Stopped-flow Spectroscopy Research Articles

A Long‐Lived Halogen‐Bonding Anion Triple Helicate Accommodates Rapid Guest Exchange

Anion-templated helical structures are emerging as a dynamic and tractable class of supramolecules that exhibit anion-switchable self-assembly. We present the first kinetic studies of an anion helicate by utilizing halogen-bonding m-arylene-ethynylene oligomers. These ligands formed high-fidelity triple helicates in solution with surprisingly long lifetimes on the order of seconds even at elevated temperatures. We propose an associative ligand-exchange mechanism that proceeded slowly on the same timescale. In contrast, intrachannel anion exchange occurred rapidly within milliseconds or faster as determined by stopped-flow visible spectroscopy. Additionally, the helicate accommodated bromide in solution and the solid state, while the thermodynamic stability of the triplex favored larger halide ions (bromide≈iodide≫chloride). Taken together, we elucidate a new class of kinetically stable helicates. These anion-switchable triplexes maintain their architectures while accommodating fast intrachannel guest exchange.

InhA, the enoyl-thioester reductase from Mycobacterium tuberculosis forms a covalent adduct during catalysis

The enoyl-thioester reductase InhA catalyzes an essential step in fatty acid biosynthesis of Mycobacterium tuberculosis and is a key target of antituberculosis drugs to combat multidrug-resistant M. tuberculosis strains. This has prompted intense interest in the mechanism and intermediates of the InhA reaction. Here, using enzyme mutagenesis, NMR, stopped-flow spectroscopy, and LC–MS, we found that the NADH cofactor and the CoA thioester substrate form a covalent adduct during the InhA catalytic cycle. We used the isolated adduct as a molecular probe to directly access the second half-reaction of the catalytic cycle of InhA (i.e. the proton transfer), independently of the first half-reaction (i.e. the initial hydride transfer) and to assign functions to two conserved active-site residues, Tyr-158 and Thr-196. We found that Tyr-158 is required for the stereospecificity of protonation and that Thr-196 is partially involved in hydride transfer and protonation. The natural tendency of InhA to form a covalent C2-ene adduct calls for a careful reconsideration of the enzyme's reaction mechanism. It also provides the basis for the development of effective tools to study, manipulate, and inhibit the catalytic cycle of InhA and related enzymes of the short-chain dehydrogenase/reductase (SDR) superfamily. In summary, our work has uncovered the formation of a covalent adduct during the InhA catalytic cycle and identified critical residues required for catalysis, providing further insights into the InhA reaction mechanism important for the development of antituberculosis drugs.

Kaempferol Binding to Zinc(II), Efficient Radical Scavenging through Increased Phenol Acidity.

Zinc(II) enhances radical scavenging of the flavonoid kaempferol (Kaem) most significantly for the 1:1 Zn(II)-Kaem complex in equilibrium with the 1:2 Zn(II)-Kaem complex both with high affinity at 3-hydroxyl and 4-carboxyl coordination. In methanol/chloroform (7/3, v/v), 1:1 Zn(II)-Kaem complex reduces β-carotene radical cation, β-Car•+, with a second-order rate constant, 1.88 × 108 L·mol-1·s-1, while both Kaem and 1:2 Zn(II)-Kaem complex are nonreactive, as determined by laser flash photolysis. In ethanol, 1:1 Zn(II)-Kaem complex reduces the 2,2-diphenyl-1-picrylhydrazyl radical, DPPH•, with a second-order rate constant, 2.48 × 104 L·mol-1·s-1, 16 times and 2 times as efficient as Kaem and 1:2 Zn(II)-Kaem complex, respectively, as determined by stopped-flow spectroscopy. Density functional theory calculation results indicate significantly increased acidity of Kaem as ligand in 1:1 Zn(II)-Kaem complex other than in 1:2 Zn(II)-Kaem complex. Kaem in 1:1 Zn(II)-Kaem complex loses two protons (one from 3-hydroxyl and one from phenolic hydroxyl) forming 1:1 Zn(II)-(Kaem-2H) during binding with Zn(II), while Kaem in 1:2 Zn(II)-Kaem complex loses one proton in each ligand forming Zn(II)-(Kaem-H)2, as confirmed by UV-vis absorption spectroscopy. Zn(II)-(Kaem-2H) is a far stronger reductant than Kaem and Zn(II)-(Kaem-H)2 as determined by cyclic voltammetry. Significant rate increases for the 1:1 complex in both β-Car•+ scavenging by electron transfer and DPPH• scavenging by hydrogen atom transfer were ascribed to decreases of ionization potential and of bond dissociation energy of 4'-OH for deprotonated Zn(II)-(Kaem-2H), respectively. Increased phenol acidity of plant polyphenols by 1:1 coordination with Zn(II) may explain the unique function of Zn(II) as a biological antioxidant and may help to design nontoxic metal-based drugs derived from natural bioactive molecules.

