Stopped-flow UV-visible Spectroscopy Research Articles

High Rates of Quinone-Alkyne Cycloaddition Reactions are Dictated by Entropic Factors.

Reaction rates of strained cycloalkynes and cycloalkenes with 1,2-quinone were quantified by stopped flow UV-Vis spectroscopy and computational analysis. We found that the strained alkyne BCN-OH 3 (k2 1824 M-1s-1) reacts >150 times faster than the strained alkene TCO-OH 5 (k2 11.56 M-1s-1). Also, the 8-membered strained alkyne BCN-OH 3 reacts 16 times faster than the more strained 7-membered THS 2 (k2 110.6 M-1s-1). Using the linearized Eyring equation we determined the thermodynamic activation parameters of these two strained alkynes, revealing that the SPOCQ reaction of quinone 1 with THS 2 is associated with ΔH‡ of 0.80 kcal/mol, ΔS‡ = -46.8 cal/K·mol, and ΔG‡ = 14.8 kcal/mol (at 25 °C), whereas the same reaction with BCN-OH 3 is associated with, ΔH‡ = 2.25 kcal/mol, ΔS‡ = -36.3 cal/K·mol, and ΔG‡ = 13.1 kcal/mol (at 25 °C). Computational analysis supported the values obtained by the stopped-flow measurements, with calculated ΔG‡ of 15.6 kcal/mol (in H2O) for the SPOCQ reaction with THS 2, and with ΔG‡ of 14.7 kcal/mol (in H2O) for the SPOCQ reaction with BCN-OH 3. With these empirically determined thermodynamic parameters, we set an important step towards a more fundamental understanding of this set of rapid biogenic click reactions.

Oxidation of bisphenol A (BPA) and related compounds by the multifunctional catalytic globin dehaloperoxidase

Dehaloperoxidase (DHP) from the marine polychaete Amphitrite ornata is a multifunctional enzyme that possesses peroxidase, peroxygenase, oxidase and oxygenase activities. Herein, we investigated the reactivity of DHP B with bisphenol A (BPA) and related compounds (bisphenol E, bisphenol F, tetrachlorobisphenol A, 2,2′-biphenol, 3,3′-biphenol, 4,4′-biphenol, and 3,3′-dibromo-4,4′-biphenol). As a previously unknown substrate for DHP B, BPA (as a representative substrate) is an endocrine disruptor widely used in polycarbonate and epoxy resins, thus resulting in human exposure. Reactivity studies with these substrates were investigated using high performance liquid chromatography (HPLC), and their corresponding oxidation products were determined by mass spectrometry (GC–MS/ LC-MS). BPA undergoes oxidation in the presence of DHP B and hydrogen peroxide yielding two cleavage products (4-isopropenylphenol and 4-(2-hydroxypropan-2-yl)phenol), and oligomers with varying degrees of oxidation. 18O-labeling studies confirmed that the O-atom incorporated into the products was derived exclusively from water, consistent with substrate oxidation via a peroxidase-based mechanism. The X-ray crystal structures of DHP bound with bisphenol E (1.48 Å), bisphenol F (1.75 Å), 2,2′-biphenol (1.90 Å) and 3,3′-biphenol (1.30 Å) showed substrate binding sites are in the distal pocket of the heme cofactor, similar to other previously studied DHP substrates. Stopped-flow UV–visible spectroscopy was utilized to investigate the mechanistic details and enzyme oxidation states during substrate turnover, and a reaction mechanism is proposed. The data presented here strongly suggest that DHP B can catalyze the oxidation of bisphenols and biphenols, thus providing evidence of how infaunal invertebrates can contribute to the biotransformation of these marine pollutants.

