{Fe(TPTZ)}}_{ 2}^{{ 2 { + }}} \)-terpy system, supporting the proposed mechanism.

Comprehensive Kinetic and Thermodynamic Study of the Reactions of CO2(aq) and HCO3– with Monoethanolamine (MEA) in Aqueous Solution

The temperature dependence of the reversible reaction between CO(2)(aq) and monoethanolamine (MEA) has been investigated using stopped-flow spectrophotometry by following the pH changes during the reactions with colored acid-base indicators. Multivariate global analysis of both the forward and backward kinetic measurements for the reaction of CO(2)(aq) with MEA yielded the rate and equilibrium constants, including the protonation constant of MEA carbamate, for the temperature range of 15-45 °C. Analysis of the rate and equilibrium constants in terms of the Arrhenius, Eyring, and van't Hoff relationships gave the relevant thermodynamic parameters. In addition, the rate and equilibrium constants for the slow, reversible reaction of bicarbonate with MEA are reported at 25.0 °C. At high pH, reactions of the amine with CO(2) and with bicarbonate are significant.

Electron Transfer within Nitrogenase: Evidence for a Deficit-Spending Mechanism

The reduction of substrates catalyzed by nitrogenase utilizes an electron transfer (ET) chain comprised of three metalloclusters distributed between the two component proteins, designated as the Fe protein and the MoFe protein. The flow of electrons through these three metalloclusters involves ET from the [4Fe-4S] cluster located within the Fe protein to an [8Fe-7S] cluster, called the P cluster, located within the MoFe protein and ET from the P cluster to the active site [7Fe-9S-X-Mo-homocitrate] cluster called FeMo-cofactor, also located within the MoFe protein. The order of these two electron transfer events, the relevant oxidation states of the P-cluster, and the role(s) of ATP, which is obligatory for ET, remain unknown. In the present work, the electron transfer process was examined by stopped-flow spectrophotometry using the wild-type MoFe protein and two variant MoFe proteins, one having the β-188(Ser) residue substituted by cysteine and the other having the β-153(Cys) residue deleted. The data support a "deficit-spending" model of electron transfer where the first event (rate constant 168 s(-1)) is ET from the P cluster to FeMo-cofactor and the second, "backfill", event is fast ET (rate constant >1700 s(-1)) from the Fe protein [4Fe-4S] cluster to the oxidized P cluster. Changes in osmotic pressure reveal that the first electron transfer is conformationally gated, whereas the second is not. The data for the β-153(Cys) deletion MoFe protein variant provide an argument against an alternative two-step "hopping" ET model that reverses the two ET steps, with the Fe protein first transferring an electron to the P cluster, which in turn transfers an electron to FeMo-cofactor. The roles for ATP binding and hydrolysis in controlling the ET reactions were examined using βγ-methylene-ATP as a prehydrolysis ATP analogue and ADP + AlF(4)(-) as a posthydrolysis analogue (a mimic of ADP + P(i)).

Spectral and kinetic evidence for reaction of superoxide with compound I of myeloperoxidase

Superoxide and myeloperoxidase (MPO) are essential for the oxidative killing of bacteria by neutrophils. Previously, we developed a kinetic model to demonstrate that within the confines of neutrophil phagosomes, superoxide should react exclusively with MPO and be converted to hypochlorous acid. The model consists of all known reactions and rate constants for reactions of superoxide, hydrogen peroxide, and chloride ions with MPO, except for the reaction of superoxide with compound I, which could only be estimated. Compound I is a transitory redox intermediate of MPO that is responsible for oxidizing chloride ions to hypochlorous acid. To tackle the challenge of observing the reaction between two transient species, we combined stopped-flow spectrophotometry with pulse radiolysis. Using this technique, we directly observed the reduction of compound I by superoxide. The rate constant for the reaction was determined to be 5.6±0.3×106M−1s−1. This value establishes superoxide as one of the best substrates for compound I. Based on this value, the rate constant for reduction of compound II by superoxide was determined to be 1.2±0.1×106M−1s−1. Within phagosomes, the reduction of compound I by superoxide will compete with the oxidation of chloride ions so that the relative concentrations of these two substrates will affect the yield of hypochlorous acid. Characterization of this reaction confirms that superoxide is a physiological substrate for MPO and that their interactions are central to an important host defense mechanism.