Reconstitution of a thermophilic Cu+ importer in vitro reveals intrinsic high-affinity slow transport driving accumulation of an essential metal ion

Acquisition of the trace element copper (Cu) is critical to drive essential eukaryotic processes such as oxidative phosphorylation, iron mobilization, peptide hormone biogenesis, and connective tissue maturation. The Ctr1/Ctr3 family of Cu importers, first discovered in fungi and conserved in mammals, are critical for Cu+ movement across the plasma membrane or mobilization from endosomal compartments. Whereas ablation of Ctr1 in mammals is embryonic lethal, and Ctr1 is critical for dietary Cu absorption, cardiac function, and systemic iron distribution, little is known about the intrinsic contribution of Ctr1 for Cu+ permeation through membranes or its mechanism of action. Here, we identify three members of a Cu+ importer family from the thermophilic fungus Chaetomium thermophilum: Ctr3a and Ctr3b, which function on the plasma membrane, and Ctr2, which likely functions in endosomal Cu mobilization. All three proteins drive Cu and isoelectronic silver (Ag) uptake in cells devoid of Cu+ importers. Transport activity depends on signature amino acid motifs that are conserved and essential for all Ctr1/3 transporters. Ctr3a is stable and amenable to purification and was incorporated into liposomes to reconstitute an in vitro Ag+ transport assay characterized by stopped-flow spectroscopy. Ctr3a has intrinsic high-affinity metal ion transport activity that closely reflects values determined in vivo, with slow turnover kinetics. Given structural models for mammalian Ctr1, Ctr3a likely functions as a low-efficiency Cu+ ion channel. The Ctr1/Ctr3 family may be tuned to import essential yet potentially toxic Cu+ ions at a slow rate to meet cellular needs, while minimizing labile intracellular Cu+ pools.

Spin-Doctoring Cobalt Redox Shuttles for Dye-Sensitized Solar Cells.

A new low-spin (LS) cobalt(II) outer-sphere redox shuttle (OSRS) [Co(PY5Me2)(CN)]+, where PY5Me2 represents the pentadentate ligand 2,6-bis(1,1-bis(2-pyridyl)ethyl)pyridine, has been synthesized and characterized for its potential application in dye-sensitized solar cells (DSSCs). Introduction of the strong field CN- ligand into the open axial coordination site forced the cobalt(II) complex, [Co(PY5Me2)(CN)]+, to become LS based upon the complex's magnetic susceptibility (1.91 ± 0.02 μB), determined by the Evans method. Interestingly, dimerization and subsequent cobalt hexacyanide cluster formation of the [Co(PY5Me2)(CN)]+ monomer was observed upon long-term solvent exposure or addition of a supporting electrolyte for electrochemical characterization. Although long-term stability of the [Co(PY5Me2)(CN)]+ complex made it difficult to fabricate liquid electrolytes for DSSC applications, short-term stability in neat solvent afforded the opportunity to isolate the self-exchange kinetics of [Co(PY5Me2)(CN)]2+/+ via stopped-flow spectroscopy. Use of Marcus theory provided a smaller than expected self-exchange rate constant of 20 ± 5.5 M-1 s-1 for [Co(PY5Me2)(CN)]2+/+, which we attribute to a Jahn-Teller effect observed from the collected monomer crystallographic data. When compared side-by-side to cobalt tris(2,2'-bipyridine), [Co(bpy)3]3+, DSSCs employing [Co(PY5Me2)(CN)]2+ are expected to achieve superior charge collection, which result from a smaller rate constant, ket, for recombination based upon simple dark J- E measurements of the two redox shuttles. Given the negative redox potential (0.254 V vs NHE) of [Co(PY5Me2)(CN)]2+/+ and the slow recombination kinetics, [Co(PY5Me2)(CN)]2+/+ becomes an attractive OSRS to regenerate near IR absorbing sensitizers in solid-state DSSC devices.