Conformational selection is present in ligand binding to cytochrome P450 19A1 lipoprotein nanodiscs

Cytochromes P450 (CYPs) display remarkable plasticity in their ability to bind substrates and catalyze a broad array of chemical reactions. Herein we evaluate binding of androstenedione, testosterone, and 7-hydroxyflavone to CYP19A1, also known as aromatase, in phospholipid nanodiscs by stopped-flow UV–vis spectroscopy. Exponential fitting of the kinetic traces supports the possibility of a multi-step binding mechanism. Subsequent global fitting of the data to the solutions of the coupled differential equations describing the fundamental mechanisms of induced fit and conformational selection, consistently support presence of the latter. To our knowledge, this is the first discrimination of conformational selection from induced fit for a mono-disperse CYP in a native-like membrane environment. In addition, 7-hydroxyflavone binds to CYP19A1 nanodiscs with comparable affinity to the substrates and induces an unusual spectral response likely attributable to hydrogen bonding to, rather than displacement of the heme-coordinated water molecule.

Biophysical Characterization of Aptenodytes forsteri Cytochrome P450 19 (Aromatase)

Cytochrome P450 19 (aromatase) catalyzes the conversion of androgens to estrogens in a sequence of three reactions that each depend on NADPH and O2. As the terminal enzyme in estrogen biosynthesis, it is critical for the maintenance of myriad tissues and has emerged as a successful target for the treatment of estrogen‐dependent breast cancer. Aromatase is a phylogenetically‐ancient enzyme and its breadth of expression in other species has highlighted distinct physiological functions. In songbirds, estrogen production is required for programming of neural circuits controlling song. Aromatase also plays a role in sex determination of fish, reptiles, and amphibians. Biophysical characterization of the human enzyme has been limited by low yields from recombinant expression systems. Accordingly, we sought to identify an aromatase homolog with enhanced expression properties in the absence of stabilizing ligands. This work describes the expression, purification, and biophysical characterization of Aptenodytes forsteri (Emperor penguin) aromatase (afCYP19A1). Using human cytochrome P450 reductase as a redox partner, the enzyme kinetic parameters for turnover of androstenedione were like the human enzyme and mass spectrometry analysis conformed that afCYP19A1 likewise catalyzes the transformation through 19‐hydroxy‐ and 19‐oxoandrostenedione intermediates. Equilibrium binding studies revealed that androstenedione and anastrozole had the highest affinity for the enzyme and were closely followed by 19‐hydroxy‐androstenedione and testosterone. The affinity of 19‐oxoandrostenedione was an order‐of‐magnitude lower. Ligand‐binding kinetics were determined by stopped‐flow UV‐visible spectroscopy. Both androstenedione and testosterone where determined to bind to the enzyme in a step‐wise fashion with a spectroscopically‐detectable intermediate. In summary, these studies describe the first biophysical characterization of a non‐human aromatase that displays strikingly similar enzyme kinetics and ligand binding properties to the human enzyme despite bearing 75% sequence identity. In addition, they provide access to an alternative model to study the catalytic mechanism of the enzyme using methods requiring substantial amounts of ligand‐free enzyme.Support or Funding InformationNational Institutes of Health National Institute of General Medical Sciences [Grant R01GM114168]This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Long-range proton-coupled electron transfer in the Escherichia coli class Ia ribonucleotide reductase.

Escherichia coli class Ia ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to 2'-deoxynucleotides using a radical mechanism. Each turnover requires radical transfer from an assembled diferric tyrosyl radical (Y•) cofactor to the enzyme active site over 35 Å away. This unprecedented reaction occurs via an amino acid radical hopping pathway spanning two protein subunits. To study the mechanism of radical transport in RNR, a suite of biochemical approaches have been developed, such as site-directed incorporation of unnatural amino acids with altered electronic properties and photochemical generation of radical intermediates. The resulting variant RNRs have been investigated using a variety of time-resolved physical techniques, including transient absorption and stopped-flow UV-Vis spectroscopy, as well as rapid freeze-quench EPR, ENDOR, and PELDOR spectroscopic methods. The data suggest that radical transport occurs via proton-coupled electron transfer (PCET) and that the protein structure has evolved to manage the proton and electron transfer co-ordinates in order to prevent 'off-pathway' reactivity and build-up of oxidised intermediates. Thus, precise design and control over the factors that govern PCET is key to enabling reversible and long-range charge transport by amino acid radicals in RNR.

Modulation of the CO2 fixation in dinickel azacryptands.

