Biomedical subjects

Stephen J Lippard

Publications and source records attributed to Stephen J Lippard.

At least 91 records · Page 5Linked to original sources

PubMed2003

Modeling the syn disposition of nitrogen donors at the active sites of carboxylate-bridged diiron enzymes. Enforcing dinuclearity and kinetic stability with a 1,2-diethynylbenzene-based ligand.

The syn coordination of histidine residues at the active sites of several carboxylate-rich non-heme diiron enzymes has been difficult to reproduce with small molecule model compounds. In this study, ligands derived from 1,8-naphthyridine, phthalazine, and 1,2-diethynylbenzene were employed to mimic this geometric feature. The preassembled diiron(II) complex [Fe(2)(micro-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)(THF)(2)] (1), where Ar(Tol)CO(2)(-) is the sterically hindered carboxylate 2,6-di(p-tolyl)benzoate, served as a convenient starting material for the preparation of iron(II) complexes, all of which were crystallographically characterized. Use of the ligand 2,7-dimethyl-1,8-naphthyridine (Me(2)-napy) afforded the mononuclear complex [Fe(O(2)CAr(Tol))(2)(Me(2)-napy)] (2), whereas dinuclear [Fe(2)(micro-DMP)(micro-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)(THF)] (3) resulted when 1,4-dimethylphthalazine (DMP) was employed. The dinuclear core of compound 3 is kinetically labile, as evidenced by the formation of [Fe(O(2)CAr(Tol))(2)(vpy)(2)] (4) upon addition of 2-vinylpyridine (vpy). The diiron analogue of 4, [Fe(2)(micro-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)(vpy)(2)] (5), was prepared directly from 1. When the sterically more demanding ligand 2,6-di(4-tert-butylphenyl)benzoate (Ar(4-tBuPh)CO(2)(-)) was used, mononuclear [Fe(O(2)CAr(4)(-)(tBuPh))(2)(THF)(2)] (6) and [Fe(O(2)CAr(4)(-)(tBuPh))(2)(DMP)(2)] (7) formed. The difficulty in stabilizing a dinuclear core with these simple (N)(2)-donor ligands was circumvented by preparing a family of 1,2-diethynylbenzene-based ligands, from which were readily assembled the complexes [Fe(2)(Et(2)BCQEB(Et))(micro-O(2)CAr(Tol))(3)](OTf) (15) and [Cu(2)(Et(2)BCQEB(Et))(micro-I)(2)] (16), where Et(2)BCQEB(Et) is 1,2-bis(3-ethynyl-8-carboxylatequinoline)benzene ethyl ester. The Et(2)BCQEB(Et) framework provides both structural flexibility and the desired syn nitrogen donor geometry, thus serving as a good first-generation ligand in this class.

Alkynes↗

PubMed2003

Synthesis, characterization, and dioxygen reactivity of tetracarboxylate-bridged Diiron(II) complexes with coordinated substrates.

The synthesis and characterization of [Fe(2)(micro-O(2)CAr(Tol))(4)L(2)] complexes, where L is benzylamine or 4-methoxybenzylamine (BA(p)()(-)(OMe)), are described. The reaction of the latter diiron(II) complex with dioxygen at -78 degrees C affords a metastable mixed-valent Fe(II)Fe(III) green intermediate. When O(2) is introduced at ambient temperature, N-dealkyation occurs to yield anisaldehyde, eliminating N-oxidation as a viable pathway for the reaction. Use of [Fe(2)(micro-O(2)CAr(T)(omicron)(l))(4)(alpha-d(1)-BA(p)()(-)(OMe))(2)] allowed a deuterium kinetic isotope of approximately 3 to be determined.

Benzylamines↗

PubMed2003

cis-[Pt(NH3)2(L)]2+/+ (L = Cl, H2O, NH3) binding to purines and CO: does pi-back-donation play a role?