Synthesis, characterization and biological activity of copper(II) complexes with ligands derived from β-amino acids

Two copper(II) complexes with ligands derived from β-amino acids, 2-(1-aminocyclohexyl)acetic acid L1 and 2-(1-amino-4-(tert-butyl)cyclohexyl)acetic acid L2, were synthesized and characterized by microanalysis, infrared, UV–Vis and EPR spectra. The spectroscopically predicted structure of the square-planar copper(II) complex with 2-(1-aminocyclohexyl)-acetic acid C1 was confirmed by single-crystal X-ray analysis. The biological activities (antitumor activities and interaction with DNA) of the compounds were also investigated. The interactions of both complexes with calf thymus (CT) and herring testes (HT) DNA were examined by stopped-flow spectroscopy, by absorption (UV–Vis) and by emission spectral studies (ethidium bromide displacement studies). Both complexes were found to react a bit faster with HT-DNA than with CT-DNA. The obtained binding constants suggested a moderate intercalative binding mode between the complexes and DNA. In addition, fluorescence spectrometry of bovine serum albumin with the complexes showed a good fluorescence quenching of the complexes. The obtained copper(II) complexes have a relatively low cytotoxic effect on murine mammary carcinoma cell line, 4T1, a moderate effect on murine colon carcinoma cell line, CT26, and a relatively high cytotoxicity toward murine lung cancer cells, LLC1.

Cytochrome b5 enhances androgen synthesis by rapidly reducing the CYP17A1 oxy-complex in the lyase step.

Cytochrome P450 17A1 (CYP17A1) catalyzes the synthesis of androgens from the steroid precursors pregnenolone and progesterone in a two-step reaction process: allylic hydroxylation and carbo-carbon bond scission. Cytochrome b5 (Cyt-b5 ) is a stimulator of the second lyase reaction, but the chemical mechanism is unclear. We have shown previously that this stimulatory effect requires redox active Cyt-b5 . To investigate the origin of the lyase reaction enhancement by electron transfer from Cyt-b5 , we measured the reduction rates of oxy-ferrous substrate-bound CYP17A1 by Cyt-b5 and by cytochrome P450 reductase (CPR) coincorporated in Nanodiscs using stopped flow spectroscopy. We observed that Cyt-b5 reduces oxy-ferrous CYP17A1 10-fold faster than CPR, with the rate similar to that observed in a ternary complex of all three proteins.

P-376 - Crosstalk between gasotransmitters at the H2S-synthesizing human cystathionine β-synthase: heme-based regulation by CO and NO

Cystathionine β-synthase (CBS) plays a central role in hydrogen sulfide (H2S) metabolism in human (patho) physiology. Each CBS monomer is composed of a central catalytic domain containing a pyridoxal 5’-phosphate active site flanked by two regulatory domains: an N-terminal domain harboring a b-type heme, and a C-terminal S-adenosyl-L-methionine (AdoMet)-binding domain. CBS is intricately regulated at different levels, its function depending on the redox state and availability of exogenous ligands, such as AdoMet and the gasotransmitters nitric oxide (NO) and carbon monoxide (CO). Herein, we employed static and stopped-flow absorption spectroscopy to investigate the kinetics of CO and NO binding to the CBS heme. NO binds faster and with higher affinity than CO, each gasotransmitter initially binding to opposite sides of the heme plane. AdoMet surprisingly enhances CO inhibition of H2S production, as well as CO and NO binding. In the clinically relevant p.P49L CBS variant, we observed much higher CO affinity with respect to WT CBS, likely resulting from increased flexibility of the heme surroundings. Overall, the binding kinetics attest the in vivo relevance of CO and NO inhibition of CBS-catalyzed H2S production, an underlying mechanism in the crosstalk between gasotransmitters.

Bifunctionality of Iminodiacetic Acid-Modified Lysozyme on Inhibiting Zn2+-Mediated Amyloid β-Protein Aggregation.