While bimetallic azacryptands are known to selectively coordinate CO2, there is little knowledge on how different substitution patterns of the azacryptand cage structure influence CO2 coordination. Stopped-flow UV-vis spectroscopy, electrochemical analysis and DFT calculations were performed on a series of dinickel azacryptands and showed different rates of CO2 coordination to the complexes. We herein present data showing that the different flexibility of the azacryptands is directly responsible for the difference in the CO2 uptake capability of dinickel azacryptand complexes.

Reactivity of the copper(iii)-hydroxide unit with phenols.

Kinetic studies of the reactions of two previously characterized copper(iii)-hydroxide complexes (LCuOH and NO2 LCuOH, where L = N,N'-bis(2,6-diisopropylphenyl)-2,6-pyridine-dicarboxamide and NO2 L = N,N'-bis(2,6-diisopropyl-4-nitrophenyl)pyridine-2,6-dicarboxamide) with a series of para substituted phenols (XArOH where X = NMe2, OMe, Me, H, Cl, NO2, or CF3) were performed using low temperature stopped-flow UV-vis spectroscopy. Second-order rate constants (k) were determined from pseudo first-order and stoichiometric experiments, and follow the trends CF3 < NO2 < Cl < H < Me < OMe < NMe2 and LCuOH < NO2 LCuOH. The data support a concerted proton-electron transfer (CPET) mechanism for all but the most acidic phenols (X = NO2 and CF3), for which a more complicated mechanism is proposed. For the case of the reactions between NO2 ArOH and LCuOH in particular, competition between a CPET pathway and one involving initial proton transfer followed by electron transfer (PT/ET) is supported by multiwavelength global analysis of the kinetic data, formation of the phenoxide NO2 ArO- as a reaction product, observation of an intermediate [LCu(OH2)]+ species derived from proton transfer from NO2 ArOH to LCuOH, and thermodynamic arguments indicating that initial PT should be competitive with CPET.

A spectroscopic (stopped-flow UV–Vis and 1H NMR Evans method) and DFT thermodynamic study of the comproportionation reaction of [OsVIIIO4(OH)n]n− (n = 1, 2) and [OsVIO2(OH)4]2−

From a mole ratio 1H NMR Evans method experiment it is found that, in a 2.0M NaOH aqueous matrix, diamagnetic [OsVIIIO4(OH)n]n− (n=1, 2) (of d0 electron configuration) and trans-[OsVIO2(OH)4]2− species (d2) react in a 1:1 mol ratio to form two paramagnetic OsVII oxido/hydroxido product species (d1). This result is further validated as the chemical reaction model that best fitted stopped-flow UV–Vis spectroscopy kinetic data is given by OsVIII+OsVI⇌kCOMkCOM2OsVII. From non-linear least squares fits of stopped-flow UV–Vis spectroscopy kinetic traces the comproportionation reaction rate constants, activation energies (forward: ΔH‡(obs), ΔS‡(obs) and ΔG‡(obs) are 10.3±0.5kcalmol−1, −2.6±1.6calmol−1K−1 and 11.1±0.9kcalmol−1, respectively; and reverse are −6.7±1.0kcalmol−1, −63.6±3.4calmol−1K−1 and 12.2±2.0kcalmol−1 respectively) and standard reaction energies (ΔH°rxn(obs), ΔS°rxn(obs) and ΔG°rxn(obs) are 17.1±1.2kcalmol−1, 61.0±4.3calmol−1K−1 and −1.1±2.5kcalmol−1, respectively) at 298.15K were determined. Stopped-flow kinetic isotope experiments provide evidence that these redox reactions coincide with the transfer of a proton. A systematic DFT speciation study of OsVI and OsVII oxido/hydroxido complexes in a simulated aqueous phase (COSMO) yield that the thermodynamically most stable OsVI species is the singlet spin state trans-[OsVIO2(OH)4]2− complex and the thermodynamically most stable paramagnetic OsVII product species are a combination of trans-[OsVIIO3(OH)2]− and mer-[OsVIIO3(OH)3]2− species. Using the DFT results, the OsVI & OsVIII comproportionation reaction is now proposed to be [OsVIIIO4(OH)2]2− reacts with trans-[OsVIO2(OH)4]2− to yield two trans-[OsVIIO3(OH)2]− species and two hydroxide anions. The DFT-calculated standard reaction energies for this reaction at 298.15K (e.g. with the PBE functional, ΔH°rxn, ΔS°rxn and ΔG°rxn are 21.13kcalmol−1, 71.06calmol−1K−1 and −0.06kcalmol−1, respectively) compare exceptionally well with the obtained experimental data.

