Chemical probes into the active centre of a heme thiolate monoxygenase

Linalool-8-monoxygenase, a typical bacterial P-450 heme thiolase, shows a high degree of substrate specificity towards linalool. The active site of the pure enzyme has been probed with a large number of substrate analogues with systematic alterations or conformational variations in the linalool molecule. The comparison of three parameters, the mo→mos conversion of the enzyme as a result of substrate binding monitored at 392 nm, theK D of the analogues giving information about energies of association and the relative turnover as substrate have given information about the space-filling characteristics of the substrates in the enzyme cleft, the number of contacts the molecules make with the respective domains of the enzyme and the distance of the site undergoing hydroxylation from the oxygen site, respectively. The data permit the conclusion that linalool makes contact with the enzyme by hydrogen bonding with the hydroxyl group as well through hydrophobic association with all the eight carbons carrying hydrogen in the molecules.

Synthesis and characterization of Pd(0), PdS, and Pd@PdO core-shell nanoparticles by solventless thermolysis of a Pd-thiolate cluster

Colloids of palladium nanoparticles have been prepared by the solvated metal atom dispersion (SMAD) method. The as-prepared Pd colloid consists of particles with an average diameter of 2.8 +/- 0.1 nm. Digestive ripening of the as-prepared Pd colloid, a process involving refluxing the as-prepared colloid at or near the boiling point of the solvent in the presence of a passivating agent, dodecanethiol resulted in a previously reported Pd-thiolate cluster, Pd(SC12H25)(2)](6) but did not render the expected narrowing down of the particle size distribution. Solventless thermolysis of the Pd-thiolate complex resulted in various Pd systems such as Pd(0), PdS, and Pd@PdO core-shell nanoparticles thus demonstrating its versatility. These I'd nanostructures have been characterized using high-resolution electron microscopy and powder X-ray diffraction methods. (C) 2010 Elsevier Inc. All rights reserved.

Remarkable photocytotoxicity in hypoxic HeLa cells by a dipyridophenazine copper(II) Schiff base thiolate

Copper(II) complexes Cu(satp)(L)] (1-3) of a Schiff base thiolate (salicylidene-2-aminothiophenol, H(2)satP) and phenanthroline bases (L), viz. 1,10-phenanthroline (phen in 1), dipyrido3,2-d:2',3'-f]quinoxaline (dpq in 2) and dipyrido3,2-a:2',3'-c]phenazine (dppz in 3), were prepared, characterized and their anaerobic DNA photocleavage activity and hypoxic photocytotoxicity studied. The redox active complexes show the Cu(II)-Cu(I) couple near -0.5 V for 1 and near 0.0 V vs. SCE (saturated calomel electrode) for 2 and 3. The one-electron paramagnetic complexes (similar to 1.85 mu(B)) are avid DNA binders giving K(b) values within 1.0 x 10(5) - 8.0 x 10(5) M(-1). Thermal melting and viscosity data along with molecular docking calculations suggest DNA groove and/or partial intercalative binding of the complexes. The complexes show anaerobic DNA cleavage activity in red light under argon via type-I pathway, while DNA photocleavage in air proceeds via hydroxyl radical pathway. The DFT (density functional theory) calculations reveal a thyil radical pathway for the anaerobic DNA photocleavage activity and suggest the possibility of generation of a transient copper(I) species due to bond breakage between the copper and sulfur to generate the thyil radical. An oxidation of the copper(I) species is likely by oxygen in an aerobic medium or by the buffer medium in an anaerobic condition. Complex 3 exhibits significant photocytotoxicity in HeLa cells (IC(50) = 8.3(+/- 1.0) mu M) in visible light, while showing lower dark toxicity (IC(50) = 17.2(+/- 1.0) mu M). A significant reduction in the dark toxicity is observed under hypoxic cellular conditions (IC(50) = 30.0(+/- 1.0) mu M in dark), while retaining its photocytotoxicity (IC(50) = 8.0(+/- 1.0) mu M). (C) 2011 Elsevier Inc. All rights reserved.