Aggregation of amyloid β-proteins (Aβ) mediated by metal ions such as Zn2+ has been suggested to be implicated in the progression of Alzheimer's disease (AD). Hence, development of bifunctional agents capable of inhibiting Aβ aggregation and modulating metal-Aβ species is an effective strategy for the treatment of AD. In this work, we modified iminodiacetic acid (IDA) onto human lysozyme (hLys) surface to create an inhibitor of Zn2+-mediated Aβ aggregation and cytotoxicity. The IDA-modified hLys (IDA-hLys) retained the stability and biocompatibility of native hLys. Extensive biophysical and biological analyses indicated that IDA-hLys significantly attenuated Zn2+-mediated Aβ aggregation and cytotoxicity due to its strong binding affinity for Zn2+, whereas native hLys showed little effect. Stopped-flow fluorescence spectroscopy showed that IDA-hLys could protect Aβ from Zn2+-induced aggregation and rapidly depolymerize Zn2+-Aβ aggregates. The research indicates that IDA-hLys is a bifunctional agent capable of inhibiting Aβ fibrillization and modulating Zn2+-mediated Aβ aggregation and cytotoxicity as a strong Zn2+ chelator.

PABP and eIF4B Effects Binding Affinity and Kinetic Rates of Genome Linked Viral Protein (VPg) with Eukaryotic Initiation Factor 4F

Translation initiation is a critical early step in the viral replication cycle. Interaction of eukaryotic translational initiation factors with VPg of Turnip mosaic virus (TuMV) plays an important role in the cap‐independent initiation of protein synthesis. To understand this process in potyviruses, we investigated the equilibrium binding, thermodynamic parameters and rate constants for the interaction of VPg with eIF4F in the presence of PABP and eIF4B using fluorescence and stopped‐flow spectroscopy. Equilibrium studies showed that PABP increased VPg binding to eIF4F about 3‐fold; however, both PABP and eIF4B together enhanced VPg binding to eIF4F about 4‐fold, showing an additive effect rather than the large increase in affinity shown for cap binding. Temperature dependent studies showed the increase in VPg binding to eIF4F in the presence of PABP and eIF4B was enthalpy‐driven and entropy‐favorable. PABP and eIF4B decreased the entropic contribution for VPg binding to eIF4F. The decrease in entropy for the formation of eIF4F·4Bmiddot;PABP‐VPg complex suggested weakened hydrophobic interactions for complex formation and an overall conformational change. Rate constant for eIF4F interaction with VPg in the presence of PABP and eIF4B was 4‐fold faster; and dissociation rate was 3‐fold slower as compared to eIF4F alone. The temperature dependence of the VPg binding to eIF4F was significantly reduced in activation energy in the presence of PABP and eIF4B. When both PABP and eIF4B were present, not only was the energy barrier reduced but the binding rate was faster and dissociation rate was slower for the formation of eIF4F‐4B‐PABP‐VPg complex suggests more stable platform for further initiation complex formation. These results further support the importance of PABP and eIF4B interaction to eIF4F with VPg for efficient viral translation.Support or Funding InformationNational Science Foundation MCB‐1513737 (to D.J.G.) and Alfaisal University Research Support IRG‐16414 (to M.A.K.)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

“Thermodynamic Analysis of ATP Dissociation in a DEAD‐Box Protein”

RNA plays an essential role in many cellular functions. Similar to proteins, RNA function is strictly determined by the overall structure and dynamics of the RNA macromolecule. Thus, many cellular proteins or accessory factors are associated with RNA folding. One example of these RNA binding proteins are DEAD‐Box proteins. DEAD‐Box proteins utilize ATP hydrolysis to unwind and reanneal RNA duplexes to promote correct folding during RNA processing. DEAD‐Box proteins possess two conserved RecA‐like domains that bind ATP and RNA. In addition, these proteins also contain N‐ and/or C‐ terminal domains (NTD and CTD, respectively). Peripheral domains in DEAD‐Box proteins are proposed to regulate ATP hydrolysis as well as confer RNA specificity. Thus, the NTD and/or CTD can alter the ATP binding pocket and subsequently alter ATP association and/or dissociation. For example, Rok1p, a yeast DEAD‐Box protein, contains both a NTD and a CTD. The NTD in Rok1p is suggested to affect the structure and stability of the protein. Therefore, the dissociation rate of a fluorescence ATP analog was determined through transient kinetics and stopped‐flow spectroscopy under various thermal conditions. Resulting trends conform to double exponential fits suggesting a two‐step mechanism. The observed slow rate was consistent with a conformation change in the protein that allows rapid dissociation. The rate in which the conformation change occurs was observed to be temperature dependent. The current hypothesis centers on the ability, of the Rok1p peripheral domains, to affect the slow structural change needed for ATP dissociation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

Myeloperoxidase-catalyzed oxidation of cyanide to cyanate: A potential carbamylation route involved in the formation of atherosclerotic plaques?