The ability of cis-[Pt(NH(3))(2)(L)](2+/+), a molecular fragment of the anticancer drug cisplatin, to bind to purines and CO by pi-back-donation from Pt to the ligand was examined computationally. Optimized geometries and computed vibrational frequencies suggest that cis-[Pt(NH(3))(2)(L)](2+/+) (L = Cl, H(2)O, NH(3)) is a poor pi-donor and that pi-back-donation does not play an important role for Pt(II)-ligand interactions in general.

Carbon Monoxide↗

PubMed2003

A "turn-on" fluorescent sensor for the selective detection of mercuric ion in aqueous media.

A water-soluble turn-on fluorescent sensor for Hg(II) is described. Incorporation of soft thioether donors into an aniline-derived ligand framework that can be linked to a fluorescein platform affords sensor MS1, which shows a approximately 5-fold increase in integrated emission upon addition of 1 equiv of Hg(II). The synthesis and metal-binding properties of MS1 are discussed, and its ability to detect environmentally relevant concentrations of Hg(II) is demonstrated.

Alkanes↗

PubMed2003

Theoretical study of cisplatin binding to purine bases: why does cisplatin prefer guanine over adenine?

The thermodynamics and kinetics for the monofunctional binding of the antitumor drug cisplatin, cis-diamminedichloroplatinum(II), to a purine base site of DNA were studied computationally using guanine and adenine as model reactants. A dominating preference for initial attack at the N7-position of guanine is established experimentally, which is a crucial first step for the formation of a 1,2-intrastrand cross-link of adjacent guanine bases that leads to bending and unwinding of DNA. These structural distortions are proposed ultimately to be responsible for the anticancer activity of cisplatin. Utilizing density functional theory in combination with a continuum solvation model, we developed a concept for the initial Pt-N7 bond formation to atomic detail. In good agreement with experiments that suggested DeltaG++ = approximately 23 kcal/mol for the monofunctional platination of guanine, our model gives DeltaG++ = 24.6 kcal/mol for guanine, whereas 30.2 kcal/mol is computed when adenine is used. This result predicts that guanine is 3-4 orders of magnitude more reactive toward cisplatin than adenine. A detailed energy decomposition and molecular orbital analysis was conducted to explain the different barrier heights. Two effects are equally important to give the preference for guanine over adenine: First, the transition state is characterized by a strong hydrogen bond between the ammine-hydrogen of cisplatin and the O=C6 moiety of guanine in addition to a stronger electrostatic interaction between the two reacting fragments. When adenine binds, only a weak hydrogen bond forms between the chloride ligand of cisplatin and the H(2)N-C6 group of adenine. Second, a significantly stronger molecular orbital interaction is identified for guanine compared to adenine. A detailed MO analysis is presented to provide an intuitive view into the different electronic features governing the character of the Pt-N7 bond in platinated purine bases.

Adenine↗

PubMed2003

Domain engineering of the reductase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath).

Soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath) is a three-component enzyme system that catalyzes the conversion of methane to methanol. A reductase (MMOR), which contains [2Fe-2S] and FAD cofactors, facilitates electron transfer from NADH to the hydroxylase diiron active sites where dioxygen activation and substrate hydroxylation take place. By separately expressing the ferredoxin (MMORFd, MMOR residues 1-98) and FAD/NADH (MMOR-FAD, MMOR residues 99-348) domains of the reductase, nearly all biochemical properties of full-length MMOR are retained, except for interdomain electron transfer rates. To investigate the extent to which rapid electron transfer between domains might be restored and further to explore the modularity of MMOR, MMOR-Fd and MMOR-FAD were connected in a non-native fashion. Four different linker sequences were employed to create MMOR reversed-domain (MMOR-RD) constructs, MMOR(99-342)-linker-MMOR(2-98), with a domain connectivity observed in other homologous oxidoreductases. The optical, redox, and electron transfer properties of the four MMOR-RD proteins were characterized and compared with those of wild-type MMOR. The linker sequence plays a key role in controlling solvent accessibility to the FAD cofactor, as evidenced by perturbed flavin optical spectra, decreased FADox/FADsq redox potentials, and increased steady-state oxidase activities in three of the constructs. Stopped-flow optical spectroscopy revealed slow interdomain electron transfer (k < 0.04 s(-1) at 4 degrees C, compared with 90 s(-1) for wild-type MMOR) for all three MMOR-RD proteins with 7-residue linkers. A long (14-residue), flexible linker afforded much faster electron transfer between the FAD and [2Fe-2S] cofactors (k = 0.9 s(-1) at 4 degrees C).