Synthesis and characterization of some gold(I)-thiolate complexes having N-methylimidazole moiety

Antithyroid drugs inhibit the thyroid hormone synthesis by inactivating the thyroid peroxidase and/or iodothyronine deiodinase, which are involved in iodination and deiodination reactions. Gold(I) compounds also inhibit the thyroid hormone synthesis by interacting with the selenocysteine residue of iodothyronine deiodinase. However, the chemical reactions between these two different classes of compounds have not been studied. In this paper, we describe the interaction of therapeutic gold(I) compounds with the commonly used thiourea-based antithyroid drug, methimazole. It is observed that the gold(I) phosphine complexes (R(3)PAuCl, where R = Me, Et, Ph) react with methimazole only upon deprotonation to produce the corresponding gold(I)-thiolate complexes. Addition of PPh(3) to the gold(I)-thiolates produces (R(3)PAuPPh(3))(+) (R = Me or Et), indicating the possibility of ligand exchange reactions.

Formation of nickel-thiolate aggregates via reaction with CH2Cl2

Reaction of the mononuclear nickel-thiolate complex [Ni(L-1)(dppe)] with CH2Cl2 affords the novel pentanuclear complex Ni5Cl2(L-1)(4)(dppe)(2)], while [Ni(L-1)(dcpe)] reacts with CH2Cl2 to give the binuclear species [Ni2Cl2(L-2)(dcpe)(2)] in which two L-1 units are linked by a methylene group derived from CH2Cl2.

1,3-dipolar cycloaddition of CS2 to the coordinated azide in the cyclopalladated [Pd(bzan)(mu-N-3)](2). Crystal and molecular structure of di(mu-N,S-1,2,3,4-thiatriazole-5-thiolate)bis[(benzylideneaniline-C-2,N)palladium(II)]

The compound [Pd(bzan)(mu -N-3)](2) 1, bzan = benzylideneaniline, was prepared from [Pd(bzan) (mu -OOCCH3)](2) by an anion exchange reaction. The 1,3-dipolar cycloaddition of carbon disulfide to the bridged coordinated azide in the cyclometallated compound I was investigated. The species resulting from this reaction, di(mu -N,S-1,2,3,4-thiatriazol-5-thiolate)bis[(benzylideneaniline)palladium(II)] 2, was characterized by IR spectroscopy and X-ray diffraction. The compound 2 is a dimer containing two [Pd(benzylideneaniline)] moieties connected by two vicinal bridging N,S-1,2,3,4-thiatriazole-5-thiolate anions in a square-planar coordination geometry for the palladium atoms.

Cycloaddition reaction of the azido-bridged cyclometallated complex [Pd(dmba)N-3](2) with CS2. Crystal and molecular structure of DI(mu, N,S-1,2,3,4-thiatriazole-5-thiolate) bis[(N,N-dimethylbenzyl-amine-C-2,N)palladium(II)]

The 1,3-dipolar cycloaddition of carbon disulfide to the coordinated azide in the cyclometallated compound [Pd(dmba)(N-3)](2) (1), dmba = N,N-dimethylbenzylamine, was investigated. The compound obtained di(mu, N,S-1,2,3,4-thiatriazole-5-thiolate)-bis[(N,N-dimethylbenzylamine-C-2,N)palladium(II)] (2), was characterized by IR spectroscopy and X-ray diffraction. Complex (2) is dimeric with the two [Pd(N,N-dimethylbenzylamine)] moieties being connected by the two vicinal bridging N,S-1,2,3,4-thiatriazole-5-thiolate anions in a square-planar coordination for the palladium atoms.

SYNTHESIS AND CRYSTAL-STRUCTURE OF THE NOVEL CYCLOPALLADATED COMPLEX DI(MU,N-S-ETA(2)-QUINOLINE-2-THIOLATE)-BIS[(N,N-DIMETHYLBENZYLAMINE-C(2),N) PALLADIUM(II)]

The compound di(mu,N-Seta2-2-quinoline-2-thiolate)-bis[(N,N-dimethylbenzylamine-C2,N)palladium(II)] was synthesized and studied by IR, NMR and X-ray diffraction: monoclinic, a = 20.138(3), b = 10.831(1), c = 14.973(2) angstrom, beta = 98.04(1)-degrees, Z = 4, space group P2(1)/c, R = 0.032. The compound is dimeric with the two [Pd(N,N-dimethylbenzylamine)]moieties being connected by the two vicinal bridging eta2-N,S-quinoline-2-thiolate anions in a square-planar coordination geometry for the palladium atoms.