Protein carbamylation by cyanate is a post-translational modification associated with several (patho)physiological conditions, including cardiovascular disorders. However, the biochemical pathways leading to protein carbamylation are incompletely characterized. This work demonstrates that the heme protein myeloperoxidase (MPO), which is secreted at high concentrations at inflammatory sites from stimulated neutrophils and monocytes, is able to catalyze the two-electron oxidation of cyanide to cyanate and promote the carbamylation of taurine, lysine, and low-density lipoproteins. We probed the role of cyanide as both electron donor and low-spin ligand by pre-steady-state and steady-state kinetic analyses and analyzed reaction products by MS. Moreover, we present two further pathways of carbamylation that involve reaction products of MPO, namely oxidation of cyanide by hypochlorous acid and reaction of thiocyanate with chloramines. Finally, using an in vivo approach with mice on a high-fat diet and carrying the human MPO gene, we found that during chronic exposure to cyanide, mimicking exposure to pollution and smoking, MPO promotes protein-bound accumulation of carbamyllysine (homocitrulline) in atheroma plaque, demonstrating a link between cyanide exposure and atheroma. In summary, our findings indicate that cyanide is a substrate for MPO and suggest an additional pathway for in vivo cyanate formation and protein carbamylation that involves MPO either directly or via its reaction products hypochlorous acid or chloramines. They also suggest that chronic cyanide exposure could promote the accumulation of carbamylated proteins in atherosclerotic plaques.

Toward Understanding the Mechanism of Calcium-Inhibited Membrane Binding of the SLP-2 C2A Domain

Synaptotagmin-like proteins (Slps) are membrane-binding proteins that play key roles in trafficking of large dense-core vesicles for exocytosis. In particular, Slp-2 is responsible for docking hormone secretory vesicles to sites of exocytosis in pancreatic alpha cells and other cell types. Like all Slp family members, Slp-2 contains an N-terminal Slp-homology domain, a linker region of undefined structure, and two C-terminal membrane-binding C2 domains (C2A and C2B). The C2A domain of Slp-2 has been shown to have a unique response to Ca2+ ions, in that it binds anionic liposome membranes in the absence of Ca2+ but dissociates upon addition of sufficient Ca2+. This Ca2+ dependence contrasts with most other C2 domains, which bind lipid membranes in either a Ca2+-dependent or Ca2+-independent fashion. To investigate the mechanism of this unusual Ca2+ inhibition, a series of biophysical and spectroscopic measurements has been completed. First, the affinity of the well-purified C2A domain for liposomes containing different compositions of key anionic lipids was measured using steady-state and stopped-flow fluorescence spectroscopy. Next, the effect of Ca2+ on C2A domain binding to each of the liposome compositions was measured. It was found that Ca2+ does inhibit membrane binding, albeit at significantly higher concentrations than previously reported. Although the protein-liposome affinity varies by >10-fold among the different lipid compositions tested, the Ca2+ concentration required for inhibition varies relatively little, suggesting a nonspecific mechanism. Current efforts are directed toward quantifying Ca2+ and other metal binding affinities using a luminescence-based Tb3+ displacement assay, and measuring effects of Ca2+ and lipids on protein structure using EPR and NMR.

Mutual synergy between catalase and peroxidase activities of the bifunctional enzyme KatG is facilitated by electron hole-hopping within the enzyme

KatG is a bifunctional, heme-dependent enzyme in the front-line defense of numerous bacterial and fungal pathogens against H2O2-induced oxidative damage from host immune responses. Contrary to the expectation that catalase and peroxidase activities should be mutually antagonistic, peroxidatic electron donors (PxEDs) enhance KatG catalase activity. Here, we establish the mechanism of synergistic cooperation between these activities. We show that at low pH values KatG can fully convert H2O2 to O2 and H2O only if a PxED is present in the reaction mixture. Stopped-flow spectroscopy results indicated rapid initial rates of H2O2 disproportionation slowing concomitantly with the accumulation of ferryl-like heme states. These states very slowly returned to resting (i.e. ferric) enzyme, indicating that they represented catalase-inactive intermediates. We also show that an active-site tryptophan, Trp-321, participates in off-pathway electron transfer. A W321F variant in which the proximal tryptophan was replaced with a non-oxidizable phenylalanine exhibited higher catalase activity and less accumulation of off-pathway heme intermediates. Finally, rapid freeze-quench EPR experiments indicated that both WT and W321F KatG produce the same methionine-tyrosine-tryptophan (MYW) cofactor radical intermediate at the earliest reaction time points and that Trp-321 is the preferred site of off-catalase protein oxidation in the native enzyme. Of note, PxEDs did not affect the formation of the MYW cofactor radical but could reduce non-productive protein-based radical species that accumulate during reaction with H2O2 Our results suggest that catalase-inactive intermediates accumulate because of off-mechanism oxidation, primarily of Trp-321, and PxEDs stimulate KatG catalase activity by preventing the accumulation of inactive intermediates.