Catalysis↗

PubMed2003

Multiple states of stalled T7 RNA polymerase at DNA lesions generated by platinum anticancer agents.

Transcription inhibition by DNA adducts of cisplatin is considered to be one of the major routes by which this anticancer drug kills cancer cells. Stalled RNA polymerases at platinum-DNA lesions evoke various cellular responses such as nucleotide excision repair, polymerase degradation, and apoptosis. T7 RNA polymerase and site-specifically platinated DNA templates immobilized on a solid support were used to study stalled transcription elongation complexes. In vitro transcription studies were performed in both a promoter-dependent and -independent manner. An elongation complex is strongly blocked by cisplatin 1,2-intrastrand d(GpG) and 1,3-intrastrand d(GpTpG) cross-links located on the template strand. Polymerase action is inhibited at multiple sites in the vicinity of the platinum lesion, the nature of which can be altered by the choice and concentration of NTPs. The [(1R,2R-diaminocyclohexane)Pt]2+ DNA adducts formed by oxaliplatin, which carries a stereochemically more demanding spectator ligand than the ammine groups in cisplatin, also strongly block the polymerase with measurable differences compared with cis-[(NH3)2Pt]2+ lesions. Elongation complexes stopped at sites of platinum damage were isolated and characterized. The stalled polymerase can be dissociated from the DNA by subsequent polymerases initiated from the same template. We also discovered that a polymerase stalled at the platinum-DNA lesion can resume transcription after the platinum adduct is chemically removed from the template.

Antineoplastic Agents↗

PubMed2003

Modeling features of the non-heme diiron cores in O2-activating enzymes through the synthesis, characterization, and oxidation of 1,8-naphthyridine-based complexes.

Multidentate naphthyridine-based ligands were used to prepare a series of diiron(II) complexes. The compound [Fe(2)(BPMAN)(mu-O(2)CPh)(2)](OTf)(2) (1), where BPMAN = 2,7-bis[bis(2-pyridylmethyl)aminomethyl]-1,8-naphthyridine, exhibits two reversible oxidation waves with E(1/2) values at +310 and +733 mV vs Cp(2)Fe(+)/Cp(2)Fe, as revealed by cyclic voltammetry. Reaction with O(2) or H(2)O(2) affords a product with optical and Mössbauer properties that are characteristic of a (mu-oxo)diiron(III) species. The complexes [Fe(2)(BPMAN)(mu-OH)(mu-O(2)CAr(Tol))](OTf)(2) (2) and [Fe(2)(BPMAN)(mu-OMe)(mu-O(2)CAr(Tol))](OTf)(2) (3) were synthesized, where Ar(Tol)CO(2)(-) is the sterically hindered ligand 2,6-di(p-tolyl)benzoate. Compound 2 has a reversible redox wave at +11 mV, and both 2 and 3 react with O(2), via a mixed-valent Fe(II)Fe(III) intermediate, to give final products that are also consistent with (mu-oxo)diiron(III) species. The paddle-wheel compound [Fe(2)(BBAN)(mu-O(2)CAr(Tol))(3)](OTf) (4), where BBAN = 2,7-bis(N,N-dibenzylaminomethyl)-1,8-naphthyridine, reacts with dioxygen to yield benzaldehyde via oxidative N-dealkylation of a benzyl group on BBAN, an internal substrate. In the presence of bis(4-methylbenzyl)amine, the reaction also produces p-tolualdehyde, revealing oxidation of an external substrate. A structurally related compound, [Fe(2)(BEAN)(mu-O(2)CAr(Tol))(3)](OTf) (5), where BEAN = 2,7-bis(N,N-diethylaminomethyl)-1,8-naphthyridine, does not undergo N-dealkylation, nor does it facilitate the oxidation of bis(4-methylbenzyl)amine. The contrast in reactivity of 4 and 5 is attributed to a difference in accessibility of the substrate to the diiron centers of the two compounds. The Mössbauer spectroscopic properties of the diiron(II) complexes were also investigated.