The superoxide reductase from the early diverging eukaryote Giardia intestinalis

Unlike superoxide dismutases (SODs), superoxide reductases (SORs) eliminate superoxide anion (O2•−) not through its dismutation, but via reduction to hydrogen peroxide (H2O2) in the presence of an electron donor. The microaerobic protist Giardia intestinalis, responsible for a common intestinal disease in humans, though lacking SOD and other canonical reactive oxygen species-detoxifying systems, is among the very few eukaryotes encoding a SOR yet identified. In this study, the recombinant SOR from Giardia (SORGi) was purified and characterized by pulse radiolysis and stopped-flow spectrophotometry. The protein, isolated in the reduced state, after oxidation by superoxide or hexachloroiridate(IV), yields a resting species (Tfinal) with Fe3+ ligated to glutamate or hydroxide depending on pH (apparent pKa=8.7). Although showing negligible SOD activity, reduced SORGi reacts with O2•− with a pH-independent second-order rate constant k1=1.0×109M−1s−1 and yields the ferric-(hydro)peroxo intermediate T1; this in turn rapidly decays to the Tfinal state with pH-dependent rates, without populating other detectable intermediates. Immunoblotting assays show that SORGi is expressed in the disease-causing trophozoite of Giardia. We propose that the superoxide-scavenging activity of SOR in Giardia may promote the survival of this air-sensitive parasite in the fairly aerobic proximal human small intestine during infection.

Notable Effects of the Metal Salts on the Formation and Decay Reactions of α-Tocopheroxyl Radical in Acetonitrile Solution. The Complex Formation between α-Tocopheroxyl and Metal Cations

The measurement of the UV-vis absorption spectrum of α-tocopheroxyl (α-Toc(•)) radical was performed by reacting aroxyl (ArO(•)) radical with α-tocopherol (α-TocH) in acetonitrile solution including four kinds of alkali and alkaline earth metal salts (MX or MX(2)) (LiClO(4), LiI, NaClO(4), and Mg(ClO(4))(2)), using stopped-flow spectrophotometry. The maximum wavelength (λ(max)) of the absorption spectrum of the α-Toc(•) at 425.0 nm increased with increasing concentration of metal salts (0-0.500 M) in acetonitrile, and it approached constant values, suggesting an [α-Toc(•)-M(+) (or M(2+))] complex formation. The stability constants (K) were determined to be 9.2, 2.8, and 45 M(-1) for LiClO(4), NaClO(4), and Mg(ClO(4))(2), respectively. By reacting ArO(•) with α-TocH in acetonitrile, the absorption of ArO(•) disappeared rapidly, while that of α-Toc(•) appeared and then decreased gradually as a result of the bimolecular self-reaction of α-Toc(•) after passing through the maximum. The second-order rate constants (k(s)) obtained for the reaction of α-TocH with ArO(•) increased linearly with an increasing concentration of metal salts. The results indicate that the hydrogen transfer reaction of α-TocH proceeds via an electron transfer intermediate from α-TocH to ArO(•) radicals followed by proton transfer. Both the coordination of metal cations to the one-electron reduced anions of ArO(•) (ArO:(-)) and the coordination of counteranions to the one-electron oxidized cations of α-TocH (α-TocH(•)(+)) may stabilize the intermediate, resulting in the acceleration of electron transfer. A remarkable effect of metal salts on the rate of bimolecular self-reaction (2k(d)) of the α-Toc(•) radical was also observed. The rate constant (2k(d)) decreased rapidly with increasing concentrations of the metal salts. The 2k(d) value decreased at the same concentration of the metal salts in the following order: no metal salt > NaClO(4) > LiClO(4) > Mg(ClO(4))(2). The complex formation between α-Toc(•) and metal cations may stabilize the energy level of the reactants (α-Toc(•) + α-Toc(•)), resulting in the decrease of the rate constant (2k(d)). The alkali and alkaline earth metal salts having a smaller ionic radius of cation and a larger charge of cation gave larger K and k(s) values and a smaller 2k(d) value.

