Biomedical subjects

Stephen J Lippard

Publications and source records attributed to Stephen J Lippard.

At least 55 records · Page 3Linked to original sources

PubMed2005

Conjugated polymer-based fluorescence turn-on sensor for nitric oxide.

A turn-on fluorescent sensor for NO (g) in solution was synthesized using a bipyridyl-substituted poly(p-phenylene vinylene) derivative (CP1) as the sensory scaffold. The action of NO (g) upon the CP1-Cu(II) complex reduces it to the CP1-Cu(I) complex with a concomitant 2.8-fold increase in emission intensity. The reagent is selective for NO (g) versus other biological reactive nitrogen species, except for nitroxyl, and has a detection sensitivity limit of 6.3 nM. [structure: see text]

Copper↗

PubMed2005

X-ray absorption spectroscopic study of the reduced hydroxylases of methane monooxygenase and toluene/o-xylene monooxygenase: differences in active site structure and effects of the coupling proteins MMOB and ToMOD.

The diiron active sites of the reduced hydroxylases from methane monooxygenase (MMOH(red)) and toluene/o-xylene monooxygenase (ToMOH(red)) have been investigated by X-ray absorption spectroscopy (XAS). Results of Fe K-edge and extended X-ray absorption fine structure analysis reveal subtle differences between the hydroxylases that may be correlated to access of the active site. XAS data were also recorded for each hydroxylase in the presence of its respective coupling protein. MMOB affects the outer-shell scattering contributions in the diiron site of MMOH(red), whereas ToMOD exerts its main effect on the first-shell ligation of ToMOH(red); it also causes a slight decrease in the Fe-Fe separation. These results provide an initial step toward delineating the differences in structure and reactivity in bacterial multicomponent monooxygenase proteins.

Bacteria↗

PubMed2005

Water affects the stereochemistry and dioxygen reactivity of carboxylate-rich diiron(II) models for the diiron centers in dioxygen-dependent non-heme enzymes.

Carboxylate-bridged high-spin diiron(II) complexes with distinctive electronic transitions were prepared by using 4-cyanopyridine (4-NCC(5)H(4)N) ligands to shift the charge-transfer bands to the visible region of the absorption spectrum. This property facilitated quantitation of water-dependent equilibria in the carboxylate-rich diiron(II) complex, [Fe(2)(mu-O(2)CAr(Tol))(4)(4-NCC(5)H(4)N)(2)] (1), where (-)O(2)CAr(Tol) is 2,6-di-(p-tolyl)benzoate. Addition of water to 1 reversibly shifts two of the bridging carboxylate ligands to chelating terminal coordination positions, converting the structure from a paddlewheel to a windmill geometry and generating [Fe(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)(4-NCC(5)H(4)N)(2)(H(2)O)(2)] (3). This process is temperature dependent in solution, rendering the system thermochromic. Quantitative treatment of the temperature-dependent spectroscopic changes over the temperature range from 188 to 298 K in CH(2)Cl(2) afforded thermodynamic parameters for the interconversion of 1 and 3. Stopped flow kinetic studies revealed that water reacts with the diiron(II) center ca. 1000 time faster than dioxygen and that the water-containing diiron(II) complex reacts with dioxygen ca. 10 times faster than anhydrous analogue 1. Addition of {H(OEt(2))(2)}{B}, where B(-) is tetrakis(3,5-di(trifluoromethyl)phenyl)borate, to 1 converts it to [Fe(2)(mu-O(2)CAr(Tol))(3)(4-NCC(5)H(4)N)(2)](B) (5), which was also structurally characterized. Mossbauer spectroscopic investigations of solid samples of 1, 3, and 5, in conjunction with several literature values for high-spin iron(II) complexes in an oxygen-rich coordination environment, establish a correlation between isomer shift, coordination number, and N/O composition. The products of oxygenating 1 in CH(2)Cl(2) were identified crystallographically to be [Fe(2)(mu-OH)(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)(4-NCC(5)H(4)N)(2)].2(HO(2)CAr(Tol)) (6) and [Fe(6)(mu-O)(2)(mu-OH)(4)(mu-O(2)CAr(Tol))(6)(4-NCC(5)H(4)N)(4)Cl(2)] (7).

Carboxylic Acids↗

PubMed2005

Reactions of the peroxo intermediate of soluble methane monooxygenase hydroxylase with ethers.