Crystallography, X-Ray↗

PubMed2003

A novel two-fluorophore approach to ratiometric sensing of Zn(2+).

We present a small molecule ratiometric Zn2+-sensing system based on two fluorophores excited by visible light, a Zn2+-insensitive reporter fluorophore, coumarin 343, and a Zn2+-sensitive fluorescein-based compound, ZPA-1. The two fluorophores are linked by an ester to give Coumazin-1, a membrane-permeable, essentially nonfluorescent compound. Upon exposure to esterases, Coumazin-1 is hydrolyzed to its constituent fluorophores. Measurement of the ratio of coumarin emission at 488 nm (lambdaexc = 445 nm) and comparison with ZPA-1 emission at 534 nm (lambdaexc = 505 nm) affords information about the amount of sensor present as well as the amount of Zn2+ present. A generally applicable synthetic route to amide-functionalized ZP1 sensors is also described. The Zn2+-sensing properties of one member of this class are similar to those of the parent ZP1 sensor, with slightly tighter binding and lower background signal.

Animals↗

PubMed2003

Stopped-flow Fourier transform infrared spectroscopy of nitromethane oxidation by the diiron(IV) intermediate of methane monooxygenase.

The hydroxylase component (MMOH) of soluble methane monooxygenase from Methylococcus capsulatus (Bath) was reduced to the diiron(II) form and then allowed to react with dioxygen to generate the diiron(IV) intermediate Q in the first phase of a double-mixing stopped-flow experiment. CD3NO2 was then introduced in the second phase of the experiment, which was carried out in D2O at 25 degrees C. The kinetics of the reaction of the substrate with Q were monitored by stopped-flow Fourier transform infrared spectroscopy, observing the disappearance of the asymmetric NO2 bending vibration at 1548 cm-1. The data were fit to a single-exponential function, which yielded a kobs of 0.45 +/- 0.07 s-1. This result is in quantitative agreement with a kobs of 0.39 +/- 0.01 s-1 obtained by observing the disappearance of Q by double-mixing stopped-flow optical spectroscopy at its absorption maximum of 420 nm. These results provide for the first time direct monitoring of the hydroxylation of a methane-derived substrate in the MMOH reaction pathway and demonstrate that Q decay occurs concomitantly with substrate consumption.

Escherichia coli↗

PubMed2003

Synthesis and metal-binding properties of chelating fluorescein derivatives.

[reaction: see text] Two routes to highly functionalized metal-chelating fluorescein derivatives have been pursued. Compound 3 is partially quenched by a variety of first-row transition metal ions in aqueous solution, with EC(50) values ranging from 0.4 to 60 microM. Compounds of this type may find application in biological sensing.

Chelating Agents↗

PubMed2003

Nucleotide excision repair from site-specifically platinum-modified nucleosomes.

Nucleotide excision repair is a major cellular defense mechanism against the toxic effects of the anticancer drug cisplatin and other platinum-based chemotherapeutic agents. In this study, mononucleosomes were prepared containing either a site-specific cis-diammineplatinum(II)-DNA intrastrand d(GpG) or a d(GpTpG) cross-link. The ability of the histone core to modulate the excision of these defined platinum adducts was investigated as a model for exploring the cellular response to platinum-DNA adducts in chromatin. Comparison of the extent of repair by mammalian cell extracts of free and nucleosomal DNA containing the same platinum-DNA adduct reveals that the nucleosome significantly inhibits nucleotide excision repair. With the GTG-Pt DNA substrate, the nucleosome inhibits excision to about 10% of the level observed with free DNA, whereas with the less efficient GG-Pt DNA substrate the nucleosome inhibited excision to about 30% of the level observed with free DNA. The effects of post-translational modification of histones on excision of platinum damage from nucleosomes were investigated by comparing native and recombinant nucleosomes containing the same intrastrand d(GpTpG) cross-link. Excision from native nucleosomal DNA is approximately 2-fold higher than the level observed with recombinant material. This result reveals that post-translational modification of histones can modulate nucleotide excision repair from damaged chromatin. The in vitro system established in this study will facilitate the investigation of platinum-DNA damage by DNA repair processes and help elucidate the role of specific post-translational modification in NER of platinum-DNA adducts at the physiologically relevant nucleosome level.