Palladacycle-Promoted Solvolytic Cleavage of O,O-Dimethyl O-Aryl Phosphorothioates. Converting a Phosphorane-Like Transition State to an Observable Intermediate

The mechanism of cleavage of a series of seven O,O-dimethyl O-aryl phosphorothioates (6a-g) promoted by a C,N-palladacycle, (2-[N,N-dimethylamino(methyl)phenyl]-C(1),N)(pyridine) palladium(II) triflate (5:OTf) in methanol at 25 °C was investigated with the aim of identifying catalytically important intermediates. Complete (s)(s)pH/rate profiles (in methanol) were conducted for the cleavage of 6a-g in the presence of 0.08 mM 5. The log k(obs) for the catalyzed methanolysis of 6a increases linearly with (s)(s)pH with a plateau above the (s)(s)pK(a)(1) of 11.16 for formation of 5:(-)OCH3. The profiles for 6b-g are bell-shaped, depending on the apparent ionizations of two acidic groups, with the rate constant maximum of the bell and the (s)(s)pK(a)(1) values shifting to higher (s)(s)pH values as the (s)(s)pK(a)(HOAr) of the leaving group phenol increases. A Brønsted plot of the log k(obs)(max) (the maximum rate constants for cleavage of 6a-g) vs (s)(s)pK(a)(HOAr) exhibits a downward break at ~ (s)(s)pK(a)(HOAr) 13, with the two wings having β(lg) values of 0.01 and -0.96. A model describing the kinetically important species involves a complex series of equilibria: 5:(HOCH(3)):pyr <=> 5:((-)OCH3):pyr + H(+) <=>(6) 5:((-)OCH3):6 + pyr <=> phosphorane 7 → product, where the rate limiting steps change from formation of 5:((-)OCH3):6 to formation of thiophosphorane 7 and then to product formation as the aryloxy leaving groups of 6 get progressively worse. Kinetic experiments indicate that the reaction of 5 with 6e, having a 4-chlorophenoxy leaving group, rapidly produces a transient intermediate, postulated to be the palladacycle-bound 5-coordinate thiophosphorane (7e) that exists long enough to obtain its UV/vis spectrum by stopped-flow spectrophotometry. Detailed analysis of the data sheds light on the origins of a previously reported anomalously large β(lg) of -1.93 for the descending wing of a Brønsted plot (J. Am. Chem. Soc. 2010, 132, 16599). Finally, energetics analysis indicates that the binding of palladacycle to the transition state comprising attack of methoxide on 6e, [MeO(-) + 6e](++), stabilizes the latter by 34.9 kcal/mol, converting that transition state into an observable intermediate.

Polymorphic Variants of Cytochrome P450 2B6 (CYP2B6.4–CYP2B6.9) Exhibit Altered Rates of Metabolism for Bupropion and Efavirenz: A Charge-Reversal Mutation in the K139E Variant (CYP2B6.8) Impairs Formation of a Functional Cytochrome P450-Reductase Complex

In this study, metabolism of bupropion, efavirenz, and 7-ethoxy-4-trifluoromethylcoumarin (7-EFC) by CYP2B6 wild type (CYP2B6.1) and six polymorphic variants (CYP2B6.4 to CYP2B6.9) was investigated in a reconstituted system to gain a better understanding of the effects of the mutations on the catalytic properties of these naturally occurring variants. All six variants were successfully overexpressed in Escherichia coli, including CYP2B6.8 (the K139E variant), which previously could not be overexpressed in mammalian COS-1 cells (J Pharmacol Exp Ther 311:34-43, 2004). The steady-state turnover rates for the hydroxylation of bupropion and efavirenz and the O-deethylation of 7-EFC showed that these mutations significantly alter the catalytic activities of CYP2B6. It was found that CYP2B6.6 exhibits 4- and 27-fold increases in the K(m) values for the hydroxylation of bupropion and efavirenz, respectively, and CYP2B6.8 completely loses its ability to metabolize any of the substrates under normal turnover conditions. However, compared with CYP2B6.1, CYP2B6.8 retains 77% of its 7-EFC O-deethylase activity in the presence of tert-butyl hydroperoxide as an alternative oxidant, indicating that the heme and the active site are catalytically competent. Presteady-state measurements of the rate of electron transfer from NADPH-dependent cytochrome P450 reductase (CPR) to CYP2B6.8 using stopped-flow spectrophotometry revealed that CYP2B6.8 is incapable of accepting electrons from CPR. These observations provide conclusive evidence suggesting that the charge-reversal mutation in the K139E variant prevents CYP2B6.8 from forming a functional complex with CPR. Results from this work provide further insights to better understand the genotype-phenotype correlation regarding CYP2B6 polymorphisms and drug metabolism.

