A de novo binuclear zinc enzyme with DNA cleavage activity.

Metallohydrolases are broadly used throughout biology, often to catalyze the degradation of macromolecules such as DNA and proteins. Many of these enzymes function with zinc in their active site, and an important subset of these enzymes utilize a binuclear zinc active site. Mimics of these enzymes have been developed, some of which catalyze the digestion of DNA. However, the majority of the mimics that utilize zinc are small molecules, and most are mononuclear. Herein, we report DNA cleavage activity by the de novo designed Due Ferri single-chain (DFsc) protein containing a binuclear zinc active site. This binuclear zinc-protein complex is able to digest plasmid DNA at rates up to 50 ng/h, and these cleavage rates are affected by changes to amino acid residues near the zinc-binding site. These results indicate that the DFsc scaffold is a good model system to carry out careful structure-function relationship studies to understand key structural features that influence reactivity in natural binuclear zinc hydrolases, as it is the first report of a binuclear model system in a protein scaffold.

DNA Cleavage by a De Novo Designed Protein-Titanium Complex.

Titanium is one of the most abundant elements on Earth but is commonly thought to have no role in biology because of its propensity to hydrolyze. Nature stabilizes hard Lewis acidic metals from hydrolysis using a variety of mechanisms, providing inspiration for how titanium can be stabilized using biological ligands. The well-characterized Due Ferri single-chain (DFsc) de novo designed protein was developed to bind and stabilize iron and provides a binding site with hard Lewis basic residues able to bind two metal ions. We demonstrate that the DFsc scaffold stably binds 2 equiv of titanium and protects them from unwanted hydrolysis. The Ti4+-DFsc protein complex was tested for its ability to hydrolytically cleave DNA, where it was seen to linearize plasmid DNA in an overnight reaction. Ti4+-DFsc is thus the first example of a functional, soluble titanium-protein complex.

Titanium(IV) and vitamin C: aqueous complexes of a bioactive form of Ti(IV).

Ascorbic acid is among the biorelevant ligands that render Ti(IV) stable in aqueous solution. A series of pH-dependent titanium(IV) coordination complexes of L-ascorbic acid is described. Directed by spectropotentiometric methods, important aspects of the aqueous interactions in this system are investigated, including ligand binding mode, pH-dependent metal-ligand stoichiometry, and the importance of metal ion-promoted hydrolysis and the binding of hydroxide. Stability constants are determined for all metal ion-ligand-proton complexes by a process of model optimization and nonlinear least-squares fitting of the combined spectropotentiometric titration data to the log ß(MLH) values in the model. A speciation diagram is generated from the set of stability constants described in the model. In the range pH 3-10, the aqueous speciation is characterized by the sequential appearance of the following complexes as a function of added base: Ti(asc)(2)(0) → Ti(asc)(3)(2-) → Ti(asc)(2)(OH)(2)(2-) → Ti(asc)(OH)(4)(2-). These species dominate the speciation at pH < 3, pH 4-5, pH ~ 8, and pH > 10, respectively, with minimum log stability constants (ß values) of 25.70, 36.91, 16.43, and -6.91. Results from electrospray mass spectrometry, metal-ligand binding experiments, and kinetics measurements support the speciation, which is characterized by bidentate chelation of the ascorbate dianion to the titanium(IV) ion via proton displacement, and a pH-dependent metal-ligand binding motif of ligand addition followed by metal ion-promoted hydrolysis, stepwise ligand dissociation, and the concomitant binding of hydroxide ion. Additionally, the kinetics of ligand exchange of titanium ascorbate with citrate are reported to understand better the possible fate of titanium ascorbate under biologically relevant conditions.

Pharmaceutical formulation affects titanocene transferrin interactions.

Since the discovery of the anticancer activity of titanocene dichloride (TDC), many derivatives have been developed and evaluated. MKT4, a soluble, water-stable formulation of TDC, was used for both Phase I and Phase II human clinical trials. This formulation is investigated here by using (1)H and (13)C NMR, FT-ICR mass spectrometry, and UV/vis-detected pH-dependent speciation. DFT calculations are also utilized to assess the likelihood of proposed species. Human serum transferrin has been identified as a potential vehicle for the Ti anticancer drugs; these studies examine whether and how formulation of TDC as MKT4 may influence its interactions, both thermodynamic and kinetic, with human serum transferrin by using UV/vis absorption and fluorescence quenching. MKT4 binds differently than TDC to transferrin, showing different kinetics of binding as well as a different molar absorptivity of binding (7500 M(-1) cm(-1) per site). Malate, used in the buffer for MKT4 administration, acts as a synergistic anion for Ti binding, shifting the tyrosine to Ti charge transfer energy and decreasing the molar absorptivity to 5000 M(-1) cm(-1) per site. These differences may have had consequences after the change from TDC to MKT4 in human clinical trials.