Animals↗

PubMed2003

Peripheral heme substituents control the hydrogen-atom abstraction chemistry in cytochromes P450.

We elucidate the hydroxylation of camphor by cytochrome P450 with the use of density functional and mixed quantum mechanics/molecular mechanics methods. Our results reveal that the enzyme catalyzes the hydrogen-atom abstraction step with a remarkably low free-energy barrier. This result provides a satisfactory explanation for the experimental failure to trap the proposed catalytically competent high-valent heme Fe(IV) oxo (oxyferryl) species responsible for this hydroxylation chemistry. The primary and previously unappreciated contribution to stabilization of the transition state is the interaction of positively charged residues in the active-site cavity with carboxylate groups on the heme periphery. A similar stabilization found in dioxygen binding to hemerythrin, albeit with reversed polarity, suggests that this mechanism for controlling the relative energetics of redox-active intermediates and transition states in metalloproteins may be widespread in nature.

Camphor↗

PubMed2003

Reversible dioxygen binding to hemerythrin.

A combination of conventional quantum chemical methods and a recently developed mixed quantum mechanical/molecular mechanical (QM/MM) method (QSite) is used to determine the different energetic components involved in reversible binding of O(2) to hemerythrin. The use of an accurate quantum chemical description of the active site and the inclusion of effects from the surrounding protein environment are both essential to achieve reversibility and thus to model accurately the binding of O(2) to the carboxylate-bridged diiron center in the protein. The major contributions from the protein environment stabilizing dioxygen binding are (1) the van der Waals interaction between the bound dioxygen and the protein atoms and (2) an increase in the hydrogen bonding energy of an imidazole group, ligated to one of the iron atoms, and a neighboring carboxylate side chain in the second coordination sphere. The protein strain energy for this system is negligible. The calculated total O(2) binding free energy is in good agreement with that derived from the experimental equilibrium constant.

Hemerythrin↗

PubMed2003

Structural features of covalently cross-linked hydroxylase and reductase proteins of soluble methane monooxygenase as revealed by mass spectrometric analysis.

Soluble methane monooxygenase requires complexes between its three component proteins for efficient catalysis. The hydroxylase (MMOH) must bind both to the reductase (MMOR) and to the regulatory protein (MMOB) to facilitate oxidation of methane to methanol. Although structures of MMOH, MMOB, and one domain of MMOR have been determined, less geometric information is available for the complexes. To address this deficiency, MMOH and MMOR were cross-linked by a carbodiimide reagent and analyzed by specific proteolysis, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and capillary high performance liquid chromatography mass spectrometry. Tandem mass spectra conclusively identified two amine-to-carboxylate cross-linked sites involving the alpha subunit of MMOH and the [2Fe-2S] domain of MMOR (MMOR-Fd). In particular, the N terminus of the MMOH alpha subunit forms cross-links to the side chains of MMOR-Fd residues Glu-56 and Glu-91. These Glu residues are close to one another on the surface of MMOR-Fd and >25 A from the [2Fe-2S] cluster. Because the N terminus of the alpha subunit of MMOH was not located in the crystal structure of MMOH, a detailed structural model of the complex based on the cross-link was precluded; however, a previously proposed binding site for MMOR on MMOH could be ruled out. Based on the cross-linking results, a MMOR E56Q/E91Q double mutant was generated. The mutant retains >80% of MMOR NADH oxidase activity but reduces sMMO activity to approximately 65% of the level supported by the wild type reductase. Cross-linking to MMOH was diminished but not abolished in the double mutant, indicating that other residues of MMOR also form cross-links to MMOH.