Soluble methane monooxygenase (sMMO) isolated from Methylococcus capsulatus (Bath) utilizes a carboxylate-bridged diiron center and dioxygen to catalyze the conversion of methane to methanol. Previous studies revealed that a di(mu-oxo)diiron(IV) intermediate termed Q is responsible for the catalytic activity with hydrocarbons. In addition, the peroxodiiron(III) intermediate (H(peroxo)) that precedes Q formation in the catalytic cycle has been demonstrated to react with propylene, but its reactivity has not been extensively investigated. Given the burgeoning interest in the existence of multiple oxidants in metalloenzymes, a more exhaustive study of the reactivity of H(peroxo) was undertaken. The kinetics of single turnover reactions of the two intermediates with ethyl vinyl ether and diethyl ether were monitored by single- and double-mixing stopped-flow optical spectroscopy. For both substrates, the rate constants for reaction with H(peroxo) are greater than those for Q. An analytical model for explaining the transient kinetics is described and used successfully to fit the observed data. Activation parameters were determined through temperature-dependent studies, and the kinetic isotope effects for the reactions with diethyl ether were measured. The rate constants indicate that H(peroxo) is a more electrophilic oxidant than Q. We propose that H(peroxo) reacts via two-electron transfer mechanisms, and that Q reacts by single-electron transfer steps.

Catalysis↗

PubMed2005

Esterase-activated two-fluorophore system for ratiometric sensing of biological zinc(II).

Intracellular ester hydrolysis by cytosolic esterases is a common strategy used to trap fluorescent sensors within the cell. We have prepared analogues of Zinpyr-1 (ZP1), an intensity-based fluorescent sensor for Zn2+, that are linked via an amido-ester or diester moiety to a calibrating fluorophore, coumarin 343. These compounds, designated Coumazin-1 and -2, are nonpolar and are quenched by intramolecular interactions between the two fluorophores. Esterase-catalyzed hydrolysis generates a Zn2+-sensitive ZP1-like fluorophore and a Zn2+-insensitive coumarin as a calibrating fluorophore. Upon excitation of the fluorophores, coumarin 343 emission relays information concerning sensor concentration whereas ZP1 emission indicates the relative concentration of Zn2+-bound sensor. This approach enables intracellular monitoring of total sensor concentration and provides a ratiometric system for sensing biological zinc ion.

Animals↗

PubMed2005

Product bound structures of the soluble methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath): protein motion in the alpha-subunit.

The soluble methane monooxygenase hydroxylase (MMOH) alpha-subunit contains a series of cavities that delineate the route of substrate entrance to and product egress from the buried carboxylate-bridged diiron center. The presence of discrete cavities is a major structural difference between MMOH, which can hydroxylate methane, and toluene/o-xylene monooxygenase hydroxylase (ToMOH), which cannot. To understand better the functions of the cavities and to investigate how an enzyme designed for methane hydroxylation can also accommodate larger substrates such as octane, methylcubane, and trans-1-methyl-2-phenylcyclopropane, MMOH crystals were soaked with an assortment of different alcohols and their X-ray structures were solved to 1.8-2.4 A resolution. The product analogues localize to cavities 1-3 and delineate a path of product exit and/or substrate entrance from the active site to the surface of the protein. The binding of the alcohols to a position bridging the two iron atoms in cavity 1 extends and validates previous crystallographic, spectroscopic, and computational work indicating this site to be where substrates are hydroxylated and products form. The presence of these alcohols induces perturbations in the amino acid side-chain gates linking pairs of cavities, allowing for the formation of a channel similar to one observed in ToMOH. Upon binding of 6-bromohexan-1-ol, the pi helix formed by residues 202-211 in helix E of the alpha-subunit is extended through residue 216, changing the orientations of several amino acid residues in the active site cavity. This remarkable secondary structure rearrangement in the four-helix bundle has several mechanistic implications for substrate accommodation and the function of the effector protein, MMOB.

Alcohols↗

PubMed2005

Hydroxylation of C-H bonds at carboxylate-bridged diiron centres.