Creation of a Thermally Tolerant Peroxidase.

An artificial peroxidase with thermal tolerance and high catalytic activity has been successfully prepared by mutagenesis of an electron transfer protein, cytochrome c552 from Thermus thermophilus. The mutant enzymes were rationally designed based on the general peroxidase mechanism and spectroscopic analyses of an active intermediate formed in the catalytic reaction. Stopped flow UV-vis spectroscopy and EPR spectroscopy with a rapid freezing sample technique revealed that the initial double mutant, V49D/M69A, which was designed to reproduce the peroxidase mechanism, formed an active oxo-ferryl heme intermediate with a protein radical predominantly localized on Tyr45 during the catalytic reaction. The magnetic power saturation measurement obtained from EPR studies showed little interaction between the oxo-ferryl heme and the tyrosyl radical. Kinetics studies indicated that the isolated oxo-ferryl heme component in the active intermediate was a possible cause of heme degradation during the reaction with H2O2. Strong interaction between the oxo-ferryl heme and the radical was achieved by replacing Tyr45 with tryptophan (resulting in the Y45W/V49D/M69A mutant), which was similar to a tryptophanyl radical found in active intermediates of some catalase-peroxidases. Compared to the protein radical intermediates of V49D/M69A mutant, those of the Y45W/V49D/M69A mutant showed higher reactivity to an organic substrate than to H2O2. The Y45W/V49D/M69A mutant exhibited improved peroxidase activity and thermal tolerance.

Mechanistic Investigations of Water Oxidation by a Molecular Cobalt Oxide Analogue: Evidence for a Highly Oxidized Intermediate and Exclusive Terminal Oxo Participation.

Artificial photosynthesis (AP) promises to replace society's dependence on fossil energy resources via conversion of sunlight into sustainable, carbon-neutral fuels. However, large-scale AP implementation remains impeded by a dearth of cheap, efficient catalysts for the oxygen evolution reaction (OER). Cobalt oxide materials can catalyze the OER and are potentially scalable due to the abundance of cobalt in the Earth's crust; unfortunately, the activity of these materials is insufficient for practical AP implementation. Attempts to improve cobalt oxide's activity have been stymied by limited mechanistic understanding that stems from the inherent difficulty of characterizing structure and reactivity at surfaces of heterogeneous materials. While previous studies on cobalt oxide revealed the intermediacy of the unusual Co(IV) oxidation state, much remains unknown, including whether bridging or terminal oxo ligands form O2 and what the relevant oxidation states are. We have addressed these issues by employing a homogeneous model for cobalt oxide, the [Co(III)4] cubane (Co4O4(OAc)4py4, py = pyridine, OAc = acetate), that can be oxidized to the [Co(IV)Co(III)3] state. Upon addition of 1 equiv of sodium hydroxide, the [Co(III)4] cubane is regenerated with stoichiometric formation of O2. Oxygen isotopic labeling experiments demonstrate that the cubane core remains intact during this stoichiometric OER, implying that terminal oxo ligands are responsible for forming O2. The OER is also examined with stopped-flow UV-visible spectroscopy, and its kinetic behavior is modeled, to surprisingly reveal that O2 formation requires disproportionation of the [Co(IV)Co(III)3] state to generate an even higher oxidation state, formally [Co(V)Co(III)3] or [Co(IV)2Co(III)2]. The mechanistic understanding provided by these results should accelerate the development of OER catalysts leading to increasingly efficient AP systems.

Strong Inhibition of O-Atom Transfer Reactivity for Mn(IV)(O)(π-Radical-Cation)(Lewis Acid) versus Mn(V)(O) Porphyrinoid Complexes.