New Insight into and Characterization of the Aqueous Metal-Enol(ate) Complexes of (Acetonedicarboxylato)copper.

Nearly 50 years have passed since the classic studies by Larson and Lister [LarsonD. W.; ListerM. W.Can. J. Chem.1968, 46, 823]and Hay and Leong [HayR. W.; LeongK. N.J. Chem. Soc. A1971, 0, 3639]on the copper-catalyzed decarboxylation of acetonedicarboxylic acid (H3Acdica). Although the authors laid the foundations for what we know about this reaction; still very little information exists regarding the underlying aqueous metal-enol(ate)s of (acetonedicarboxylato)copper. In this study, UV–visible titrations revealed three pK values, pK[Cu(H2A)], pK[Cu(HA)], and pK[Cu(A)]. We associated the first two with ionization of α-carbon CH2 groups in [CuII(H2Acdica)keto]1+ and [CuII(HAcdica)keto]0 to form unstable metal-enolates, {[CuII(HAcdica)enolate]} and {[CuII(Acdica)enolate]}, which through β-carbonyl oxygen protonation can form metal-enols [CuII(H2Acdica)enol]1+ and [CuII(HAcdica)enol]0. The square-planar CuII center (electron paramagnetic resonance results) plays a dual role of stabilizing negative electron density at the β-carbonyl oxygen and as an electron sink in [[CuI(HAcdica)enolate]0]‡ and [[CuI(Acdica)enolate]1–]‡ (confirmed through cyclic voltammetry as two single 1e– transfers). The π → π* transition associated with [CuII(HAcdica)enol]0 was used to determine pK[Cu(A)] (deprotonation of enol OH) and enolization rate constant (stopped-flow spectroscopy) but also exhibited a time-dependent decrease in absorbance (on the order of min–1), suggesting a new method to possibly obtain experimental values for the estimated “kCuL” decarboxylation rate constant of metal-enolate [CuL]1– calculated by Larson and Lister. On the basis of our results, we postulate that decarboxylation takes place primarily through {[CuII(HAcdica)enolate]} and [CuII(HAcdica)enol]0. These results add to our understanding of aqueous metal-enol(ate)s, which contain underlying CuII/I redox chemistry, “active methylenes” and enol tautomers and enolate anions, which play roles in many catalytic reactions of interdisciplinary importance.

Correlation among Singlet-Oxygen Quenching, Free-Radical Scavenging, and Excited-State Intramolecular-Proton-Transfer Activities in Hydroxyflavones, Anthocyanidins, and 1-Hydroxyanthraquinones.

Singlet-oxygen (1O2) quenching, free-radical scavenging, and excited-state intramolecular proton-transfer (ESIPT) activities of hydroxyflavones, anthocyanidins, and 1-hydroxyanthraquinones were studied by means of laser, stopped-flow, and steady-state spectroscopies. In hydroxyflavones and anthocyanidins, the 1O2 quenching activity positively correlates to the free-radical scavenging activity. The reason for this correlation can be understood by considering that an early step of each reaction involves electron transfer from the unfused phenyl ring (B-ring), which is singly bonded to the bicyclic chromen or chromenylium moiety (A- and C-rings). Substitution of an electron-donating OH group at B-ring enhances the electron transfer leading to activation of the 1O2 quenching and free-radical scavenging. In 3-hydroxyflavones, the OH substitution at B-ring reduces the activity of ESIPT within C-ring, which can be explained in terms of the nodal-plane model. As a result, the 1O2 quenching and free-radical scavenging activities negatively correlate to the ESIPT activity. A catechol structure at B-ring is another factor that enhances the free-radical scavenging in hydroxyflavones. In contrast to these hydroxyflavones, 1-hydroxyanthraquinones having an electron-donating OH substituent adjacent to the O-H---O═C moiety susceptible to ESIPT do not show a simple correlation between their 1O2 quenching and ESIPT activities, because the OH substitution modulates these reactions.