Nature uses carboxylate-bridged diiron centres at the active sites of enzymes that catalyse the selective hydroxylation of hydrocarbons to alcohols. The resting diiron(III) state of the hydroxylase component of soluble methane monooxygenase enzyme is converted by two-electron transfer from an NADH-requiring reductase into the active diiron(II) form, which subsequently reacts with O2 to generate a high-valent diiron(IV) oxo species (Q) that converts CH4 into CH3OH. In this step, C-H bond activation is achieved through a transition state having a linear C...H...O unit involving a bound methyl radical. Kinetic studies of the reaction of Q with substrates CH3X, where X=H, D, CH3, NO2, CN or OH, reveal two classes of reactivity depending upon whether binding to the enzyme or C-H bond activation is rate-limiting. Access of substrates to the carboxylate-bridged diiron active site in the hydroxylase (MMOH) occurs through a series of hydrophobic pockets. In the hydroxylase component of the closely related enzyme toluene/o-xylene monooxygenase (ToMOH), substrates enter through a wide channel in the alpha-subunit of the protein that tracks a course identical to that found in the structurally homologous MMOH. Synthetic models for the carboxylate-bridged diiron centres in MMOH and ToMOH have been prepared that reproduce the stoichiometry and key geometric and physical properties of the reduced and oxidized forms of the proteins. Reactions of the diiron(II) model complexes with dioxygen similarly generate reactive intermediates, including high-valent species capable not only of hydroxylating pendant C-H bonds but also of oxidizing phosphine and sulphide groups.

Carbon↗

PubMed2005

Fourier transform infrared characterization of the azido complex of methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath).

The azido complex formed in oxidized methane monooxygenase from Methylococcus capsulatus (Bath) was investigated with resonance Raman and FTIR techniques. These experiments show the presence of a nuas(NNN) at approximately 2077 cm-1 which splits to two components at 2059 and 2073 cm-1 with 15N14N2. The vibrational data are assigned to an azido complex bound terminally to one iron(III) at the diiron center. When the azido complex is illuminated at 15 K, a new nuas(NNN) is observed at 2136 cm-1 which is assigned to a photodissociated HN3 within the substrate pocket. We propose a model where an aqua ligand engages a hydrogen bond interaction with the 1N atom of the azido group and acts as at a proton donor during the photolysis process.

Azides↗

PubMed2005

Substrate hydroxylation in methane monooxygenase: quantitative modeling via mixed quantum mechanics/molecular mechanics techniques.

Using broken-symmetry unrestricted density functional theory quantum mechanical (QM) methods in concert with mixed quantum mechanics/molecular mechanics (QM/MM) methods, the hydroxylation of methane and substituted methanes by intermediate Q in methane monooxygenase hydroxylase (MMOH) has been quantitatively modeled. This protocol allows the protein environment to be included throughout the calculations and its effects (electrostatic, van der Waals, strain) upon the reaction to be accurately evaluated. With the current results, recent kinetic data for CH3X (X = H, CH3, OH, CN, NO2) substrate hydroxylation in MMOH (Ambundo, E. A.; Friesner, R. A.; Lippard, S. J. J. Am. Chem. Soc. 2002, 124, 8770-8771) can be rationalized. Results for methane, which provide a quantitative test of the protocol, including a substantial kinetic isotope effect (KIE), are in reasonable agreement with experiment. Specific features of the interaction of each of the substrates with MMO are illuminated by the QM/MM modeling, and the resulting effects upon substrate binding are quantitatively incorporated into the calculations. The results as a whole point to the success of the QM/MM methodology and enhance our understanding of MMOH catalytic chemistry. We also identify systematic errors in the evaluation of the free energy of binding of the Michaelis complexes of the substrates, which most likely arise from inadequate sampling and/or the use of harmonic approximations to evaluate the entropy of the complex. More sophisticated sampling methods will be required to achieve greater accuracy in this aspect of the calculation.

Acetonitriles↗

PubMed2005

Cellular processing of platinum anticancer drugs.

Cisplatin, carboplatin and oxaliplatin are platinum-based drugs that are widely used in cancer chemotherapy. Platinum-DNA adducts, which are formed following uptake of the drug into the nucleus of cells, activate several cellular processes that mediate the cytotoxicity of these platinum drugs. This review focuses on recently discovered cellular pathways that are activated in response to cisplatin, including those involved in regulating drug uptake, the signalling of DNA damage, cell-cycle checkpoints and arrest, DNA repair and cell death. Such knowledge of the cellular processing of cisplatin adducts with DNA provides valuable clues for the rational design of more efficient platinum-based drugs as well as the development of new therapeutic strategies.

Animals↗

PubMed2004

Water-dependent reactions of diiron(II) carboxylate complexes.

Carboxylate-rich diiron(II) compounds with varying numbers of water ligands have been characterized, including the first complex with a {Fe2(mu-OH2)2(mu-O2CArTol)}3+ unit. The isolation of these complexes reveals how water can alter the structural properties of carboxylate-bridged diiron(II) core similar to those that occur in a variety of dioxygen-activating metalloenzyme cores. Mössbauer and variable temperature, variable field magnetic susceptibility experiments indicate that the compound [Fe2(mu-OH2)2(mu-O2CAr4F-Ph)(O2CAr4F-Ph)3(THF)2(OH2)] has a high-spin diiron(II) core with little significant magnetic exchange coupling.