The oxygen atom transfer (OAT) reactivity of two valence tautomers of a Mn(V)(O) porphyrinoid complex was compared. The OAT kinetics of Mn(V)(O)(TBP8Cz) (TBP8Cz = octakis(p-tert-butylphenyl)corrolazinato(3-)) reacting with a series of triarylphosphine (PAr3) substrates were monitored by stopped-flow UV-vis spectroscopy, and revealed second-order rate constants ranging from 16(1) to 1.43(6) × 10(4) M(-1) s(-1). Characterization of the OAT transition state analogues Mn(III)(OPPh3)(TBP8Cz) and Mn(III)(OP(o-tolyl)3)(TBP8Cz) was carried out by single-crystal X-ray diffraction (XRD). A valence tautomer of the closed-shell Mn(V)(O)(TBP8Cz) can be stabilized by the addition of Lewis and Brønsted acids, resulting in the open-shell Mn(IV)(O)(TBP8Cz(•+)):LA (LA = Zn(II), B(C6F5)3, H(+)) complexes. These Mn(IV)(O)(π-radical-cation) derivatives exhibit dramatically inhibited rates of OAT with the PAr3 substrates (k = 8.5(2) × 10(-3) - 8.7 M(-1) s(-1)), contrasting the previously observed rate increase of H-atom transfer (HAT) for Mn(IV)(O)(TBP8Cz(•+)):LA with phenols. A Hammett analysis showed that the OAT reactivity for Mn(IV)(O)(TBP8Cz(•+)):LA is influenced by the Lewis acid strength. Spectral redox titration of Mn(IV)(O)(TBP8Cz(•+)):Zn(II) gives Ered = 0.69 V vs SCE, which is nearly +700 mV above its valence tautomer Mn(V)(O)(TBP8Cz) (Ered = -0.05 V). These data suggest that the two-electron electrophilicity of the Mn(O) valence tautomers dominate OAT reactivity and do not follow the trend in one-electron redox potentials, which appear to dominate HAT reactivity. This study provides new fundamental insights regarding the relative OAT and HAT reactivity of valence tautomers such as M(V)(O)(porph) versus M(IV)(O)(porph(•+)) (M = Mn or Fe) found in heme enzymes.

Kinetic and structural studies, origins of selectivity, and interfacial charge transfer in the artificial photosynthesis of CO

The effective design of an artificial photosynthetic system entails the optimization of several important interactions. Herein we report stopped-flow UV-visible (UV-vis) spectroscopy, X-ray crystallographic, density functional theory (DFT), and electrochemical kinetic studies of the Re(bipy-tBu)(CO)(3)(L) catalyst for the reduction of CO(2) to CO. A remarkable selectivity for CO(2) over H(+) was observed by stopped-flow UV-vis spectroscopy of [Re(bipy-tBu)(CO)(3)](-1). The reaction with CO(2) is about 25 times faster than the reaction with water or methanol at the same concentrations. X-ray crystallography and DFT studies of the doubly reduced anionic species suggest that the highest occupied molecular orbital (HOMO) has mixed metal-ligand character rather than being purely doubly occupied d(z)(2), which is believed to determine selectivity by favoring CO(2) (σ + π) over H(+) (σ only) binding. Electrocatalytic studies performed with the addition of Brönsted acids reveal a primary H/D kinetic isotope effect, indicating that transfer of protons to Re -CO(2) is involved in the rate limiting step. Lastly, the effects of electrode surface modification on interfacial electron transfer between a semiconductor and catalyst were investigated and found to affect the observed current densities for catalysis more than threefold, indicating that the properties of the electrode surface need to be addressed when developing a homogeneous artificial photosynthetic system.