Stretching of BDT-gold molecular junctions: thiol or thiolate termination?

It is often assumed that the hydrogen atoms in the thiol groups of a benzene-1,4-dithiol dissociate when Au-benzene-1,4-dithiol-Au junctions are formed. We demonstrate, by stability and transport property calculations, that this assumption cannot be made. We show that the dissociative adsorption of methanethiol and benzene-1,4-dithiol molecules on a flat Au(111) surface is energetically unfavorable and that the activation barrier for this reaction is as high as 1 eV. For the molecule in the junction, our results show, for all electrode geometries studied, that the thiol junctions are energetically more stable than their thiolate counterparts. Due to the fact that density functional theory (DFT) within the local density approximation (LDA) underestimates the energy difference between the lowest unoccupied molecular orbital and the highest occupied molecular orbital by several electron-volts, and that it does not capture the renormalization of the energy levels due to the image charge effect, the conductance of the Au-benzene-1,4-dithiol-Au junctions is overestimated. After taking into account corrections due to image charge effects by means of constrained-DFT calculations and electrostatic classical models, we apply a scissor operator to correct the DFT energy level positions, and calculate the transport properties of the thiol and thiolate molecular junctions as a function of the electrode separation. For the thiol junctions, we show that the conductance decreases as the electrode separation increases, whereas the opposite trend is found for the thiolate junctions. Both behaviors have been observed in experiments, therefore pointing to the possible coexistence of both thiol and thiolate junctions. Moreover, the corrected conductance values, for both thiol and thiolate, are up to two orders of magnitude smaller than those calculated with DFT-LDA. This brings the theoretical results in quantitatively good agreement with experimental data.

Reaction of pentacarbonyliron with a nitrogen heterocycle. X-ray crystal structure of bis[(carbonyl)(quinoline-2-thiolate-N,S)]iron(II)

The monomeric compound bis[(carbonyl)(quinoline-2-thiolate-N,S)]iron(II) was synthesized and studied by IR and Mossbauer spectroscopy, cyclic voltammetry and X-ray diffraction. The molecule has two terminal carbonyl groups and two quinoline-2-thiolate anions coordinated as N,S-donor chelates, and the iron atom shows an octahedral coordination geometry.

Replacement of the proximal heme thiolate ligand in chloroperoxidase with a histidine residue

Chloroperoxidase is a versatile heme enzyme which can cross over the catalytic boundaries of other oxidative hemoproteins and perform multiple functions. Chloroperoxidase, in addition to catalyzing classical peroxidative reactions, also acts as a P450 cytochrome and a potent catalase. The multiple functions of chloroperoxidase must be derived from its unique active site structure. Chloroperoxidase possesses a proximal cysteine thiolate heme iron ligand analogous to the P450 cytochromes; however, unlike the P450 enzymes, chloroperoxidase possesses a very polar environment distal to its heme prosthetic group and contains a glutamic acid residue in close proximity to the heme iron. The presence of a thiolate ligand in chloroperoxidase has long been thought to play an essential role in its chlorination and epoxidation activities; however, the research reported in this paper proves that hypothesis to be invalid. To explore the role of Cys-29, the amino acid residue supplying the thiolate ligand in chloroperoxidase, Cys-29 has been replaced with a histidine residue. Mutant clones of the chloroperoxidase genome have been expressed in a Caldariomyces fumago expression system by using gene replacement rather than gene insertion technology. C. fumago produces wild-type chloroperoxidase, thus requiring gene replacement of the wild type by the mutant gene. To the best of our knowledge, this is the first time that gene replacement has been reported for this type of fungus. The recombinant histidine mutants retain most of their chlorination, peroxidation, epoxidation, and catalase activities. These results downplay the importance of a thiolate ligand in chloroperoxidase and suggest that the distal environment of the heme active site plays the major role in maintaining the diverse activities of this enzyme.