Binding Sites↗

PubMed2003

Meeting of the minds: metalloneurochemistry.

Metalloneurochemistry is the study of metal ion function in the brain and nervous system at the molecular level. Research in this area is exemplified through discussion of several forefront areas where significant progress has been made in recent years. The structure and function of ion channels have been elucidated through high-resolution x-ray structural work on the bacterial K(+) ion channel. Selection of potassium over sodium ions is achieved by taking advantage of key principles of coordination chemistry. The role of calcium ions in neuronal signal transduction is effected by several Ca(2+)-binding protein such as calmodulin, calcineurin, and synaptotagmin. Structural changes in response to calcium ion concentrations allow these proteins to function in memory formation and other neurochemical roles. Metallochaperones help to achieve metal ion homeostasis and thus prevent neurological diseases because of metal ion imbalance. Much detailed chemical information about these systems has become available recently. Zinc is another important metal ion in neuroscience. Its concentration in brain is in part controlled by metallothionein, and zinc is released in the hippocampus at glutamatergic synapses. New fluorescent sensors have become available to help track such zinc release.

Animals↗

PubMed2003

Nature of full-length HMGB1 binding to cisplatin-modified DNA.

HMGB1, a highly conserved non-histone DNA-binding protein, interacts with specific DNA structural motifs such as those encountered at cisplatin damage, four-way junctions, and supercoils. The interaction of full-length HMGB1, containing two tandem HMG box domains and a C-terminal acidic tail, with cisplatin-modified DNA was investigated by hydroxyl radical footprinting and electrophoretic gel mobility shift assays. The full-length HMGB1 protein binds to DNA containing a 1,2-intrastrand d(GpG) cross-link mainly through domain A, as revealed by footprinting, with a dissociation constant K(d) of 120 nM. Site-directed mutagenesis of intercalating residues in both HMG domains A and B in full-length HMGB1 further supports the conclusion that only one HMG box domain is bound to the site of cisplatin damage. Interaction of the C-terminal tail with the rest of the HMGB1 protein was examined by EDC cross-linking experiments. The acidic tail mainly interacts with domain B and linker regions rather than domain A in HMGB1. These results illuminate the respective roles of the tandem HMG boxes and the C-terminal acidic tail of HMGB1 in binding to DNA and to the major DNA adducts formed by the anticancer drug cisplatin.

Alanine↗

PubMed2003

Zinc-bound thiolate-disulfide exchange: a strategy for inhibiting metallo-beta-lactamases.

The mononuclear zinc thiolate complexes [(Tp(PhMe))Zn(S-R)], where Tp(PhMe) is hydrotris((3-methyl-5-phenyl)pyrazolyl)borate and (S-R) is benzyl thiolate, 4-nitrophenylthiolate, 4-trifluoromethylphenylthiolate, 4-chlorophenylthiolate, phenylthiolate, 2-methylphenylthiolate, 4-methylphenylthiolate, 4-methoxyphenylthiolate, or 4-hydroxyphenylthiolate, were synthesized. Representative members of the class were also characterized structurally. The benzyl thiolate complex undergoes a thiolate-disulfide exchange reaction with a variety of diphenyl and dipyridyl disulfides. Kinetic studies revealed that the reaction shows saturation behavior in both complex and disulfide for most of the disulfides studied. Combined with studies of the lability of the coordinated thiolate, a mechanism is proposed where the reactive species is the zinc-coordinated thiolate. When the free benzyl thiol was allowed to react with the same disulfides, the reaction was slower by a factor of 20-200 than that for the zinc-thiolate complex, depending on the particular disulfide employed. Since most metallo-beta-lactamases contain one or more cysteine residues, the one in the active site being coordinated to zinc, the present study was extended to examine whether disulfides can be used as inhibitors of these enzymes by selective oxidation of the metal-bound cysteine. Several disulfides allowed to react with metallo-beta-lactamase CcrA from Bacteroides fragilis were moderate to potent irreversible inhibitors of the enzyme.

Bacterial Proteins↗