Encapsulation of hemoglobin inside liposomes surface conjugated with poly(ethylene glycol) attenuates their reactions with gaseous ligands and regulates nitric oxide dependent vasodilation

Acellular hemoglobin (Hb)-based O2 carriers (HBOCs) are being investigated as red blood cell (RBC) substitutes for use in transfusion medicine. However, commercial acellular HBOCs elicit both vasoconstriction and systemic hypertension which hampers their clinical use. In this study, it is hypothesized that encapsulation of Hb inside the aqueous core of liposomes should regulate the rates of NO dioxygenation and O2 release, which should in turn regulate its vasoactivity. To test this hypothesis, poly(ethylene glycol) (PEG) conjugated liposome-encapsulated Hb (PEG-LEHs) dispersions were prepared using human and bovine Hb. In this study, the rate constants for O2 dissociation, CO association, and NO dioxygenation were measured for free Hb and PEG-LEH dispersions using stopped-flow UV-visible spectroscopy, while vasoactivity was assessed in rat aortic ring strips using both endogenous and exogenous sources of NO. It was observed that PEG-LEH dispersions had lower O2 release and NO dioxygenation rate constants compared with acellular Hbs. However, no difference was observed in the CO association rate constants between free Hb and PEG-LEH dispersions. Furthermore, it was observed that Hb encapsulation inside vesicles prevented Hb dependent inhibition of NO-mediated vasodilation. In addition, the magnitude of the vasoconstrictive effects of Hb and PEG-LEH dispersions correlated with their respective rates of NO dioxygenation and O2 release. Overall, this study emphasizes the pivotal role Hb encapsulation plays in regulating gaseous ligand binding/release kinetics and the vasoactivity of Hb.

Reaction Mechanism of Heme-Based Dioxygenases

Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) are two heme-containing enzymes that catalyze the oxidative cleavage of tryptophan (Trp) to N-formyl kynurenine (NFK), the initial and rate-limiting step of the kynurenine pathway. Until recently it was generally believed that the heme dioxygenase reaction follows a base-catalyzed mechanism. Based on this mechanism, the reaction is initiated by deprotonation of the indoleamine group of Trp by an active site base. It is followed by electrophilic addition of the heme-bound dioxygen to the C2=C3 bond of the indole moiety of Trp, leading to a heme-bound 3-indolenylperoxo intermediate, which subsequently converts to the product NFK, via a dioxetane intermediate or a Criegee type of rearrangement. In this work, we sought to use continuous-flow resonance Raman spectroscopy, combined with stopped-flow UV-Vis spectroscopy, to investigate the dioxygenase reaction carried out by IDO and TDO. Surprisingly, a ferryl intermediate was detected during the IDO reaction at 0.2 s. The presence of this intermediate supports a new mechanism, in which the two atoms of dioxygen are sequentially incorporated into the substrate via a two-step reaction. The ferryl intermediate is not observable during the TDO reaction, highlighting the structural differences between the two types of dioxygenases, as well as the importance of the stereoelectronic factors in modulating the reactions.

Nitric oxide dioxygenation reaction in DevS and the initial response to nitric oxide in Mycobacterium tuberculosis.

DevS and DosT from Mycobacterium tuberculosis (MTB) are paralogous heme-based sensor kinases that respond to hypoxia and to low concentrations of nitric oxide (NO). Both proteins work with the response regulator DevR as a two-component regulatory system to induce the dormancy regulon in MTB. While DevS and DosT are inactive when dioxygen is bound to the heme Fe(II) at their sensor domain, autokinase activity is observed in their heme Fe(II)-NO counterparts. To date, the conversion between active and inactive states and the reactivity of the heme-oxy complex toward NO have not been investigated. Here, we use stopped-flow UV-vis spectroscopy and rapid freeze quench resonance Raman spectroscopy to probe these reactions in DevS. Our data reveal that the heme-O(2) complex of DevS reacts efficiently with NO to produce nitrate and the oxidized Fe(III) heme through an NO dioxygenation reaction that parallels the catalytic reactions of bacterial flavohemoglobin and truncated hemoglobins. Autophosphorylation activity assays show that the Fe(III) heme state of DevS remains inactive but exhibits a high affinity for NO and forms an Fe(III)-NO complex that is readily reduced by ascorbate, a mild reducing agent. On the basis of these results, we conclude that upon exposure to low NO concentrations, the inactive oxy-heme complex of DevS is rapidly converted to the Fe(II)-NO complex in the reducing environment of living cells and triggers the initiation of dormancy.