Tailoring heme -thiolate proteins into efficient biocatalysts with high specificity and selectivity

Cytochrome P450 monooxygenases, one of the most important classes of heme-thiolate proteins, have attracted considerable interest in the biochemical community because of its catalytic versatility, substrate diversity and great number in the superfamily. Although P450s are capable of catalyzing numerous difficult oxidation reactions, the relatively low stability, low turnover rates and the need of electron-donating cofactors have limited their practical biotechnological and pharmaceutical applications as isolated enzymes. The goal of this study is to tailor such heme-thiolate proteins into efficient biocatalysts with high specificity and selectivity by protein engineering and to better understand the structure-function relationship in cytochromes P450. In the effort to engineer P450cam, the prototype member of the P450 superfamily, into an efficient peroxygenase that utilizes hydrogen peroxide via the “peroxide-shunt” pathway, site-directed mutagenesis has been used to elucidate the critical roles of hydrophobic residues in the active site. Various biophysical, biochemical and spectroscopic techniques have been utilized to investigate the wild-type and mutant proteins. Three important P450cam variants were obtained showing distinct structural and functional features. In P450camV247H mutant, which exhibited almost identical spectral properties with the wild-type, it is demonstrated that a single amino acid switch turned the monooxygenase into an efficient preoxidase by increasing the peroxidase activity nearly one thousand folds. In order to tune the distal pocket of P450cam with polar residues, Leu 246 was replaced with a basic residue, lysine, resulting in a mutant with spectral features identical to P420, the inactive species of P450. But this inactive-species-like mutant showed catalytic activities without the facilitation of any cofactors. By substituting Gly 248 with a histidine, a novel Cys-Fe-His ligation set was obtained in P450cam which represented the very rare case of His ligation in heme-thiolate proteins. In addition to serving as a convenient model for hemoprotein structural studies, the G248H mutant also provided evidence about the nature of the axial ligand in cytochrome P420 and other engineered hemoproteins with thiolate ligations. Furthermore, attempts have been made to replace the proximal ligand in sperm whale myoglobin to construct a heme-thiolate protein model by mimicking the protein environment of cytochrome P450cam and chloroperoxidase.

Tailoring Heme-Thiolate Proteins into Efficient Biocatalysts with High Specificity and Selectivity

Cytochrome P450 monooxygenases, one of the most important classes of heme-thiolate proteins, have attracted considerable interest in the biochemical community because of its catalytic versatility, substrate diversity and great number in the superfamily. Although P450s are capable of catalyzing numerous difficult oxidation reactions, the relatively low stability, low turnover rates and the need of electron-donating cofactors have limited their practical biotechnological and pharmaceutical applications as isolated enzymes. The goal of this study is to tailor such heme-thiolate proteins into efficient biocatalysts with high specificity and selectivity by protein engineering and to better understand the structure-function relationship in cytochromes P450. In the effort to engineer P450cam, the prototype member of the P450 superfamily, into an efficient peroxygenase that utilizes hydrogen peroxide via the “peroxide-shunt” pathway, site-directed mutagenesis has been used to elucidate the critical roles of hydrophobic residues in the active site. Various biophysical, biochemical and spectroscopic techniques have been utilized to investigate the wild-type and mutant proteins. Three important P450cam variants were obtained showing distinct structural and functional features. In P450camV247H mutant, which exhibited almost identical spectral properties with the wild-type, it is demonstrated that a single amino acid switch turned the monooxygenase into an efficient preoxidase by increasing the peroxidase activity nearly one thousand folds. In order to tune the distal pocket of P450cam with polar residues, Leu 246 was replaced with a basic residue, lysine, resulting in a mutant with spectral features identical to P420, the inactive species of P450. But this inactive-species-like mutant showed catalytic activities without the facilitation of any cofactors. By substituting Gly 248 with a histidine, a novel Cys-Fe-His ligation set was obtained in P450cam which represented the very rare case of His ligation in heme-thiolate proteins. In addition to serving as a convenient model for hemoprotein structural studies, the G248H mutant also provided evidence about the nature of the axial ligand in cytochrome P420 and other engineered hemoproteins with thiolate ligations. Furthermore, attempts have been made to replace the proximal ligand in sperm whale myoglobin to construct a heme-thiolate protein model by mimicking the protein environment of cytochrome P450cam and chloroperoxidase.