Electron transfer and half-reactivity in nitrogenase

Nitrogenase is a globally important enzyme that catalyses the reduction of atmospheric dinitrogen into ammonia and is thus an important part of the nitrogen cycle. The nitrogenase enzyme is composed of a catalytic molybdenum-iron protein (MoFe protein) and a protein containing an [Fe4-S4] cluster (Fe protein) that functions as a dedicated ATP-dependent reductase. The current understanding of electron transfer between these two proteins is based on stopped-flow spectrophotometry, which has allowed the rates of complex formation and electron transfer to be accurately determined. Surprisingly, a total of four Fe protein molecules are required to saturate one MoFe protein molecule, despite there being only two well-characterized Fe-protein-binding sites. This has led to the conclusion that the purified Fe protein is only half-active with respect to electron transfer to the MoFe protein. Studies on the electron transfer between both proteins using rapid-quench EPR confirmed that, during pre-steady-state electron transfer, the Fe protein only becomes half-oxidized. However, stopped-flow spectrophotometry on MoFe protein that had only one active site occupied was saturated by approximately three Fe protein equivalents. These results imply that the Fe protein has a second interaction during the initial stages of mixing that is not involved in electron transfer.

Kinetic study of aroxyl radical-scavenging action of vitamin E in membranes of egg yolk phosphatidylcholine vesicles

Vitamin E is localized in membranes and functions as an efficient inhibitor of lipid peroxidation in biological systems. In this study, we measured the reaction rates of vitamin E (α-, β-, γ-, δ-tocopherols, TocH) and tocol with aroxyl radical (ArO ) as model lipid peroxyl radicals in membranes by stopped-flow spectrophotometry. Egg yolk phosphatidylcholine (EYPC) vesicles were used as a membrane model. EYPC vesicles were prepared in the aqueous methanol solution (MeOH:H 2O = 7:3, v/v) that gave the lowest turbidity in samples. The second-order rate constants ( k s) for α-TocH in MeOH/H 2O solution with EYPC vesicles were apparently 3.45 × 10 5 M −1 s −1, which was about 8 times higher than that (4.50 × 10 4 M −1 s −1) in MeOH/H 2O solution without EYPC vesicles. The corrected k s of α-TocH in vesicles, which was calculated assuming that the concentration of α-TocH was 133 times higher in membranes of 10 mM EYPC vesicles than in the bulk MeOH/H 2O solution, was 2.60 × 10 3 M −1 s −1, which was one-seventeenth that in MeOH/H 2O solution because of the lower mobility of α-TocH in membranes. Similar analyses were performed for other vitamin E analogues. The k s of vitamin E in membranes increased in the order of tocol < δ-TocH < γ-TocH ∼ β-TocH < α-TocH. There was not much difference in the ratios of reaction rates in vesicles and MeOH/H 2O solution among vitamin E analogues [ k s(vesicle)/ k s (MeOH/H 2O) = 7.7, 10.0, 9.5, 7.4, and 5.1 for α-, β-, γ-, δ-TocH, and tocol, respectively], but their reported ratios in solutions of micelles and ethanol were quite different [ k s(micelle)/ k s(EtOH) = 100, 47, 41, 15, and 6.3 for α-, β-, γ-, δ-TocH, and tocol, respectively]. These results indicate that the reaction sites of vitamin E analogues were similar in vesicle membranes but depended on hydrophobicity in micelle membranes, which increased in the order of tocol < δ-TocH < γ-TocH ∼ β-TocH < α-TocH.