Occurrence and mammalian cell toxicity of iodinated disinfection byproducts in drinking water.

An occurrence study was conducted to measure five iodo-acids (iodoacetic acid, bromoiodoacetic acid, (Z)-3-bromo-3-iodo-propenoic acid, (E)-3-bromo-3-iodo-propenoic acid, and (E)-2-iodo-3-methylbutenedioic acid) and two iodo-trihalomethanes (iodo-THMs), (dichloroiodomethane and bromochloroiodomethane) in chloraminated and chlorinated drinking waters from 23 cities in the United States and Canada. Since iodoacetic acid was previouslyfound to be genotoxic in mammalian cells, the iodo-acids and iodo-THMs were analyzed for toxicity. A gas chromatography (GC)/negative chemical ionization-mass spectrometry (MS) method was developed to measure the iodo-acids; iodo-THMs were measured using GC/high resolution electron ionization-MS with isotope dilution. The iodo-acids and iodo-THMs were found in waters from most plants, at maximum levels of 1.7 microg/L (iodoacetic acid), 1.4 microg/L (bromoiodoacetic acid), 0.50 microg/L ((Z)-3-bromo-3-iodopropenoic acid), 0.28 microg/L ((E)-3-bromo-3-iodopropenoic acid), 0.58 microg/L ((E)-2-iodo-3-methylbutenedioic acid), 10.2 microg/L (bromochloroiodomethane), and 7.9 microg/L (dichloroiodomethane). Iodo-acids and iodo-THMs were highest at plants with short free chlorine contact times (< 1 min), and were lowest at a chlorine-only plant or at plants with long free chlorine contact times (> 45 min). Iodide levels in source waters ranged from 0.4 to 104.2 microg/L (when detected), but there was not a consistent correlation between bromide and iodide. The rank order for mammalian cell chronic cytotoxicity of the compounds measured in this study, plus other iodinated compounds, was iodoacetic acid > (E)-3-bromo-2-iodopropenoic acid > iodoform > (E)-3-bromo-3-iodo-propenoic acid > (Z)-3-bromo-3-iodo-propenoic acid > diiodoacetic acid > bromoiodoacetic acid > (E)-2-iodo-3-methylbutenedioic acid > bromodiiodomethane > dibromoiodomethane > bromochloroiodomethane approximately chlorodiiodomethane > dichloroiodomethane. With the exception of iodoform, the iodo-THMs were much less cytotoxic than the iodo-acids. Of the 13 compounds analyzed, 7 were genotoxic; their rank order was iodoacetic acid >> diiodoacetic acid > chlorodiiodomethane > bromoiodoacetic acid > E-2-iodo-3-methylbutenedioic acid > (E)-3-bromo-3-iodo-propenoic acid > (E)-3-bromo-2-iodopropenoic acid. In general, compounds that contain an iodo-group have enhanced mammalian cell cytotoxicity and genotoxicity as compared to their brominated and chlorinated analogues.

Occurrence, synthesis, and mammalian cell cytotoxicity and genotoxicity of haloacetamides: an emerging class of nitrogenous drinking water disinfection byproducts.

The haloacetamides, a class of emerging nitrogenous drinking water disinfection byproduct (DBPs), were analyzed for their chronic cytotoxicity and for the induction of genomic DNA damage in Chinese hamster ovary cells. The rank order for cytotoxicity of 13 haloacetamides was DIAcAm > IAcAm > BAcAm > TBAcAm > BIAcAm > DBCAcAm > CIAcAm > BDCAcAm > DBAcAm > BCAcAm > CAcAm > DCAcAm > TCAcAm. The rank order of their genotoxicity was TBAcAm > DIAcAm approximately equal to IAcAm > BAcAm > DBCAcAm > BIAcAm > BDCAcAm > CIAcAm > BCAcAm > DBAcAm > CAcAm > TCAcAm. DCAcAm was not genotoxic. Cytotoxicity and genotoxicity were primarily determined by the leaving tendency of the halogens and followed the order I > Br > > Cl. With the exception of brominated trihaloacetamides, most of the toxicity rank order was consistent with structure-activity relationship expectations. For di- and trihaloacetamides, the presence of at least one good leaving halogen group (I or Br but not Cl) appears to be critical for significant toxic activity. Log P was not a factor for monohaloacetamides but may play a role in the genotoxicity of trihaloacetamides and possible activation of dihaloacetamides by intracellular GSH and -SH compounds.