Electron transfer through arsenite oxidase: Insights into Rieske interaction with cytochrome c

Arsenic is a widely distributed environmental toxin whose presence in drinking water poses a threat to >140 million people worldwide. The respiratory enzyme arsenite oxidase from various bacteria catalyses the oxidation of arsenite to arsenate and is being developed as a biosensor for arsenite. The arsenite oxidase from Rhizobium sp. str. NT-26 (a member of the Alphaproteobacteria) is a heterotetramer consisting of a large catalytic subunit (AioA), which contains a molybdenum centre and a 3Fe-4S cluster, and a small subunit (AioB) containing a Rieske 2Fe-2S cluster. Stopped-flow spectroscopy and isothermal titration calorimetry (ITC) have been used to better understand electron transfer through the redox-active centres of the enzyme, which is essential for biosensor development. Results show that oxidation of arsenite at the active site is extremely fast with a rate of >4000s−1 and reduction of the electron acceptor is rate-limiting. An AioB-F108A mutation results in increased activity with the artificial electron acceptor DCPIP and decreased activity with cytochrome c, which in the latter as demonstrated by ITC is not due to an effect on the protein-protein interaction but instead to an effect on electron transfer. These results provide further support that the AioB F108 is important in electron transfer between the Rieske subunit and cytochrome c and its absence in the arsenite oxidases from the Betaproteobacteria may explain the inability of these enzymes to use this electron acceptor.

Mechanistic study of the substitution reactions of [Pt(II)(bis(2-pyridylmethyl)amine)H2O](ClO4)2 and [Pt(II)(bis(2-pyridylmethyl)sulfide)H2O](ClO4)2 with azole Nucleophiles. Crystal structure of [Pt(II)(bis(2-pyridylmethyl)sulfide)Cl]ClO4

The substitution kinetics of the complexes [Pt(II)(bis(2-pyridylmethyl)amine)H2O](ClO4)2, Ptdpa and [Pt(II)(bis(2-pyridylmethyl)sulfide)H2O](ClO4)2, Ptdps, with a series of azole nucleophiles: Imidazole (Im), 1-methylimidazole (MIm), 1,2-Dimethylimidazole (DIm), 1,2,4-triazole (Trz) and pyrazole (Pyz), were studied in an aqueous medium at constant ionic strength (0.1M NaClO4). The substitution of the coordinated water ligand on the Pt(II) complexes by the azoles was studied under pseudo-first order conditions as a function of the incoming nucleophiles concentration and temperature using either stopped-flow techniques or UV-Vis spectroscopy. Ptdps was found to be more reactive (three magnitude higher) than Ptdpa. The second-order rate constant, k2, for all the nucleophiles ranged between 0.087±0.005 and 0.926±0.05M−1s−1 for Ptdpa and between 146±4 and 1458±10M−1s−1 for Ptdps. The rate of substitution of the aqua ligand is dependent on the strength of the σ-donor character and the π-acceptability of the trans atom to the leaving group. The observed reactivity trend for the azoles followed the trend, MIm>Im>DIm>Trz>Pyz. This reactivity trend correlates with the basicity, steric and electrophilic effects of the nucleophiles. The X-ray crystal structure of Ptdps–Cl is reported.

Crystal structure and functional analysis of Leishmania major pseudoperoxidase.

Leishmania major pseudoperoxidase (LmPP) is a recently discovered heme protein expressed by the human pathogen. Previous in vivo and in vitro studies suggest that LmPP is a crucial element of the pathogen's defense mechanism against the reactive nitrogen species peroxynitrite produced during the host immune response. To shed light on the potential mechanism of peroxynitrite detoxification, we have determined the 1.76-Å X-ray crystal structure of LmPP, revealing a striking degree of homology with heme peroxidases. The most outstanding structural feature is a Cys/His heme coordination, which corroborates previous spectroscopic and mutagenesis studies. We also used a combination of stopped-flow and electron paramagnetic spectroscopies that together suggest that peroxynitrite is not a substrate for LmPP catalysis, leaving the function of LmPP an open question.