Kinetic and Theoretical Studies on the Protonation of [Ni(2-SC6H4N){PhP(CH2CH2PPh2)2}]+: Nitrogen versus Sulfur as the Protonation Site

The complexes [Ni(4-Spy)(triphos)]BPh(4) and [Ni(2-Spy)(triphos)]BPh(4) {triphos = PhP(CH(2)CH(2)PPh(2))(2), 4-Spy = 4-pyridinethiolate, 2-Spy = 2-pyridinethiolate} have been prepared and characterized both spectroscopically and using X-ray crystallography. In both complexes the triphos is a tridentate ligand. However, [Ni(4-Spy)(triphos)](+) comprises a 4-coordinate, square-planar nickel with the 4-Spy ligand bound to the nickel through the sulfur while [Ni(2-Spy)(triphos)](+) contains a 5-coordinate, trigonal-bipyramidal nickel with a bidentate 2-Spy ligand bound to the nickel through both sulfur and nitrogen. The kinetics of the reactions of [Ni(4-Spy)(triphos)](+) and [Ni(2-Spy)(triphos)](+) with lutH(+) (lut = 2,6-dimethylpyridine) in MeCN have been studied using stopped-flow spectrophotometry, and the two complexes show very different reactivities. The reaction of [Ni(4-Spy)(triphos)](+) with lutH(+) is complete within the deadtime of the stopped-flow apparatus (2 ms) and corresponds to protonation of the nitrogen. However, upon mixing [Ni(2-Spy)(triphos)](+) and lutH(+) a reaction is observed (on the seconds time scale) to produce an equilibrium mixture. The mechanistic interpretation of the rate law has been aided by the application of MSINDO semiempirical and ADF calculations. The kinetics and calculations are consistent with the reaction between [Ni(2-Spy)(triphos)](+) and lutH(+) involving initial protonation of the sulfur followed by dissociation of the nitrogen and subsequent transfer of the proton from sulfur to nitrogen. The factors affecting the position of protonation and the coupling of the coordination state of the 2-pyridinethiolate ligand to the site of protonation are discussed.

Product-assisted Catalysis as the Basis of the Reaction Specificity of Threonine Synthase

Threonine synthase (TS), which is a pyridoxal 5'-phosphate (PLP)-dependent enzyme, catalyzes the elimination of the γ-phosphate group from O-phospho-L-homoserine (OPHS) and the subsequent addition of water at Cβ to form L-threonine. The catalytic course of TS is the most complex among the PLP enzymes, and it is an intriguing problem how the elementary steps are controlled in TS to carry out selective reactions. When L-vinylglycine was added to Thermus thermophilus HB8 TS in the presence of phosphate, L-threonine was formed with k(cat) and reaction specificity comparable with those when OPHS was used as the substrate. However, in the absence of phosphate or when sulfate was used in place of phosphate, only the side reaction product, α-ketobutyrate, was formed. Global analysis of the spectral changes in the reaction of TS with L-threonine showed that compared with the more acidic sulfate ion, the phosphate ion decreased the energy levels of the transition states of the addition of water at the Cβ of the PLP-α-aminocrotonate aldimine (AC) and the transaldimination to form L-threonine. The x-ray crystallographic analysis of TS complexed with an analog for AC gave a distinct electron density assigned to the phosphate ion derived from the solvent near the Cβ of the analog. These results indicated that the phosphate ion released from OPHS by γ-elimination acts as the base catalyst for the addition of water at Cβ of AC, thereby providing the basis of the reaction specificity. The phosphate ion is also considered to accelerate the protonation/deprotonation at Cγ.

Reduction Kinetics of a Flavin Oxidoreductase LuxG from Photobacterium leiognathi (TH1): Half-Sites Reactivity