Ascorbate peroxidase activity of cytochrome c

The peroxidase-type reactivity of cytochrome c is proposed to play a role in free radical production and/or apoptosis. This study describes cytochrome c catalysis of peroxide consumption by ascorbate. Under conditions where the sixth coordination position at the cytochrome c heme iron becomes more accessible for exogenous ligands (by carboxymethylation, cardiolipin addition or by partial denaturation with guanidinium hydrochloride) this peroxidase activity is enhanced. A reaction intermediate is detected by stopped-flow UV-vis spectroscopy upon reaction of guanidine-treated cytochrome c with peroxide, which resembles the spectrum of globin Compound II species and is thus proposed to be a ferryl species. The ability of physiological levels of ascorbate (10–60 µM) to interact with this species may have implications for mechanisms of cell signalling or damage that are based on cytochrome c/peroxide interactions.

Spectroscopic Probes of the Reactive Intermediates of Dehaloperoxidase from Amphitrite ornata

The enzyme intermediates of dehaloperoxidase (DHP) from the marine worm Amphitrite ornata are unique within both the globin and cytochrome c peroxidase superfamilies. DHP has been shown to oxidize trihalophenols to dihaloquinones in a dehalogenation reaction that uses hydrogen peroxide as a substrate. We show that the initially formed heme intermediate in this reaction is not Compound I as is often the case in peroxidases, but rather is a combination of Compound II and a tyrosyl radical that has similarity to the Compound ES intermediate of cytochrome c peroxidase. Using stopped-flow UV-visible spectroscopy, we provide a detailed kinetic description for the reaction of pre-formed DHP Compound II and tyrosyl radical with the substrate 2,4,6-trichlorophenol, and demonstrate the catalytic competency of this intermediate in generating the product 2,4-dichloroquinone. Furthermore, using rapid-freeze-quench electron paramagnetic resonance spectroscopy, we detected a signal at g ≈ 2.0058, confirming the presence of a protein radical in DHP Compound II, and assign it as a tyrosyl radical based upon mutagenesis studies and structural arguments. In the absence of a halophenol substrate, the DHP Compound II + tyrosyl radical intermediate decomposes to a new and significantly less active species, termed Compound RH, which is unique to dehaloperoxidase. We propose that this intermediate plays a protective role against heme bleaching. While unreactive toward further oxidation, Compound RH can be reduced and subsequently bind dioxygen, generating oxyferrous DHP, which may represent the catalytic link between the peroxidase and oxygen-transport activities in this bifunctional protein.

Oxygen Evolution Catalysis by a Dimanganese Complex and Its Relation to Photosynthetic Water Oxidation

[Mn2(III/IV)(mu-O) 2(terpy)2(OH 2)2](NO3)3 (1, where terpy = 2,2':6'2''-terpyridine) acts as a water-oxidation catalyst with HSO5(-) as the primary oxidant in aqueous solution and, thus, provides a model system for the oxygen-evolving complex of photosystem II (Limburg, J.; et al. J. Am. Chem. Soc. 2001, 123, 423-430). The majority of the starting [Mn2(III/IV)(mu-O)2](3+) complex is converted to the[Mn2(IV/IV)(mu-O)2](4+) form (2) during this reaction (Chen, H.; et al. Inorg. Chem. 2007, 46, 34-43). Here, we have used stopped-flow UV-visible spectroscopy to monitor UV-visible absorbance changes accompanying the conversion of 1 to 2 by HSO5(-). With excess HSO5(-), the rate of absorbance change was found to be first-order in [1] and nearly zero-order in [HSO5(-)]. At relatively low [HSO5(-)], the change of absorbance with time is distinctly biphasic. The observed concentration dependences are interpreted in terms of a model involving the two-electron oxidation of 1 by HSO5(-), followed by the rapid reaction of the two-electron-oxidized intermediate with another molecule of 1 to give two molecules of 2. In order to rationalize biphasic behavior at low [HSO5(-)], we propose a difference in reactivity of the [Mn2(III/)(IV)(mu-O)2](3+) complex upon binding of HSO5(-) to the Mn(III) site as compared to the reactivity upon binding HSO5(-) to the Mn(IV) site. The kinetic distinctness of the Mn(III) and Mn(IV) sites allows us to estimate upper limits for the rates of intramolecular electron transfer and terminal ligand exchange between these sites. The proposed mechanism leads to insights on the optimization of 1 as a water-oxidation catalyst. The rates of terminal ligand exchange and electron transfer between oxo-bridged Mn atoms in the oxygen-evolving complex of photosystem II are discussed in light of these results.