Biomimetic Materials↗

PubMed2004

Preparation and X-ray structures of metal-free, dicobalt and dimanganese forms of soluble methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath).

A three-component soluble methane monooxygenase (sMMO) enzyme system catalyzes the hydroxylation of methane to methanol at a carboxylate-bridged diiron center housed in the alpha-subunit of the hydroxylase (MMOH). Catalysis is facilitated by the presence of a regulatory protein (MMOB) and inhibited by MMOD, a protein of unknown function encoded in the sMMO operon. Both MMOB and MMOD are presumed to bind to the same region of the MMOH alpha-subunit. A colorimetric method for monitoring removal of Fe(II) from MMOH was developed using 1,10-phenanthroline and yields apo MMOH with <0.1 Fe/homodimer. With the use of this method, it was possible to investigate the X-ray structure of the apoenzyme and to perform metal reconstitution studies. Using MMOH from Methylococccus capsulatus (Bath), the effects of MMOB and MMOD on metal binding were studied and structural perturbations relevant to the function of this enzyme were identified. X-ray crystal structures of the apo, Mn(II)-soaked, and Co(II)-grown MMOH, determined to 2.3 A or greater resolution, reveal that the presence of metal ions is essential for the proper folding of helices E, F, and H of the alpha-subunit. The active sites of Mn(II)-soaked and Co(II)-grown MMOH are similar to that of reduced, native MMOH with notable differences in the metal-metal distances and ligand coordination sphere that may reflect how this dinuclear metal center might change in the presence of MMOB. MMOB and MMOD decrease the rate of removal of Fe(II) from the enzyme by 22- and 16-fold, respectively. On the basis of previous studies, it is hypothesized that MMOB, and perhaps MMOD, function to block solvent access to the MMOH active site. Finally, ITC studies and the observed disorder in helices E, F, and H in the apo and Mn(II)-soaked structures suggest that these regions of MMOH are critical for MMOB and MMOD binding.

Apoproteins↗

PubMed2004

The zinspy family of fluorescent zinc sensors: syntheses and spectroscopic investigations.

Four fluorescent sensors designed for Zn(II) detection that contain a fluorescein reporting group and a pyridyl-amine-thioether derivatized ligand moiety were prepared and their photophysical properties characterized. These "Zinspy" sensors are water soluble and generally display approximately 1.4- to approximately 4.5-fold fluorescence enhancement upon Zn(II) coordination, depending upon fluorescein halogenation and the number and nature of the zinc-binding appendages. The Zinspy sensors exhibit improved selectivity for zinc compared to the di-(2-picolyl)amine-based Zinpyr family.

Fluoresceins↗

PubMed2004

ZP8, a neuronal zinc sensor with improved dynamic range; imaging zinc in hippocampal slices with two-photon microscopy.

The synthesis of a difluorofluorescein monocarboxaldehyde platform and its use for preparing ZP8, a new member of the Zinpyr family of neuronal Zn(2+) sensors, are described. By combining an aniline photoinduced electron transfer (PET) switch and an electron-withdrawing fluorescein scaffold, ZP8 displays reduced background fluorescence and improved dynamic range compared to previous ZP probes. The bright sensor undergoes an 11-fold increase in fluorescence intensity upon Zn(2+) complexation (Phi = 0.03-0.35) with high selectivity over cellular concentrations of Ca(2+) and Mg(2+). In addition, sensors in the ZP family have been utilized for optical imaging in biological samples using two-photon microscopy (TPM). The cell-permeable ZP3 probe is capable of identifying natural pools of labile Zn(2+) within the mossy fiber synapses of live hippocampal slices using TPM, establishing the application of this technique for monitoring endogenous Zn(2+) stores.

Animals↗

PubMed2004

Method for identifying neuronal cells suffering zinc toxicity by use of a novel fluorescent sensor.