Bacterial bioluminescence is a phenomenon resulting from the reaction of a two-component FMN-dependent aldehyde monooxygenase system, which comprises a bacterial luciferase and a flavin reductase. Bacterial luciferase (LuxAB) is one of the most extensively investigated two-component monooxygenases, while its reductase partner, the flavin reductase (LuxG) from the same operon, has only been recently expressed in a functional form. This work reports transient kinetics identification of intermediates in the LuxG reaction using stopped-flow spectrophotometry. The results indicate that the overall reaction follows a sequential-ordered mechanism in which NADH binds first to the enzyme, followed by FMN, resulting in the formation of charge-transfer intermediate 1 (CT-1) typical of those between reduced pyridine nucleotides and oxidized flavins. The next step is the reduction of FMN as indicated by a large decrease in absorbance at 450 nm. The reduction of FMN is biphasic. The first phase of FMN reduction occurs concurrently with formation of charge-transfer intermediate 2 (CT-2), while the second phase is synchronous with the decay of CT-2. When the isotope-labeled substrate, 4(R)-[(2)H]NADH, was used, the first reduction phase showed a primary kinetic isotope effect ((D)k(red)) of ≥3.9 and resulted in greater accumulation of CT-1. These results are consistent with CT-1 being the FMN(ox):NADH complex, while CT-2 is the FMN(red):NAD(+) complex. Because CT-2 decays with a rate constant of 2.8 ± 0.2 s(-1), while the turnover number obtained from the steady-steady-state kinetics is 1.7 s(-1), it is likely that the CT-2 decay step largely controls the overall reaction rate. All kinetic data are consistent with a half-sites reactivity model in which flavin reduction occurs at only one subunit at a time. The first reduction phase is due to the reduction of FMN in the first subunit, while the second phase is due to the reduction of FMN in the second subunit. The latter phase is limited by the rate of decay of CT-2 in the first subunit. The half-sites reactivity model is also supported by detection of burst kinetics during the pre-steady-state period that is correlated with 0.5 mol of the FMN being reduced/mol of the LuxG:NADH complex. The functional importance of this half-site reactivity phenomenon is still unclear.

Spectroscopic Characterization of Neutral and Cation Radicals of α-Tocopherol and Related Molecules: A Satisfactory Denouement

Notwithstanding the facile occurrence of one-electron oxidation in α-tocopherol and its acetate (TOH and TOAc, respectively), and despite the remarkable stability, under appropriate conditions, of the oxidation products (TOH(•+), TO(•), and TOAc(•+)), their spectroscopic characterization is in an unsatisfactory state, calling for a fresh attempt to acquire reliable data. A new, model-free method is developed for analyzing time-resolved spectra showing the progress of the reaction TOH + R(•) → TO(•) + RH, where R(•) is a stable free radical. The resulting absorption coefficients of TO(•) in dichloromethane and hexane are in severe disagreement with some recent values derived from stopped-flow spectrophotometry. The discrepancy is traced to the imposition of boundary conditions that do not take proper account of the dead time of the apparatus; when multiplied by a factor of two, the stopped-flow data fall mostly in the range ε = (7.5 ± 0.5) × 10(3) M(-1) cm(-1), conforming with the results of this study and the values found by Boguth and Niemann in 1969. Absorption spectra of the radical cations produced (electro)chemically are found to be reliable only in the visible region. Incomplete conversion of the parent compound to the radical cation, an obstacle to the determination of absorption coefficients from electrochemical studies, is circumvented by combining EPR and optical spectroscopy. The absorption coefficients of TOH(•+) and TOAc(•+), determined in this manner, are found to be, respectively, 1.6 × 10(4) and 1.3 × 10(4) M(-1) cm(-1), in accord with the values found first through similar means.

ChemInform Abstract: Silver(III) Oxidation of the DL-Mandelate Ion

The oxidation of DL-mandelate (MAN–) by [Ag(OH)4]– in strongly alkaline media has been investigated by stopped-flow spectrophotometry. Product analysis shows that benzaldehyde is the sole organic product when MAN– is in excess. The rate law is kobs=(a+b[OH–])[MAN–], where a=(6.2 ± 0.2) dm3 mol–1 s–1 and b=(7.0 ± 0.3) dm6 mol–2s–1 at 25 °C and I= 1.3 mol dm–3. Oxidation of benzaldehyde (to benzoic acid) by AgIII is more than an order of magnitude slower than the mandelate reaction. Activation parameters for both hydroxide-dependent and -independent paths are ΔHa‡=(59.3 ± 2.1) kJ mol–1, ΔSb‡=(–32.0 ± 0.5) J mol–1 K–1 and ΔH‡b=(48.0 ± 2.3) kJ mol–1, ΔSb‡=(–68.5 ± 0.6) J mol–1 K–1 respectively. Two different mechanistic pathways are discussed. A base-catalysed processs, followed by C–C cleavage, seems to be the most probable mode of decarboxylation. The comparative non-reactivity of phenylglyoximate and α-methoxyphenylacetate ions underlines the importance of the α-OH group in the mandelate oxidation.