Intermolecular versus intramolecular electron-/atom- (Cl [rad]) transfer in heme-iron and copper pyridylalkylamine complexes

While outer-sphere electron-transfer reactions come with a firm experimental and theoretical basis, less is known about the redox reactions occurring by atom-transfer. In the present study, relevant reactions occur upon mixing, (F 8)Fe III(Cl) (F 8 is tetrakis(2,6-difluorophenyl)porphyrinate) with a series of [(L)Cu I] + complexes (L are tripodal tetradentate pyridylalkylamine ligands varying in effective chelate ring sizes: TMPA, PMEA, PMAP) to form (F 8)Fe II and [(L)Cu II(Cl)] +. The electron-/atom- (Cl ) transfer process is characterized by electrochemical measurements as well as UV–Vis, 1H NMR, and EPR spectroscopies. Stopped-flow UV–Vis spectroscopy in THF indicate the following relative rates ( k obs) [Cu I(pmea)] + > [Cu I(pmap)] + > [Cu I(tmpa)(thf)] +. However, the redox potentials as related to Δ G ∘ for the reaction, [ E 1/2(oxidant) − E 1/2(reductant)], predict the trend [Cu I(tmpa)(CH 3CN)] + > [Cu I(pmea)] + > [Cu I(pmap)] +. A detailed description of Cu I-to-Cu II structural changes is provided and these likely influence the observed [(L)Cu I] +/(F 8)Fe III(Cl) reaction rates. Analogous chemistry is described for a heme–copper system utilizing a heterobinucleating ligand ( 6L), which is comprised of a TMPA like moiety tethered at the 6-position of one of the pyridyl donors to a F 8-like heme. Kinetic/mechanistic insights were obtained from transient absorption spectroscopic monitoring in CH 3CN following photoejection of carbon monoxide from [( 6L)Fe II(CO)⋯Cu II(Cl)].

Pre-steady-state and steady-state kinetic analysis of E. coli class I ribonucleotide reductase.

E. coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates (NDPs) to dNDPs and is composed of two homodimeric subunits: R1 and R2. R1 binds NDPs and contains binding sites for allosteric effectors that control substrate specificity and turnover rate. R2 contains a diiron-tyrosyl radical (Y(*)) cofactor that initiates nucleotide reduction. Pre-steady-state experiments with wild type R1 or C754S/C759S-R1 and R2 were carried out to determine which step(s) are rate-limiting and whether both active sites of R1 can catalyze nucleotide reduction. Rapid chemical quench experiments monitoring dCDP formation gave k(obs) of 9 +/- 4 s(-1) with an amplitude of 1.7 +/- 0.4 equiv. This amplitude, generated in experiments with pre-reduced R1 (3 or 15 microM) in the absence of reductant, indicates that both monomers of R1 are active. Stopped-flow UV-vis spectroscopy monitoring the concentration of the Y(*) failed to reveal any changes from 2 ms to seconds under similar conditions. These pre-steady-state experiments, in conjunction with the steady-state turnover numbers for dCDP formation of 2-14 s(-1) at RNR concentrations of 0.05-0.4 microM (typical assay conditions), reveal that the rate-determining step is a physical step prior to rapid nucleotide reduction and rapid tyrosine reoxidation to Y(*). Steady-state experiments conducted at RNR concentrations of 3 and 15 microM, typical of pre-steady-state conditions, suggest that, in addition to the slow conformational change(s) prior to chemistry, re-reduction of the active site disulfide to dithiol or a conformational change accompanying this process can also be rate-limiting.