During excitotoxic brain damage, injured neurons accumulate an anomalous, pathological burden of weakly bound, rapidly exchangeable Zn(2+) that diffusely fills the soma, nucleus and proximal dendrites. Mounting evidence indicates that this Zn(2+) is a major contributing factor in the subsequent demise of the damaged neurons. Thus, identifying, imaging, and characterizing zinc-filled cells have become essential steps in understanding excitotoxicity. Here we demonstrate that a new fluorescent stain for zinc can rather selectively and quite vividly label zinc-filled neurons in frozen histologic sections. The method is more sensitive and selective than the existing stain TSQ, and simpler than the Timm-Danscher silver staining techniques. A previously unobserved population of apparently injured cells in the dentate gyrus has been discovered with the new reagent. Whereas cells viewed in situ in normal, healthy tissue virtually never display any perikaryal staining by histochemical methods for zinc, injured cells stain intensely for zinc in culture, acute slice preparations and in tissue harvested in vivo. Thus, the presence of rapidly-exchangeable, "stainable" perikaryal zinc may be taken as an indicator of cell injury.

Animals↗

PubMed2004

Fluorescence-based nitric oxide detection by ruthenium porphyrin fluorophore complexes.

The ruthenium(II) porphyrin fluorophore complexes [Ru(TPP)(CO)(Ds-R)] (TPP = tetraphenylporphinato dianion; Ds = dansyl; R = imidazole (im), 1, or thiomorpholine (tm), 2) were synthesized and investigated for their ability to detect nitric oxide (NO) based on fluorescence. The X-ray crystal structures of 1 and 2 were determined. The Ds-im or Ds-tm ligand coordinates to an axial site of the ruthenium(II) center through a nitrogen or sulfur atom, respectively. Both exhibit quenched fluorescence when excited at 368 or 345 nm. Displacement of the metal-coordinated fluorophore by NO restores fluorescence within minutes. These observations demonstrate fluorescence-based NO detection using ruthenium porphyrin fluorophore conjugates.

Crystallography, X-Ray↗

PubMed2004

NMR structure of the flavin domain from soluble methane monooxygenase reductase from Methylococcus capsulatus (Bath).

Soluble methane monooxygenase (sMMO) catalyzes the hydroxylation of methane by dioxygen to methanol, the first step in carbon assimilation by methanotrophs. This multicomponent system transfers electrons from NADH through a reductase component to the non-heme diiron center in the hydroxylase where O(2) is activated. The reductase component comprises three distinct domains, a [2Fe-2S] ferredoxin domain along with FAD- and NADH-binding domains. We report the solution structure of the reduced 27.6 kDa FAD- and NADH-binding domains (MMOR-FAD) of the reductase from Methylococcus capsulatus (Bath). The FAD-binding domain consists of a six-stranded antiparallel beta-barrel and one alpha-helix, with the first 10 N-terminal residues unstructured. In the interface between the two domains, the FAD cofactor is tightly bound in an unprecedented extended conformation. The NADH-binding domain consists of a five-stranded parallel beta-sheet with four alpha-helices packing closely around this sheet. MMOR-FAD is structurally homologous to other FAD-containing oxidoreductases, and we expect similar structures for the FAD/NADH-binding domains of reductases that occur in other multicomponent monooxygenases.

Amino Acid Sequence↗

PubMed2004

Nitric oxide reactivity of fluorophore coordinated carboxylate-bridged diiron(II) and dicobalt(II) complexes.

The synthesis, structural characterization, and NO reactivity of carboxylate-bridged dimetallic complexes were investigated. The diiron(II) complex [Fe(2)(mu-O(2)CAr(Tol))(4)(Ds-pip)(2)] (1), where O(2)CAr(Tol) = 2,6-di(p-tolyl)benzoate and Ds-pip = dansyl-piperazine, was prepared and determined by X-ray crystallography to have a paddlewheel geometry. This complex reacts with NO within 1 min with a concomitant 4-fold increase in fluorescence emission intensity ascribed to displacement of Ds-pip. Although the diiron complex reacts with NO, as revealed by infrared spectroscopic studies, its sensitivity to dioxygen renders it unsuitable as an atmospheric NO sensor. The air-stable dicobalt(II) analogue was also synthesized and its reactivity investigated. In solution, the dicobalt(II) complex exists as an equilibrium between paddlewheel [Co(2)(mu-O(2)CAr(Tol))(4)(Ds-pip)(2)] (2) and windmill [Co(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)(Ds-pip)(2)] (3) geometric isomers. Conditions for crystallizing pure samples of each of these isomers are described. Reaction of 2 with excess NO proceeds by reductive nitrosylation giving [Co(mu-O(2)CAr(Tol))(2)(NO)(4)] (5), which is accompanied by release of the Ds-pip fluorophore that is N-nitrosated in the process. This reaction affords an overall 9.6-fold increase in fluorescence emission intensity, further demonstrating the potential utility of ligand dissociation as a strategy for designing fluorescence-based sensors to detect nitric oxide in a variety of contexts.

Journal Article↗